WO2024042489A1

WO2024042489A1 - Chemical modification of guide rnas with locked nucleic acid for rna guided nuclease-mediated gene editing

Info

Publication number: WO2024042489A1
Application number: PCT/IB2023/058418
Authority: WO
Inventors: Michael Coyle; Alexandra Briner CRAWLEY; Tedd D. Elich; Drew KELSO; Hui-Chia YU-KEMP; David Wiley
Original assignee: Life Edit Therapeutics Inc
Current assignee: Life Edit Therapeutics Inc
Priority date: 2022-08-25
Filing date: 2023-08-25
Publication date: 2024-02-29
Anticipated expiration: 2025-02-25
Also published as: CA3265664A1; CN120322549A; KR20250075747A; EP4577650A1; JP2025529915A; TW202424186A; AU2023331255A1

Abstract

Compositions including engineered and/or chemically modified transactivating CRISPR RNA (tracrRNA), guide RNA (gRNA), and/or CRISPR RNA (crRNA) are provided. The chemically modified tracrRNA, gRNA, and/or crRNA incorporate bridged nucleic acid (BNA) modifications and other chemical modifications. BNA modifications include 2',4' locked nucleic acid (LNA) modifications. Additional chemical modifications include 2'-O-methyl (2'-O-Me), 2'-O-methyl 3'phosphorothioate (MS), and phosphorothioate (PS) modifications. The engineered and/or chemically modified tracrRNA, gRNA, and/or crRNA of the disclosure improves gene editing (including base editing and prime editing) efficiency of an RNA-guided nuclease (RGN) system.

Description

CHEMICAL MODIFICATION OF GUIDE RNAS WITH LOCKED NUCLEIC ACID FOR RNA GUIDED NUCLEASE-MEDIATED GENE EDITING

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/373,498, filed August 25, 2022; U.S. Provisional Application No. 63/385,887, filed December 2, 2022; and U.S. Provisional Application No. 63/517,703, filed August 4, 2023, each of which is incorporated by reference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY AS AN XML FILE

The instant application contains a Sequence Listing which has been submitted in xml format via USPTO Patent Center and is hereby incorporated by reference in its entirety. Said xml copy, created on August 24, 2023, is named L103438_1290WO_0235_5_Sequence Listing, and is 2.18 MB in size.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology and gene editing.

BACKGROUND OF THE INVENTION

Targeted genome editing or modification is rapidly becoming an important tool for basic and applied research, as it allows modification of genomes such as: cutting, deleting, and inserting nucleic acids; substituting nucleotides in nucleic acids; and regulating gene expression at specific locations in a genome, along with many other possible modifications. Genome editing systems that use RNA- guided nucleases, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- associated (Cas) proteins of the CRISPR-Cas bacterial system, function by complexing a nuclease, an enzyme that cuts a nucleic acid, with a guide RNA. The hybridization of the guide RNA to a particular target sequence allows editing at a specific location in a genome. Thus, genome editing systems that use RNA-guided nucleases (RGN) can be cost-effective and efficient for editing of genome sequences, as a guide RNA is the programmable component of an RGN system, allowing genome editing of specific target sequences by typically straightforward design of the guide RNA. RGN genome editing systems have been adapted from many microbes, and these systems are classified into two classes, 6 types, and multiple subtypes.

In microbes from which Type II and some Type V RGN systems originate, guide RNAs (gRNAs) are expressed as a two-part RNA system: a CRISPR-RNA (crRNA), containing the spacer sequence which recognizes the target genomic sequence via Watson-Crick base pairing, and scaffold transactivating crRNA (tracrRNA). This two-part guide RNA requires base pairing between regions of a crRNA molecule and a tracrRNA molecule to form a dual guide RNA (dgRNA). For many applications, a chimeric single guide RNA (sgRNA) molecule can be used, which is formed by physically linking the crRNA and tracrRNA with a short flexible loop.

Elements of a guide RNA (e.g., the phosphate backbone, the ribose sugar, the nucleobase) can be chemically modified to, for example, reduce degradation of the guide RNA. Much opportunity exists to delineate the types and/or extent of modifications to guide RNA to improve an RGN system, such as to enhance stability, editing efficiency, and specificity for a target sequence and/or to decrease inflammatory responses associated with toxicity of an RGN system.

BRIEF SUMMARY OF THE INVENTION

Provided herein are compositions including chemically modified transactivating CRISPR RNA (tracrRNA), guide RNA (gRNA), and/or CRISPR RNA (crRNA). The chemically modified tracrRNA, gRNA, and/or crRNA incorporate bridged nucleic acid (BNA) modifications and/or other chemical modifications. In some embodiments, BNA modifications include 2', 4' locked nucleic acid modifications of a nucleotide, in which the 2' oxygen is covalently linked to the 4' carbon via a methylene bridge. In some embodiments, additional modifications include 2'-O-methyl (2'-0-Me), 2'- O-methyl 3' phosphorothioate (MS), and phosphorothioate (PS) modifications. In some embodiments, the chemically modified tracrRNA, gRNA, and/or crRNA of the disclosure improves gene editing efficiency of an RNA-guided nuclease (RGN) system as compared to a reference RGN system comprising tracrRNA, gRNA, and/or crRNA having no BNA modifications. In some embodiments, the chemically modified tracrRNA, gRNA, and/or crRNA of the disclosure allows a dual guide RNA to be used in applications where otherwise a single guide RNA would be required. In some embodiments, the present disclosure provides for use of BNA modifications and/or other chemical modifications within the first stem of a stem loop 1 of a dual guide RNA to enhance the performance of an RGN system in a cell. In some embodiments, the use of BNA modifications and/or other chemical modifications allows the use of a shortened tracrRNA, gRNA, and/or crRNA. In some embodiments, the cells that are gene edited with an RGN system of the disclosure include primary cells. The chemically modified tracrRNA, gRNA, and/or crRNA of the present disclosure can be used with any model system, cell type, and target sequence where an RGN system is applied.

Also provided are methods for achieving RGN-based gene editing in cells using guide RNAs modified with BNA and for increasing gene editing efficiency.

In one aspect, the present disclosure provides a nucleic acid molecule comprising a transactivating CRISPR RNA (tracrRNA), wherein the tracrRNA comprises: (a) an anti-repeat; (b) a tail; and (c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one bridged nucleic acid (BNA) modification. In some embodiments of the above tracrRNA aspect, the at least one BNA modification is within the anti-repeat. In some embodiments of the above tracrRNA aspect, the at least one BNA modification is within the first stem of the anti-repeat. In some embodiments of the above tracrRNA aspect, the at least one BNA modification comprises at least two three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive mucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat. In some embodiments of the above tracrRNA aspect, all nucleotides within the first stem of the anti -repeat comprise BNA modifications.

In some embodiments of the above tracrRNA aspect, the at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above tracrRNA aspect, the at least one BNA modification is not within a bulge of the tracrRNA. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

In some embodiments of the above tracrRNA aspect, the at least one BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification.

In some embodiments of the above tracrRNA aspect, the tracrRNA further comprises at least one other chemical modification. In some embodiments, the at least one other chemical modification is within the anti-repeat of the tracrRNA. In some embodiments, the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA. In some embodiments, the at least one other chemical modification is within the tail of the tracrRNA.

In some embodiments of the above tracrRNA aspect, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2', 4'- di-Ca-OMe modification; 2'-O-methyl 3 'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise MS modifications. In some embodiments of the above tracrRNA aspect, three terminal nucleotides of the tail of the tracrRNA comprise MS modifications and all nucleotides of the first stem of the anti-repeat comprise BNA modifications. In some embodiments, the BNA modifications are LNA modifications.

In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of about 11 nucleotides. In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

In some embodiments of the above tracrRNA aspect, the first stem of the anti-repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence. In some embodiments of the above tracrRNA aspect, the first stem of the anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

In some embodiments of the above tracrRNA aspect, the tracrRNA comprises a total length of 60-80 nt, 80-100 nt, 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, or more than 180 nt.

In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, and 709.

In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

In some embodiments of the above tracrRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

In some embodiments of the above tracrRNA aspect, the tracrRNA is part of a gRNA that is capable of binding to an RGN. In some embodiments, the RGN is a Type II RGN.

In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93. In some embodiments of the above tracrRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

In another aspect, the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BN A) modification.

In some embodiments of the above gRNA aspect, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt. In some embodiments of the above gRNA aspect, the gRNA is a dual guide RNA (dgRNA).

In some embodiments of the above gRNA aspect, the at least one BNA modification is within the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications and phosphorothioate (PS) modifications. In some embodiments of the above gRNA aspect, the at least one BNA modification is not within the second stem of the crRNA repeat.

In some embodiments of the above gRNA aspect, the at least one BNA modification is within the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the first stem of the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat. In some embodiments of the above gRNA aspect, all nucleotides within the first stem of the anti-repeat comprises BNA modifications.

In some embodiments of the above gRNA aspect, the at least one BNA modification is not within the second stem of the anti-repeat. In some embodiments of the above gRNA aspect, the at least one BNA modification is not within a bulge of the gRNA. In some embodiments of the above gRNA aspect, the at least one BNA modification is within the tail of the tracrRNA. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise BNA modifications. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

In some embodiments of the above gRNA aspect, at least three terminal nucleotides in the 3' region of the first stem of the crRNA repeat and all nucleotides in the first stem of the anti-repeat comprise BNA modification. In some embodiments of the above gRNA aspect, all nucleotides in the first stem of the crRNA repeat lack chemical modifications and all nucleotides in the first stem of the anti -repeat comprise BNA modifications.

In some embodiments of the above gRNA aspect, the at least one BNA modification is within the spacer. In some embodiments of the above gRNA aspect, three terminal nucleotides at the 5' region of the spacer comprise BNA modifications. In some embodiments of the above gRNA aspect, the three terminal nucleotides at the 5' region of the spacer comprise both BNA modifications and phosphorothioate (PS) modifications. In some embodiments of the above gRNA aspect, the spacer is 18-30 nucleotides in length.

In some embodiments of the above gRNA aspect, the at least one BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification.

In some embodiments of the above gRNA aspect, the gRNA further comprises at least one other modification. In some embodiments of the above gRNA aspect, the at least one other modification is within the crRNA. In some embodiments of the above gRNA aspect, the at least one other modification is within the 5' region or the 3' region of the crRNA. In some embodiments of the above gRNA aspect, the at least one other modification is within the 5' region and the 3' region of the crRNA.

In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the crRNA repeat of the crRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the first stem of the crRNA repeat. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the spacer of the crRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the anti-repeat of the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA. In some embodiments of the above gRNA aspect, the at least one other chemical modification is within the tail of the tracrRNA.

In some embodiments of the above gRNA aspect, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments of the above gRNA aspect, three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications. In some embodiments of the above gRNA aspect, three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-0-Me modifications.

In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti -repeat comprises a total length of about 11 nucleotides. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat or the first stem of the anti-repeat comprises atotal length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat at the 3 ' region or the first stem of the anti -repeat at the 5 ’ region comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence. In some embodiments of the above gRNA aspect, the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5 ’ region comprises a GC-rich nucleotide sequence, wherein the first stem of the crRNA repeat at the 3 ’ region or the first stem of the anti -repeat at the 5 ’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

In some embodiments of the above gRNA aspect, three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as: (a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides; (b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides; (c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides; (d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides; (e) SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides; or (f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides. In some embodiments of the above aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, and 709.

In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as (a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides; (b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides; (c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides; (d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides; (e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides; (f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides; (g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides; (h) SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides; (i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides; j) SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides; (k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides; (1) SEQ ID NO: 389 or that differs from SEQ ID NO: 389 by 1 or 2 nucleotides; or (m) SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides. In some embodiments of the above gRNA aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as any one of: (a) SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides; (b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides; (c) SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides; (d) SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides; (e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides; (f) SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides; and (g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides. In some embodiments of the above gRNA aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337- 339. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

In some embodiments of the above gRNA aspect, the crRNA repeat has a nucleotide sequence set forth as any one of: (a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides; (b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides; (c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides; (d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides; (e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides; (f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides; (g) SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides; and (h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides. In some embodiments of the above gRNA aspect, the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519. In some embodiments of the above gRNA aspect, the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

In some embodiments of the above gRNA aspect, the crRNA and the tracrRNA are linked by a linker between 3 ’ terminal nucleotide of the crRNA repeat and 5 ’ terminal nucleotide of the antirepeat. In some embodiments, the linker comprises an azide functional group or an alkyne functional group. In some embodiments, the linker is a polynucleotide. In some embodiments, the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG. In some embodiments, the linker has a nucleotide sequence set forth as AAAG.

In some embodiments of the above gRNA aspect, the gRNA is a sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and the spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat, the linker, and the tracrRNA. In some embodiments of the above gRNA aspect, the backbone of the sgRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

In some embodiments of the above gRNA aspect, the sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

In some embodiments of the above gRNA aspect, the gRNA is capable of binding to an RGN. In some embodiments, the RGN is a Type II RGN.

In some embodiments of the above gRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

In some embodiments of the above gRNA aspect, an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

In some embodiments of the above gRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93. In some embodiments of the above gRNA aspect, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

In some embodiments of the above gRNA aspect, the gRNA further comprises an extension comprising an edit template for prime editing.

In yet another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat, wherein the crRNA repeat is capable of hybridizing to an anti -repeat of a tracrRNA to form a guide RNA (gRNA) comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5 ’ region or 3 ’ region of the crRNA.

In yet another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising: (a) a spacer; and (b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5 ’ region or 3 ’ region of the crRNA. In some embodiments, a gRNA comprising the crRNA is capable of binding to an RNA guided nuclease (RGN) that requires a tracrRNA for activity.

In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the transactivating crRNA (tracrRNA) described hereinabove; b) a crRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide. In some embodiments, the tracrRNA and the crRNA form a gRNA. In some embodiments, the RGN system binds a target sequence in a target nucleic acid molecule.

In yet another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the gRNA described hereinabove; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide. In some embodiments, the RGN system binds a target sequence in a target nucleic acid molecule.

In still another aspect, the present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) described hereinabove; b) a tracrRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide. In some embodiments, the tracrRNA and the crRNA form a gRNA. In some embodiments, the RGN system binds a target sequence in a target nucleic acid molecule.

In some embodiments of the above RGN systems aspects, the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence set forth as NNNNCC, NNGRR, NNRYA, or NGG. In some embodiments of the above RGN systems aspects, the gRNA is a sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt. In some embodiments of the above RGN systems aspects, the RGN polypeptide comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

In some embodiments of the above RGN systems aspects, the RGN polypeptide and the gRNA are not found complexed to one another in nature.

In some embodiments of the above RGN systems aspects, the target sequence is a eukaryotic target sequence. In some embodiments, the target sequence has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712. In some embodiments of the above RGN systems aspects, the target sequence is within a cell.

In some embodiments of the above RGN systems aspects, a complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence. In some embodiments, the cleavage generates a double-stranded break. In some embodiments, the cleavage generates a single-stranded break.

In some embodiments of the above RGN systems aspects, the RGN polypeptide is nuclease inactive. In some embodiments of the above RGN systems aspects, the RGN polypeptide is a nickase.

In some embodiments of the above RGN systems aspects, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase.

In some embodiments of the above RGN systems aspects, the RGN polypeptide is fused to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the above RGN systems aspects, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the above RGN systems aspects, the RGN polypeptide is fused to a detectable label. In some embodiments of the above RGN systems aspects, the RGN system further comprises a donor polynucleotide.

In some embodiments of the above RGN systems aspects, the polynucleotide comprising a nucleotide sequence encoding the RGN is an mRNA. In some embodiments, the nucleotide sequence encoding the RGN polypeptide is operably linked to a heterologous promoter. In some embodiments, the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is within a vector.

In another aspect, the present disclosure provides a ribonucleoprotein (RNP) complex comprising an RGN system as described hereinabove.

In still another aspect, the present disclosure provides a cell comprising a nucleic acid molecule comprising a tracrRNA, a gRNA, a crRNA, an RGN system, or an RNP complex as described hereinabove.

In some embodiments of the above aspect, the cell comprises a target sequence capable of being bound by a formed gRNA/RGN polypeptide complex of an RGN system, or by an RNP complex, as described hereinabove. In some embodiments, the target sequence comprises a nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a primary cell. In some embodiments, the primary cell is a T cell. In some embodiments, the eukaryotic cell is a plant cell.

In another aspect, the present disclosure provides a plant comprising a plant cell as described hereinabove.

In another aspect, the present disclosure provides a seed comprising a plant cell as described hereinabove.

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a tracrRNA, a gRNA, a crRNA, an RGN system, an RNP complex, or a cell as described hereinabove.

In another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence.

In some embodiments of the above aspect, the RGN polypeptide or the gRNA further comprises a detectable label, thereby allowing for detection of the target sequence. In some embodiments of the above aspect, the RGN polypeptide or the gRNA further comprises an expression modulator, thereby modulating expression of a target gene comprising the target sequence. In some embodiments of the above aspect, the RGN is fused to a prime editing polypeptide. In some embodiments of the above aspect, the RGN polypeptide is fused to a base-editing polypeptide. In still another aspect, the present disclosure provides a method for cleaving and/or modifying a target nucleic acid molecule that comprises a target sequence comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

In yet another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) as described hereinabove and a CRISPR RNA (crRNA); and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, the assembled RNP complex directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments of the method aspect, the prime editing polypeptide comprises a DNA polymerase. In some embodiments of the method aspect, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase.

In yet another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) as described hereinabove and a CRISPR RNA (crRNA); and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments of the method aspect, the prime editing polypeptide comprises a DNA polymerase. In some embodiments of the method aspect, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase. In some embodiments of the method aspect, the polynucleotide encoding the Type II RGN is an mRNA.

In a further aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) as described hereinabove; and ii) a Type II RNA-guided nuclease (RGN), thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, the assembled RNP complex directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing.

In still another aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) the guide RNA (gRNA) as described hereinabove; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN. In some embodiments of the method aspect, a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence. In some embodiments of the method aspect, the RGN polypeptide is fused to a base-editing polypeptide. In some embodiments, the base-editing polypeptide comprises a deaminase. In some embodiments of the method aspect, the RGN is fused to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the DNA polymerase comprises a reverse transcriptase. In some embodiments of the method aspect, the gRNA further comprises an extension comprising an edit template for prime editing. In some embodiments of the method aspect, the polynucleotide encoding the Type II RGN is an mRNA.

In a further aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) as described hereinabove and a; and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN. In a further aspect, the present disclosure provides a method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) as described hereinabove and a tracrRNA; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN. In some embodiments of the above method aspects, the target sequence comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712. In some embodiments of the above method aspects, the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

In another aspect, the present disclosure provides a method of increasing efficiency of cleaving and/or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering an RGN system or an RNP complex as described hereinabove to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering to the target sequence or to a cell comprising the target sequence a reference RGN system or RNP complex, wherein a tracrRNA, a gRNA, or a crRNA in the reference RGN system or RNP complex does not comprise a bridged nucleic acid (BNA) modification or any chemical modification.

In some embodiments of the above aspect, all nucleotides of the first stem of the anti-repeat of the tracrRNA of an RGN system or of an RNP complex as described hereinabove comprise BNA modifications. In some embodiments of the above aspect, at least three terminal nucleotides at the 3’ region of the first stem of the crRNA repeat of the crRNA comprise BNA modifications. In some embodiments of the above aspect, the BNA modifications comprise LNA modifications. In some embodiments of the above aspect, the BNA modifications comprise cEt modifications. In some embodiments of the above aspect, the efficiency of cleaving and/or modifying the target sequence is increased by 15-fold to 30-fold. In some embodiments of the above aspect, the efficiency of cleaving and/or modifying the target sequence is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence. In some embodiments of the above aspect, the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.

In another aspect, the present disclosure provides a method of engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat and the tracrRNA comprises an anti-repeat; and b) adding or substituting one or more nucleotides in the crRNA repeat and one or more nucleotides in the anti-repeat, wherein the one or more nucleotides added or substituted in the crRNA repeat and the one or more nucleotides added or substituted in the anti -repeat are capable of hybridizing to each other, wherein the 3’ region of the crRNA repeat and the 5 ’ region of the anti-repeat of the engineered gRNA comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a).

In some embodiments of the above aspect, the one or more nucleotides are 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In some embodiments of the above aspect, the added or substituted one or more nucleotides are in the 3' region of the crRNA repeat and in the 5' region of the anti-repeat, and wherein the 3' region of the crRNA repeat and the 5' region of the anti -repeat comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

In some embodiments of the above aspect, the gRNA is a dgRNA. In some embodiments of the above aspect, the gRNA is a sgRNA.

In some embodiments of the above aspect, the method further comprises c) modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA modification.

In some embodiments of the above aspect, the at least one chemical modification is in the crRNA, the tracrRNA, or both. In some embodiments of the above aspect, the at least one chemical modification is in: the crRNA repeat; the anti-repeat; a tail of the tracrRNA; the crRNA repeat and the anti-repeat; or the crRNA repeat, the anti-repeat, and the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is in: a first stem of the crRNA repeat; a first stem of the anti-repeat; a tail of the tracrRNA; the first stem of the crRNA repeat and the first stem of the anti-repeat; or the first stem of the crRNA repeat, the first stem of the anti-repeat, and the tail of the tracrRNA.

In some embodiments of the above aspect, the at least one chemical modification is on 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on consecutive nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat. In some embodiments of the above aspect, the at least one chemical modification is on alternate nucleotides in the first stem of the anti-repeat.

In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on three nucleotides at the 3’ region of the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat and on at least one nucleotide in the first stem of the crRNA repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat, on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat, and on three terminal nucleotides at the 3’ region of the tail of the tracrRNA. In some embodiments of the above aspect, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat, on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA, and on at least one nucleotide at the 3 ’ region of the first stem of the crRNA repeat.

In some embodiments of the above aspect, the at least one chemical modification comprises a BNA modification. In some embodiments, the BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification.

In some embodiments of the above aspect, the editing efficiency of the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5- fold, 10-fold, 20-fold, 50-fold, 100-fold, or more compared to the gRNA provided in step a). In some embodiments of the above aspect, the efficiency of cleaving and/or modifying a target sequence by an RGN system comprising the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold, 100-fold, or more compared to the RGN system comprising the gRNA provided in step a). In some embodiments of the above aspect, the efficiency is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence. In some embodiments of the above aspect, the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.

In another aspect, the present disclosure provides an engineered gRNA produced by the method described hereinabove.

In still another aspect, the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

In yet another aspect, the present disclosure provides a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BN A) modification.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic of a dual guide RNA (dgRNA) showing the pairing of the crRNA and the tracrRNA.

FIGs. 2A and 2B show schematics illustrating the parts of a single guide (sgRNA) (FIG. 2A), and of a dgRNA (FIG. 2B). The parts include: a spacer; a stem loop 1 including a first stem, a first bubble, and a second stem; a stem loop 2 comprising a first stem only; an inter stem loop region (ISR); a stem loop 3 including a loop, a first stem, a first bubble, a second stem, a second bubble, and a third stem; and a tail. Stem loop 1 in the sgRNA includes a loop, while stem loop 1 in the dgRNA does not include a loop. The CRISPR RNA (crRNA) repeat anneals to the anti -repeat of the transactivating CRISPR RNA (tracrRNA) to form stem loop 1.

FIGs. 3A and 3B provide a depiction of the chemical modifications of the dgRNA for APG07433.1 RNA guided nuclease. FIG. 3A shows tracrRNA modification schemes, from left to right: “Stem MS modified”, in which the first stem of stem loop 1 comprises nucleotides with 2'-O- methyl (2'-0-Me) modifications, and with 2'-O-methyl 3'phosphorothioate (MS) modifications at the three terminal nucleotides at the 5' region and MS modifications at the three terminal nucleotides at the 3' region and a 2'-0-Me modification on the fourth nucleotide from the 3’ end of the tracrRNA molecule; “Heavily MS modified” - in addition to the modifications in “Stem MS modified”, adding 2’-0-Me modifications to stem loop 3 through the tail; “Stem LNA Modified” - the first stem of stem loop 1 contains all LNA modifications, and the three terminal nucleotides at the 3' region have MS modifications and the fourth nucleotide from the 3’ end has a 2'-0-Me modification; “Heavily LNA Modified” - in addition to the modifications in “Stem LNA modified”, adding 2'-0-Me modifications throughout most of stem loop 3, with part of the first and second stems of stem loop 3 being LNA modified. FIG. 3B shows a diagram of the modification of crRNAs used in these experiments - both contain 5' and 3' MS modifications, with the version shown on the left also bearing 2'-0-Me modifications on the first stem.

FIG. 4 shows the efficiency of gene editing, measured by knockout of the CD3 surface marker using flow cytometry, in primary human T cells using different combinations of the modified crRNA and modified tracrRNA. Guide RNAs were designed to target the TRAC gene. These data demonstrate that modifications at stem loop 3 abolishes editing activity, while modifications at the stem loop 1 alone preserve editing ability of the dgRNA. The modifications are shown with the same scheme as in FIGs. 3A and 3B. TracrRNAs are shown diagrammed. The control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA. Mock indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process.

FIG. 5 demonstrates that LNA modification enhances the editing efficiency of dgRNA in a RNP complex with purified APG07433.1 protein, which reaches similar levels of editing as sgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The control sgRNA and control dgRNA each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the guide RNA.

FIG. 6 shows LNA-modified dgRNA facilitates a high rate of gene disruption, especially relative to dgRNA that is only end modified (the three terminal nucleotides at both the 5' region and 3' region of the dgRNA have MS modification) or dgRNA that has additional 2-0'-Me modifications (MS/PS mod) at stem loop 1, with an mRNA delivery method, using two exemplary spacers. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The control sgRNA and control dgRNA each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the guide RNA.

FIG. 7 demonstrates the higher potency of LNA-modified dgRNA in gene editing compared to sgRNA, using two exemplary spacers. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA.

FIGs. 8A and 8B show that LNA modification enhances the editing efficiency of sgRNAs for two different RNA-guided nucleases (RGNs), with the RGN being delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). (FIG. 8A) APG07433.1 sgRNA. ‘control_RNP’ and ‘control_mRNA’ indicate conditions without RGN and gRNA with each delivery method, where cells are mixed with nucleofection solution but do not go through the nucleofection process. (FIG. 8B) APG01604 sgRNA. ‘control TRAC’ and ‘control_B2M’ indicate conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. The two different spacer names in the controls indicate that antibodies against TRAC (2275) or B2M (1989) were used in the flow cytometry to establish a no-editing readout. The sgRNAs for each RGN used two exemplary spacers. (-): control sgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA. (+): sgRNA with additional LNA modifications as depicted in each schematic. Each guide RNA schematic shows the LNA modifications in a region of the antirepeat forming the first stem and the MS modifications of the 3 terminal nucleotides at the 5' and 3' regions of the sgRNAs. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker for editing of the TRAC target sequence or immunostaining B2M for editing of the B2M target sequence using flow cytometry.

FIGs. 9A and 9B show that MS modification of stem loop 1 does not enhance editing efficiency. FIG. 9A depicts eight scenarios of 2'-0-Me and/or MS modifications in stem loop 1 and/or stem loop 3 for APG07433.1 sgRNA. FIG. 9B shows that none of the tested sgRNAs with 2'-0-Me and/or MS modifications enhance sgRNA editing as compared to a control sgRNA. Chemical modification at stem loop 3 abolishes sgRNA activity. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery), ‘control’ indicates conditions without RGN and gRNA with each delivery method, where cells are mixed with nucleofection solution but do not go through the nucleofection process. The control sgRNA has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry.

FIGs. 10A-10C show that the amount of LNA modifications correlates with guide RNA editing efficiency in primary human T cells as measured by knockout of the CD3 surface marker (for TRAC target sequence) or immunostaining B2M (for B2M target sequence) using flow cytometry. (FIG. 10A) Gene editing efficiency of APG07433.1 dgRNA with 1, 3, 6, or 11 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. Schematics of the APG07433.1 dgRNA show: the ‘0 LNA @ stem loop 1’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA-modified. The highest editing was achieved when all nucleotides within the region of the anti-repeat forming the first stem of stem loop 1 were LNA-modified. (FIG. 10B) Gene editing efficiency of APG01604 dgRNA with 3 or 7 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. Schematics of the APG01604 dgRNA show: the ‘0 LNA, -’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA- modified. (FIG. 10C) Gene editing efficiency of APG05586 dgRNA with 4 or 9 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. Schematics of the APG05586 dgRNA show: the ‘0 LNA, -’ condition, which includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA; and the ‘LNA-mod.’ condition, which includes the MS modifications of the ‘0 LNA’ plus varying numbers of nucleotides that are LNA-modified. Gene editing was improved for the LNA-modified APG01604 dgRNA and APG05586 dgRNA as compared to the ‘0 LNA’ dgRNA. The dgRNAs for each RGN used two exemplary spacers. Each RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). ‘control_TRAC’ and ‘control_B2M’ indicate, for two different spacers in the gRNA, conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.

FIGs. 11A-11C show that LNA modification maintains or increases gene editing efficiency for shortened APG07433. 1 sgRNAs. (FIG. 11A) Top: the full-length APG07433. 1 sgRNA was shortened by a combination of truncations in various regions of the sgRNA: 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1 and 6 nt deleted from the tail (-10 first stem SL1, -6 tail); 5 nt pairs (10 nt) deleted from the first stem of stem loop 1, 4 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, -4 tail, -2 first stem SL3); and 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1, 6 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, -6 tail, -2 first stem SL3). These shortened APG07433.1 sgRNAs have MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA and serve as controls to assess additional chemical modifications introduced into the first stem of stem loop 1. Bottom: the shortened APG07433.1 sgRNAs as depicted at top but including LNA and MS modifications in the first stem of stem loop 1. (FIG. 1 IB) Gene editing efficiency for shortened APG07433.1 sgRNAs chemically modified as illustrated in FIG. 11A as compared to control full-length APG07433. 1 sgRNA or control shortened APG07433.1 sgRNA. The control full-length and shortened sgRNAs each has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not contain any chemical modifications at stem loop 1. ‘Mock’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process, for each delivery method. The sgRNAs were used at a dilution factor of 1. (FIG. 11C) LNA modification increases editing potency for shortened APG07433.1 sgRNAs. A serial dilution of the sgRNAs were conducted. Data were collected at day 4. The ‘3MS’ full-length and ‘3MS’ shortened sgRNAs are as depicted in FIG. 11A, top. The modified shortened sgRNAs are as depicted in FIG. 11A, bottom. DF = dilution factor. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).

FIGs. 12A and 12B show that LNA modification maintains or increases gene editing efficiency for shortened APG07433.1 dgRNAs with RNP delivery. The ‘M’ shortening and chemical modification scheme performs the best for sgRNA and dgRNA. (FIG. 12A) Chemical modification and shortening schemes for crRNA and tracrRNA. The crRNA is shortened by 5 terminal nt at the 3' region: left, MS modifications at the three terminal nucleotides at the 5' and 3' regions (O and Q represent two exemplary spacers used); right, MS modifications at the three terminal nucleotides at the 5' and 3' regions plus 2’-O-Me modifications within the crRNA repeat forming the first stem of stem loop 1 (P and R represent two exemplary spacers used). tracrRNA: ‘tracr(L)’, the anti-repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 6 nt, and the three terminal nucleotides at the 3' region comprise MS modifications; ‘tracr(M)’, the anti -repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 4 nt, 1 nt pair (2 nt) is deleted from the first stem of stem loop 3, and the three terminal nucleotides at the 3’ region comprise MS modifications; ‘tracr(N)’, the anti-repeat forming the first stem of stem loop 1 is shortened by 5 terminal nt at the 5' region, all nucleotides of the antirepeat forming the first stem of stem loop 1 comprise LNA modifications, the tail is shortened by 6 nt, 1 nt pair (2 nt) is deleted from the first stem of stem loop 3, and the three terminal nucleotides at the 3' region comprise MS modifications. (FIG. 12B) Gene editing efficiency for shortened APG07433.1 dgRNAs chemically modified as illustrated in FIG. 12A and for shortened APG07433. 1 sgRNAs chemically modified as illustrated in FIG. 11A. gRNAs with non-shortened backbones: 1, full-length sgRNA with 1880 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region but not having any chemical modifications elsewhere in the sgRNA; 2, dgRNA with 1880 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but not having any chemical modifications elsewhere in the dgRNA; 3, dgRNA with 1881 spacer having MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but not having any chemical modifications elsewhere in the dgRNA; 4, dgRNA comprising crRNA cr(3) and LNA-modified tracrRNA tracr(4) (see FIGs. 3A and 3B), with 1880 spacer; and 5, dgRNA comprising crRNA cr(3) and LNA-modified tracrRNA tracr(4) (see FIGs. 3A and 3B), with 1881 spacer. sgRNAs with shortened backbones (see FIG. 11A): 6, shortened (L) sgRNA without chemical modifications in the first stem of stem loop 1 ; 7, shortened (M) sgRNA without chemical modifications in the first stem of stem loop 1; 8, shortened (N) sgRNA without chemical modifications in the first stem of stem loop 1; 9, shortened (L) sgRNA with additional chemical modifications in the first stem of stem loop 1; 10, shortened (M) sgRNA with additional chemical modifications in the first stem of stem loop 1; and 11, shortened (N) sgRNA with additional chemical modifications in the first stem of stem loop 1. dgRNAs with shortened backbones: shortened and chemically modified crRNAs (O, Q, P, and R) and tracrRNAs (L, M, N) are as described in FIG. 12A. ‘control’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process, for each delivery method, ‘mock’ indicates conditions without RGN and gRNA, where cells are mixed with nucleofection solution and undergo the nucleofection process, for each delivery method. The sgRNAs and dgRNAs used two exemplary spacers. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).

FIG. 13 shows the designs for testing gene editing efficiency of a shortened (‘M’ backbone, see FIG. 11A), chemically modified APG07433.1 gRNA in a sgRNA format. Top: the shortened ‘M’ APG07433.1 sgRNA is modified with: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (3MS, 3LNA, 3LNA/PS; 3 conditions) and has no additional chemical modifications at the first stem of stem loop 1. Bottom: the shortened ‘M’ APG07433.1 sgRNA is modified with: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (3MS, 3LNA, 3LNA/PS; 3 conditions) and includes MS and/or LNA modifications at the first stem of stem loop 1.

FIG. 14 shows gene editing efficiency for shortened ‘M’ APG07433.1 sgRNAs chemically modified as illustrated in FIG. 13 as compared to a control shortened ‘M’ APG07433.1 sgRNA without any chemical modifications. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). The ‘MS/LNA’, ‘LNA’, and ‘LNA’ below the ‘Mod’ bars indicate the additional MS and/or LNA chemical modifications in the first stem of stem loop 1 in a shortened ‘M’ APG07433.1 sgRNA having 3MS, 3LNA, or 3LNA/PS modifications, respectively.

FIG. 15. shows gene editing efficiency for shortened ‘M’ APG07433.1 sgRNAs chemically modified as illustrated in FIG. 13 as compared to a control shortened ‘M’ APG07433.1 sgRNA without any chemical modifications. A serial dilution of the sgRNAs were conducted. Data were collected at day 4. The ‘3MS’, ‘3LNA’, ‘3LNA PS’ shortened ‘M’ APG07433.1 sgRNAs are as depicted in FIG. 13. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). The ‘MS/LNA’, ‘LNA’, and ‘LNA’ below the ‘Mod’ bars indicate the additional MS and/or LNA chemical modifications at the first stem of stem loop 1 in a shortened ‘M’ APG07433.1 sgRNA having 3MS, 3LNA, or 3LNA/PS modifications, respectively.

FIGs. 16A and 16B. Gene editing efficiencies of a dgRNA with various chemical modifications at the three terminal nucleotides at the 5' region and 3' region. (FIG. 16A, left) A design for testing gene editing efficiency of a wild-type (WT, full-length), chemically modified APG07433. 1 gRNA in a dgRNA format. The WT APG07433.1 gRNA is shown with possible chemical modifications: MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the crRNA; LNA modifications at all nucleotides of the first stem of the anti-repeat; and MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 3' region of the tracrRNA. (FIG. 16A, right) a table showing the 18 total conditions tested given various combinations of 3MS, 3LNA, or 3LNA+PS in the crRNA, 3MS, 3LNA, or 3LNA+PS in the tracrRNA, and two delivery modes of the RGN (RNP and mRNA). (FIG. 16B) Gene editing efficiency for APG07433. 1 dgRNAs with various combinations of chemical modifications as shown in FIG. 16A. All tested dgRNAs have LNA modifications at all nucleotides of the first stem of the anti-repeat. Two exemplary spacers (1880 and 1881) were used. ‘control_TRAC’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process, for each delivery method. The control dgRNA (‘dgl880’ and ‘dgl881’) has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but does not have any chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).

FIGs. 17A and 17B show strategies for rescuing gene editing of RGN systems having dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1. FIG. 17A shows a strategy that includes lengthening the first stem at the end distal to the first bubble of stem loop 1 of APG05586 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2 nucleotide pairs using native sequence of APG05586 pre-crRNA and LNA modification of all nucleotides in the lengthened first stem of the anti -repeat. FIG. 17B show strategies to lengthen the first stem at the end distal to the first bubble of stem loop 1 of APG05586 dgRNA or APG08167 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2 nucleotide pairs using native sequence of the respective pre-crRNAs. FIG. 17B highlights the G:C rich characteristic of the APG07433.1 nucleotide pairs most distal to the first bubble in the first stem of stem loop 1 (i.e. the nucleotides most proximal to the 3’ region of the crRNA and the 5’ region of the tracrRNA). WT APG07433.1 dgRNA having LNA modifications at all 11 nucleotides of the first stem of the anti-repeat achieves the highest gene editing (see FIG. 10A). Therefore, nucleotide sequence from APG07433. 1 most distal to the first bubble of stem loop 1 will be used to lenghthen APG05586 and APG08167 as an alternative approach.

FIG. 18 shows that gene editing is rescued for RGN systems having WT (original) dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1 by lengthening the first stem distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. Two genes were targeted for editing in the experiments, and there were two repeats per target gene. The ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘Unmod’ indicates a dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. ‘LNA’ indicates a dgRNA with 3MS plus LNA modifications at all nucleotides of the first stem of the antirepeat. ‘Native seq’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using native sequence from the respective pre-crRNA. ‘APG07433.1 seq’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA. (The indicated nucleotide lengths are for the first stem of the anti-repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.) All nucleotides of the lengthened first stem of the anti -repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA). A schematic of a APG01604 gRNA shortened in the first stem of stem loop 1 (APG01604.1, 81 nt backbone length) below the graph illustrates the ‘unmod’ APG01604.1 gRNA. The nucleotide sequences above the data points indicate the sequence of the 4 nucleotides at the 5’ terminus of the original, non-lengthened tracrRNA (for ‘unmod’ and ‘LNA’) or the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of a lengthened tracrRNA). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

FIG. 19 shows that lengthening the first stem at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) using either nucleotide sequences from a native pre-crRNA or from APG07433. 1 gRNA and modifying all nucleotides of the first stem of the anti-repeat with LNA rescues gene editing for RGN systems having WT (original) dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1. The dgRNAs were lengthened to 11 nucleotide pairs or 13 nucleotide pairs in the first stem of stem loop 1. ‘native’ indicates a dgRNA with the first stem of the anti-repeat lengthened to the indicated nucleotide length using native sequence from the respective pre-crRNA. ‘APG07433.1’ indicates a dgRNA with the first stem of the anti-repeat lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA. (The indicated nucleotide lengths are for the first stem of the anti-repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.) All nucleotides of the lengthened first stem of the anti -repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA). The nucleotide sequences above the data points indicate the sequence of the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of the lengthened tracrRNA). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

FIG. 20 shows a strategy to improve gene editing efficiency by a shortened dgRNA. Left: schematic of a WT APG07433.1 crRNA. Center: schematic of a shortened ‘M’ APG07433.1 crRNA and tracrRNA. Right: the 3’ 3 terminal nucleotides of the crRNA and the 5’ 2 terminal nucleotides of the tracrRNA are substituted with C and G nucleotides, respectively (the starred nucleotides). For both the shortened (M) and the engineered shortened (M), the first stem of the anti-repeat is LNA modified.

FIG. 21 shows that gene editing efficiency is improved for a shortened ‘M’ APG07433. 1 dgRNA by substituting nucleotides as shown in FIG. 20. Two spacers were tested. ‘Original’ indicates the shortened ‘M’ APG07433.1 dgRNA without nucleotide substitutions. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

FIGs. 22A and 22B show strategies for LNA modification of the anti-repeat forming stem loop 1 of the tracrRNA in a gRNA. FIG. 22A shows a modified APG07433.1 dgRNA that performs well in gene editing, having all 11 nucleotides LNA modified in the first stem (FS) of the anti-repeat (Tracr(J); see FIG. 10A). FIG. 22B shows APG07433.1 tracrRNA modified at all nucleotides in the FS and in the second stem (SS) of the anti-repeat (Tracr(Jb); FS+SS); modified at all nucleotides in the SS of the anti-repeat (Tracr(Jc); SS); and modified at all nucleotides in the anti-repeat, including nucleotides of the first stem, the bubble, and the second stem (Tracr(Jd); full stem loop 1).

FIG. 23 shows that having LNA modification of all nucleotides of the first stem of the antirepeat in the tracrRNA of a gRNA is most effective for gene editing as compared to other LNA modification strategies for the anti-repeat. Gene editing efficiencies are shown for APG07433.1 dgRNAs having LNA-modified tracrRNA (right-hand side of the graph; Tracr(J), Tracr(Jb), Tracr(Jc), and Tracr(Jd)), as shown in FIGs. 22A and 22B. The crRNA had MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS), as shown in FIG. 22A. Two exemplary spacers (1880 and 1881) were used. ‘control_TRAC’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. The control dgRNAs on the left-hand side of the graph has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA but does not have any chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

FIG. 24 shows that LNA modification of all nucleotides of the first stem of the crRNA repeat in the crRNA of a gRNA is effective for gene editing as long as all nucleotides of the first stem of the anti -repeat in the tracrRNA of the gRNA are also LNA-modified (see top graph, right side). Having LNA modification of all nucleotides of the second stem of the crRNA repeat in the crRNA of a gRNA worsens gene editing for a gRNA having LNA modification of all nucleotides of the first stem of the anti-repeat (see bottom graph, right side). Gene editing efficiencies are shown for APG07433.1 dgRNAs having LNA modifications at all nucleotides of the first stem or the second stem of the crRNA repeat: upper left and left side of top graph, dgRNA having crRNA with LNA modifications at all nucleotides of the first stem of the crRNA repeat and tracrRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS); upper right and right side of top graph, dgRNA having crRNA with LNA modifications at all nucleotides of the first stem of the crRNA repeat and tracrRNA with LNA modifications at all nucleotides of the first stem of the anti-repeat; lower left and left side of bottom graph, dgRNA having crRNA with LNA modifications at all nucleotides of the second stem of the crRNA repeat and tracrRNA with 3MS; and lower right and right side of bottom graph, dgRNA having crRNA with LNA modifications at all nucleotides of the second stem of the crRNA repeat and tracrRNA with LNA modifications at all nucleotides of the first stem of the anti-repeat. Two exemplary spacers (1880 and 1881) were used, ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

FIG. 25 shows that LNA modification increases editing potency for APG05586 sgRNAs. A serial dilution of the sgRNAs were conducted. The ‘unmod’ indicates sgRNAs having MS modifications at the three terminal nucleotides at both the 5' region and 3' region and no other chemical modifications (3MS). The ‘LNA @ SL1’ indicate sgRNAs having LNA modifications at all nucleotides of the first stem of the anti-repeat, along with 3MS. The ‘MS/LNA @ SL1’ indicate sgRNAs having LNA modifications at all nucleotides of the first stem of the anti-repeat, MS modifications at the three terminal nucleotides of the crRNA repeat most proximal to the loop of stem loop 1, and 3MS. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery), ‘control (TRAC)’ and ‘control (B2M)’ indicate, for two different spacers in the gRNA, conditions without RGN and gRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process.

FIG. 26 shows that the amount of LNA modification at the first stem of the anti -repeat correlates with guide RNA editing efficiency and melting temperature (Tm) of a DNA/tracrRNA antirepeat heteroduplex. Schematic of APG07433.1 dgRNA showing 1, 3, 6, or 11 LNA-modified nucleotides within a region of the anti-repeat forming the first stem of stem loop 1. The dgRNA includes MS modifications at the 5' and 3' ends of the crRNA and tracrRNA. The highest editing and highest Tm were achieved when all nucleotides within the region of the anti-repeat forming the first stem of stem loop 1 were LNA-modified. The amount of LNA modification is shown on the x-axis, and the Tm and gene editing efficiency are shown on the vertical axes. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Two spacers (1062 and 1881) were tested in the gene editing experiments.

FIG. 27 shows that gene editing is rescued for an APG07991 RGN system having WT (original) dgRNA with < 11 (6) nucleotide pairs in the first stem of stem loop 1 (see left schematic) by lengthening the first stem distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. Two genes were targeted for editing in the experiments, and there were two repeats per target gene. The ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘Unmod’ indicates a dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. ‘LNA’ indicates a dgRNA with 3MS plus LNA modifications at all nucleotides of the first stem of the anti-repeat. ‘Native’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using native sequence from the APG07991pre-crRNA. ‘APG07433.1’ indicates a dgRNA with the first stem of stem loop 1 lengthened to the indicated nucleotide length using sequence from APG07433.1 gRNA. (The indicated nucleotide lengths are for the first stem of the anti -repeat, and an identical nucleotide length would be expected on the first stem of the crRNA repeat for base pairing.) All nucleotides of the lengthened first stem of the anti-repeat are modified with LNA, and the first stem is lengthened at the end distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA). The nucleotide sequences above the data points indicate the sequence of the 6 nucleotides at the 5 ’ terminus of the original, non-lengthened tracrRNA (for ‘unmod’ and ‘LNA’) or the added 2 terminal nucleotides (the 2 nucleotides at the 5’ terminus of a lengthened tracrRNA). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

FIG. 28 shows that gene editing efficiency for APG07991 dgRNA can be rescued by lengthening the first stem distal to the first bubble of stem loop 1 to at least 11 nucleotide pairs (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘sgRNA’ indicates an APG07991 sgRNA control with the appropriate spacer (TRAC or B2M) that has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not have any chemical modifications elsewhere in the sgRNA. The numbers 6, 8, 10, 11, 12, and 13 indicate the length of the first stem of stem loop 1. ‘Native Seq’ indicates that native sequence from the APG07991 pre-crRNA was used to lengthen the first stem of stem loop 1 of a WT APG07991 dgRNA to the indicated nucleotide length. ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT APG07991 dgRNA to the indicated nucleotide length. ‘-’ and ‘+’ indicate whether all nucleotides of the first stem of the anti-repeat are modified with LNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG07991 RGN was delivered as mRNA encoding the APG07991 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

FIG. 29 shows that gene editing efficiency for Streptococcus pyogenes Cas9 (SpyCas9) dgRNA, which works with APG07991 RGN for gene editing, can also be rescued by lengthening the first stem distal to the first bubble of stem loop 1 to at least 11 nucleotide pairs (i.e. lengthening at the 3 ’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the anti-repeat with LNA. The first stem of stem loop 1 of WT (original) SpyCas9 dgRNA has 4 nucleotide pairs (see left schematic) and has very low gene editing with APG07991 RGN. ‘control’ indicates conditions without RGN and dgRNA, where cells are mixed with nucleofection solution but do not go through the nucleofection process. ‘sgRNA’ indicates a SpyCas9 sgRNA control with the appropriate spacer (TRAC or B2M) that has MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA but does not have any chemical modifications elsewhere in the sgRNA. The numbers 4, 8, 11, and 13 indicate the length of the first stem of stem loop 1. ‘Native Seq’ indicates that native sequence from the SpyCas9 pre- crRNA was used to lengthen the first stem of stem loop 1 of a WT SpyCas9 dgRNA to the indicated nucleotide length. ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT SpyCas9 dgRNA to the indicated nucleotide length. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG07991 RGN was delivered as mRNA encoding the APG07991 RGN (mRNA delivery). Two exemplary spacers (targeting the TRAC and B2M genes) were used.

FIG. 30 shows that LNA modifications of all nucleotides of the anti-repeat forming the first stem of stem loop 1 confer more stability to an sgRNA. Left schematic: (A) End modified sgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions of the sgRNA; (B) LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5 ’ and 3 ’ regions and LNA modifications at all nucleotides of the first stem of the anti-repeat; (C) MS/LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions, MS modifications of the three terminal nucleotides at the 3 ’ region of the crRNA repeat, and LNA modifications at all nucleotides of the first stem of the anti-repeat; and (D) MS/LNA modified dgRNA has MS modifications of the three terminal nucleotides at both the 5’ and 3’ regions of the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications of the three terminal nucleotides at the 3’ region of the tracrRNA. MS/LNA modified dgRNA enables effective gene editing with co-delivery of the mRNA and gRNA components, but is not as stable as the chemically modified sgRNAs, as demonstrated in the case of staggered delivery.

FIG. 31 shows that base editing efficiency is increased using gRNAs having LNA modifications. The tested gRNAs are as described in FIG. 30. The A, B, C, and D of the graphs correspond to the A, B, C, and D depicted in the left schematic.

FIG. 32 shows that base editing efficiency is increased using gRNAs having LNA modifications and also shortened gRNAs having LNA modifications. The No LNA, LNA, and MS/LNA of the graphs correspond to what is depicted in the left schematic, ‘shrt’ indicates a shortened sgRNA. gRNAs with two exemplary spacers, SGN001880 and SGN001881, were used.

FIG. 33 shows that gene editing efficiency for a dgRNA chemically modified with another bridged nucleic acid (BNA), cEt, is enhanced to comparable levels of the gene editing efficiency of a LNA-modified dgRNA. The gene editing efficiency of APG07433. 1 dgRNA with 11 LNA-modified nucleotides within the anti-repeat forming the first stem of stem loop 1 (depicted in upper left schematic, see also FIG. 10A) was compared to that of APG07433. 1 dgRNA with 11 S-constrained ethyl (cEt) -modified nucleotides within the anti-repeat forming the first stem of stem loop 1 (depicted in lower left schematic). ‘Unmod’ indicates the dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG07433.1 RGN was delivered as mRNA encoding the APG07433.1 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

FIG. 34 shows that gene editing efficiency for a dgRNA lengthened at the first stem distal to the first bubble of stem loop 1 and chemically modified with another bridged nucleic acid (BNA), cEt, is enhanced to comparable levels of the gene editing efficiency of the same lengthened dgRNA that has been LNA-modified. APG05586 dgRNA was lengthened at the first stem distal to the first bubble of stem loop 1 to 11 nucleotide pairs (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA; see FIGs. 18 and 19) and all nucleotides of the first stem of the anti-repeat were modified with either LNA (depicted in upper left schematic) or with S- constrained ethyl (cEt) (depicted in lower left schematic). ‘Native seq for APG05586’ indicates that native sequence from the APG05586 pre-crRNA was used to lengthen the first stem of stem loop 1 of a WT APG05586 dgRNA to 11 nucleotides. ‘APG07433.1 Seq’ indicates that sequence from the APG07433.1 gRNA was used to lengthen the first stem of stem loop 1 of a WT APG05586 dgRNA to 11 nucleotides. ‘Original’ indicates WT APG05586 dgRNA having 9 nucleotide pairs in the first stem of stem loop 1 that has not been lengthened. ‘Unmod’ indicates the dgRNA with MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining B2M (for editing of B2M target sequences) using flow cytometry. The APG05586 RGN was delivered as mRNA encoding the APG05586 RGN (mRNA delivery). Two exemplary spacers (TRAC and B2M) were used.

FIG. 35 shows that LNA modification improved gene editing efficiency of a dgRNA having an extension. The top of FIG. 35 shows schematics illustrating three chemically modified dgRNAs where the extensions are at the tail of the tracrRNA: (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); (b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat , and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of the stem loop most proximal to the tail of the tracrRNA (i.e. stem loop 3 for the depicted system), and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension. The left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA corresponding to a dgRNA that does not have the extension, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the guide RNA, and has no LNA modifications. The control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without the extension. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery). SL1 = stem loop 1. SL3 = stem loop 3.

FIGs. 36A-36C show schematics of guide RNAs having extensions and details of the engineering of a shortened guide RNA. FIG. 36A, a non-engineered guide RNA + extension has: a crRNA length of 46 nt (a spacer + 21 nt crRNA repeat); and a tracrRNA length of 79 nt (85 nt WT length minus 6 nt from the tail). FIG. 36B, a shortened guide RNA + extension has a shortened crRNA/tracrRNA backbone and has: a crRNA length of 41 nt (a spacer + 16 nt shortened crRNA repeat); and a tracrRNA length of 72 nt. FIG. 36C, schematic of the engineered shortened crRNA and tracrRNA (except without the extension), showing the 3' 3 terminal nucleotides of the crRNA and the 5' 2 terminal nucleotides of the tracrRNA are substituted with C and G nucleotides (the starred nucleotides). For both dgRNAs + extensions shown in FIGs. 36A and 36B, the first stem of the antirepeat is LNA modified.

FIG. 37 shows that LNA modification improved gene editing efficiency of shortened dgRNAs + extensions that are engineered with nucleotide substitutions at the 3' end of the crRNA and 5' end of the tracrRNA. a, dgRNA + extension with engineered shortened backbone and having MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); and b, dgRNA + extension with engineered shortened backbone and having 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension. The left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA that does not have an extension or shortened backbone or nucleotide engineering, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the guide RNA, and has no LNA modifications. The control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without an extension or shortened backbone or nucleotide engineering. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery). SL1 = stem loop 1. SL3 = stem loop 3.

FIG. 38 shows that LNA modification at the first stem of the anti-repeat is the key for editing efficiency of dgRNA + extension, (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends); (b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti -repeat, and MS modifications at the three terminal nucleotides at the 3 ' region of the tracrRNA + extension; (c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of the stem loop most proximal to the tail of the tracrRNA (stem loop 3 in this system), and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (d) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, and 3MS ends for the tracrRNA + extension; (e) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the antirepeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (f) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of stem loop 3, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (g) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, and 3MS ends for the tracrRNA + extension; (h) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension, (g) and (h) dgRNAs + extensions have the shortened backbone engineered as described in FIG. 36C. The left bar in the graph labeled ‘single’ indicates gene editing efficiency for a control single guide RNA that does not have an extension or shortened backbone or nucleotide engineering, has MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS) of the guide RNA, and has no LNA modifications. The control single guide RNA serves as a benchmark of gene editing efficiency for a guide RNA without an extension or shortened backbone or nucleotide engineering. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry. Data were collected at day 4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery). SL1 = stem loop 1. SL3 = stem loop 3.

DETAILED DESCRIPTION

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

I. Overview

The present disclosure provides, inter alia, compositions and methods related to modified guide RNA (gRNA) for use in RNA-guided nuclease (RGN) systems, and systems and methods related thereto. In various embodiments, inclusion of one or more bridged nucleic acid, such as, e.g., locked nucleic acid, within certain regions of the gRNA (but in some embodiments, not within other regions) improves editing efficiency of RGN systems. Additionally, such modifications unlock editing potential in mRNA-based dual guide systems not previously possible with unmodified dual guide RNAs available in the prior art. This is a critical advancement in the art, as new editing modalities, such as prime editing (also known as reverse transcriptase editing or, RT-editing), require very long gRNA templates that are difficult if not impossible to synthesize at scale in single guide format using current manufacturing processes. Thus, lack of manufacturing feasibility in the art significantly limits the commercialization potential of these mRNA-based therapeutics. These and other advancements are presented in the present disclosure.

An RGN system allows for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications. In a variety of organisms, including mammals, RGN systems have been used for creating single- or double-stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene, for example. An RGN system involves a complex of an RGN with a gRNA. The hybridization of the gRNA to a particular target sequence allows targeting of the guide RNA/RGN complex to a specific location in a genome for editing.

In RGN systems, gRNAs can exist as a two-part gRNA or as a single gRNA. The two-part gRNA system includes a CRISPR RNA (crRNA) containing a spacer sequence which recognizes the target genomic sequence via Watson-Crick base pairing, and a scaffold transactivating crRNA (tracrRNA). The crRNA hybridizes to the tracrRNA to form a dual guide RNA (dgRNA) held together as a duplex at a crRNA IracrRNA annealing region. For many applications, a chimeric single guide RNA (sgRNA) molecule can be used, which is formed by physically linking the crRNA and tracrRNA with a short flexible loop.

There are advantages and disadvantages to both the dgRNA and the sgRNA. sgRNAs generally provide relatively good editing efficiency. However, a sgRNA, while somewhat convenient as a single chemical species, is relatively long - generally at or above 100 nt. A common method to produce gRNAs is through solid phase oligonucleotide synthesis, which is a serial synthetic route. With increasing length, yield and purity of the full-length product are low despite high coupling efficiency of each individual step (Reese, Org Biomol Chem, 2005; Beaucage & Reese, Curr Protoc Nucleic Acid Chem, 2009; Beaucage, Curr Opin Drug Di De, 2008; Shiba, Nucleic Acids, 2WT, LeProust Nuc. Acids Res. 2010). Therefore, the ability to use shorter gRNAs and/or dgRNAs has some utility in reducing the maximum length of oligonucleotides to be synthesized. Another advantage of using a dgRNA is that the tracrRNA is paired with different crRNAs that contain varied spacer sequences, so tracrRNA scaffolds can be made in large batches and paired with individual crRNAs for specific genomic targets.

However, a dgRNA has relatively low gene editing efficiency, especially in a delivery method where mRNA encoding an RGN is introduced to a cell for expression of the RGN. The additional exposed 5' and 3' ends of the crRNA and tracrRNA and likely weaker duplex strength relative to sgRNA (in which intramolecular components are hybridizing) may result in lower RNA stability and hence poor performance. In some embodiments, strengthening the duplex can enhance and rescue the effectiveness of dgRNA.

A number of chemical modifications including 2'-fluoro-ribose (2'-F), 2'-O-methyl (2'-O- Me), 3' phosphorothioate (PS), 2'-O-Me 3' phosphorothioate (MS), along with other modifications, have been used to improve gRNA stability and thus increase editing efficiency in cells ex vivo and in vivo. However, these methods fail to enable effective editing when transfecting RGN-encoding mRNA with dgRNA. Instead, they require long sgRNAs to be produced. With new technologies such as prime editing using additional guide extensions, sgRNAs can be infeasible to produce at scale and with high levels of purity. Additionally, while the aforementioned modifications protect the RNA from nuclease degradation, it is possible that other mechanisms can lead to guide degradation or inactivation, notably disruption of the secondary structure. A bridged nucleic acid (BNA) includes a nucleotide analog having a restricted conformation due to an intramolecular bond or crosslink. A type of BNA, a locked nucleic acid (LNA), has been used, for example, in the design of PCR probes to shorten the length of the probes and to increase hybridization strength. An LNA includes a covalent linkage between the 2' oxygen to the 4' carbon on the ribose sugar of a nucleotide and has been shown to contribute to highly efficient complementary pairing to improve mismatch discrimination, as well as to improve nuclease resistance (Y on et al. Nucleic Acids Res., 2006; Vester & Wengel, J Biochemistry, 2004). Provided herein are methods for achieving RGN-based gene editing in cells using guide RNAs modified with BNA modifications. Data in this application demonstrate that such guide RNAs modified with BNA modifications are improved over prior art methods, significantly enhancing the potency of the guide RNA and enabling the use of dual guide RNA in applications where it otherwise would not be suitable, such as when gRNA is co-transfected with RGN-encoding mRNA. Without being limited by conjecture, we believe that these benefits are at least because the BNA modifications stabilize the stem loop formed by hybridization between a crRNA repeat and a tracr RNA anti-repeat. In some instances, the BNA is LNA. In some instances, the BNA is S- constrained ethyl (cEt). In some instances, methods are provided herein for achieving RGN-based gene editing in cells using guide RNAs modified with BNA modifications and/or other chemical modifications. In various embodiments, the present disclosure incorporates BNA (e.g., LNA and/or cEt) or other chemical modifications in tracrRNA, gRNA, and/or crRNA to generate chemically modified tracrRNA, gRNA, and/or crRNA for use in RGN-based gene editing systems. In some embodiments, the crRNA and tracrRNA both comprise BNA (e.g., LNA and/or cEt) modifications. In certain embodiments, either the crRNA or the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the tracrRNA comprises BNA modifications whereas the crRNA does not comprise BNA (e.g., LNA and/or cEt) modifications. In certain embodiments, the tracrRNA comprises BNA modifications and the crRNA comprises MS modifications. In embodiments, the chemically modified tracrRNA, gRNA, and/or crRNA of the disclosure improves gene editing efficiency of an RGN system as compared to a reference RGN system with tracrRNA, gRNA, and/or crRNA comprising only MS modifications at the three terminal nucleotides at the 5 ’ and 3’ regions. In embodiments, the modified tracrRNA, gRNA, and/or crRNA of the disclosure allows a dgRNA to be used in applications where otherwise a sgRNA would be more desirable. In some embodiments, the present disclosure provides for use of BNA (e.g., LNA and/or cEt) modifications within the crRNA: tracrRNA annealing region of a dgRNA to enhance the performance (e.g., editing efficiency) of an RGN system in a cell. In some embodiments, BNA (e.g., LNA and/or cEt) modifications allows a shortened crRNA IracrRNA annealing region. In some embodiments, BNA (e.g., LNA) modifications in combination with engineering of the crRNA:tracrRNA annealing region of a dgRNA enhances performance of an RGN system in a cell. In some embodiments, the cells that are gene edited include primary cells. The modified tracrRNA, gRNA, and/or crRNA of the present disclosure can be used with any model system, cell type, and target sequence where an RGN system is applied.

Without being bound by any one theory, chemical modifications to nucleotides in gRNAs may enhance stability by interfering with the degradation of the gRNAs by endogenous nucleases and/or stabilize RNA-RNA interactions in the chemically modified region. II. Guide RNA

The present disclosure provides guide RNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti-repeat of the tracrRNA. The term “guide RNA” is known in the art and generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a RNA-guided nuclease (RGN) and aid in targeting the RGN to a specific location within a target polynucleotide (e.g., a DNA or an mRNA molecule) such as, e.g., a genomic locus. In some embodiments, the guide RNA comprises a nucleotide sequence (i.e., a spacer) having sufficient complementarity with a target strand nucleotide sequence to hybridize with the target strand and direct sequence -specific binding of an RGN to the target nucleotide sequence. In some embodiments, when the target nucleotide sequence is double-stranded as is the case with DNA, the target nucleotide sequence comprises a non-target strand (which comprises the PAM sequence) and the target strand, which hybridizes with the spacer of the guide RNA. In these embodiments, the guide RNA has sufficient complementarity with the target strand of a double-stranded target sequence (e.g., target DNA sequence) such that the guide RNA hybridizes with the target strand and directs sequencespecific binding of an associated RGN to the target sequence (e.g., target DNA sequence). Therefore, in some embodiments, a guide RNA includes a spacer that is identical to the sequence of the nontarget strand except that uracil (U) replaces thymidine (T) in the guide RNA.

An RGN’s respective guide RNA is one or more RNA molecules (generally, one or two), that can bind to the RGN and guide the RGN to bind to a particular target sequence, and in those embodiments wherein the RGN has nickase or nuclease activity, also cleave the target strand and/or the non-target strand. In general, a guide RNA comprises a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA).

The term “guide RNA” also encompasses, collectively, a group of two or more RNA molecules, where the crRNA segment and the tracrRNA segment are located in separate RNA molecules. Native guide RNAs that comprise both a crRNA and a tracrRNA generally comprise two separate RNA molecules that hybridize to each other through the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. In certain embodiments, the crRNA and tracrRNA are linked together by a linker. A "linker" can be any kind of chemical linkage that covalently connects two molecules together, for example, a linkage formed by click chemistry or any other chemical reactions, a polynucleotide, a polymer, or any entity that can link two molecules. In some embodiments, a linker connecting a crRNA and a tracrRNA comprises a multi-nucleotide linker (e.g., a four-nucleotide linker) to form a single guide RNA molecule, wherein the crRNA and the tracrRNA hybridize to each other through the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA. Thus, a guide RNA encompasses a single-guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located in the same RNA molecule or strand.

The crRNA and tracrRNA of a guide RNA can be linked by an organic molecule, group, polymer, or chemical moiety. In some embodiments, the crRNA and tracrRNA of a guide RNA is linked by click chemistry. Click chemistry involves the rapid generation of compounds by joining small units together via heteroatom links (C-X-C). The main objective of click chemistry is to develop a set of powerful, selective, and modular “blocks” that are useful for small- and large-scale applications. Click chemistry reactions are fast, modular, efficient, often do not produce toxic waste products, can be done with water as a solvent, and can be set up to be stereospecific.

Click chemistry is a versatile reaction that can be used for the synthesis of a variety of conjugates. Virtually any biomolecules can be involved, and labeling with small molecules, such as fluorescent dyes, biotin, and other groups can be readily achieved. Click chemistry reaction takes place between two components: an azide functional group and an alkyne functional group. Azide is a linear, polyatomic anion with the formula Ns’ and structure “N=N⁺=N“. It is the conjugate base of hydrazoic acid HN3. Organic azides are organic compounds with the formula RN3, containing the azide functional group. An alkyne is an unsaturated hydrocarbon containing at least one carbon — carbon triple bond (-C=C-; e.g., terminal acetylene). The simplest acyclic alkynes with only one triple bond and no other functional groups form a homologous series with the general chemical formula CnHsn-2. Terminal alkynes have the formula RC2H. An example is methylacetylene (propyne using IUPAC nomenclature). Both azido and alkyne groups are nearly never encountered in natural biomolecules. Hence, the reaction can occur within biological systems without interfering with other cellular processes (i.e. highly bioorthogonal) and specific.

A well-known click reaction is the Huisgen 1,3-dipolar cycloaddition of azides and alkynes. This reaction, yielding triazoles, has become the gold standard of click chemistry for its reliability, specificity, and biocompatibility. Such cycloadditions need high temperatures or pressures when the reaction involves simpler alkene or azides, since the activation energies are high (AG^t»+26 kcal/mol). Cu(I) catalysts expedite the reaction of terminal alkynes and azides, thereby affording 1,4- disubstituted-I,2,3-triazoles. This reaction is an ideal click reaction and is widely employed in material science, medicinal chemistry, and chemical biology.

However, the cytotoxic nature of transition metals, employed as catalysts for the click reactions, preclude their use for in vivo applications. Alternative approaches with lower activation barriers and copper-free reactions have been established. Such reactions are referred to as “copper-free click chemistry”. Instead of using copper to activate the alkyne, the alkyne is instead introduced in a strained difluorooctyne (DIFO), in which the electron-withdrawing, propargylic, gemfluorines act together with the ring strain to greatly destabilize the alkyne (Agard et al. (2006) ACS Chem. Biol. 1(10): 644-648). This destabilization increases the reaction driving force, and the desire of the cycloalkyne to relieve its ring strain. Copper-free click chemistry proceeds as a concerted [3+2] cycloaddition in the same mechanism as the Huisgen 1,3-dipolar cycloaddition. Substituents other than fluorines, such as benzene rings, are also allowed on the cyclooctyne.

The reactive groups in click chemistry (e.g., azides, terminal alkynes, strained alkynes (e.g., dibenzocyclooctyne (DBCO)) can be introduced into any form of nucleic acid molecule and can be introduced enzymatically or chemically. Alkyne-modified and azide-modified oligonucleotides can be ordered from an oligo-synthesizing facility or company. An azide-modified version of a nucleotide can be introduced during RNA synthesis into a first RNA molecule, and an alkyne-modified version of a nucleotide can be introduced during RNA synthesis into a second RNA molecule. The resulting Click-functionalized nucleic acid molecules can be isolated and purified to remove any unreacted reagents or byproducts that might interfere with the subsequent click reaction. The purified Click- functionalized nucleic acid molecules can be mixed together in a reaction buffer that supports the click reaction. This can include a copper catalyst to facilitate the reaction between the azide and alkyne or can be copper-free. The azide and alkyne functional groups react to form a covalent bond, linking the two nucleic acid molecules together. Linked nucleic acid molecules can be further purified, and analytical techniques such as gel electrophoresis or mass spectrometry can be used to verify the successful linking of the nucleic acid molecules and assess the purity of the product.

Click chemistry is further described, for example, in: Kumar et al. (2007) J. Am. Chem. Soc, 129:6859-6864; El-Sagheer and Brown (2010) Chem. Soc. Rev. 39: 1388-1405; Haque and Peng (2014) Sci. China Chem. 57:215-231; Wittig and Krebs 1961 Chem. Ber. 1961, 94, 3260-3275; US 7,375,234; US 7,070,941; US2013/0046084, the contents of each of which are hereby incorporated by reference in its entirety.

Linking of a crRNA and a tracrRNA can proceed in the following manner. A 3' amino modifer is included on the crRNA and a 5' amino modifier is included on the tracrRNA by solid phase synthesis. After synthesis, deprotection, and purification of the RNA oligonucleotides, an NHS ester of azidobutyrate (4-azido-butan-l-oic acid A-hydroxysuccinimide ester; available from, e.g., Glen Research cat no. 50-1904-24) is used to install an azide group at the 5' region of the tracrRNA and an NHS ester of DBCO (dibenzocyclooctyne-PEG4-JV-hydroxysuccinimidyl ester; available, for example, from Sigma Aldrich cat. no. 764019) is used to install the cyclooctyne group at the 3' region of the crRNA. Assembly in aqueous media (with optionally optimization of ionic strength, pH and reagent concentration) allows the strain promoted azide-alkyne Huisgen cycloaddition ("copper free click chemistry") to proceed.

In some embodiments, a crRNA and tracrRNA linked by click chemistry are linked by chemical moieties. In some embodiments, a crRNA and tracrRNA linked by chemical moieties comprise an azide group at one or more nucleotides at the anti-repeat of the tracrRNA and an alkyne group at one or more nucleotides at the crRNA repeat of the crRNA. In some embodiments, a crRNA and tracrRNA linked by chemical moieties comprise an azide group at one or more nucleotides at the crRNA repeat of the crRNA and an alkyne group at one or more nucleotides at the anti-repeat of the tracrRNA. One or more azide-modified nucleotides or one or more alkyne-modified nucleotides can be within a stem, a bubble, or both of the crRNA repeat of the guide RNA. One or more azide- modified nucleotides or one or more alkyne-modified nucleotides can be within a stem, a bubble, or both of the anti-repeat of the guide RNA. In some embodiments, a crRNA and tracrRNA linked by chemical moieties is a single guide RNA and comprises an azide-modified nucleotide or an alkyne- modified nucleotide at one or more nucleotides in the nucleotide loop connecting the crRNA repeat and the anti-repeat.

A variety of further chemical reactions and the corresponding modifications are available to the skilled person to link nucleic acid molecules (e.g., crRNA and tracrRNA) to each other in a covalent way. These modifications include a variety of crosslinkers, such as thiol modifications, like a thioctic acid N-hydroxy succinimide (NHS) ester, chemical groups that react with primary amines ( — NH2). These primary amines are positively charged at physiologic pH and nucleophilic; this makes them easy to target for conjugation with several reactive groups. There are numerous synthetic chemical groups that will form chemical bonds with primary amines. These include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfo-NHS esters containing a sulfonate ( — SO3) group, for example, bis(sulfosuccinimidyl)suberate (BS3), sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, such as, for example l-ethyl-3-(3- dimethylaminopropyljcarbodiimide (EDC) or dicyclohexylcarbodiimide (DCC), anhydrides, and fluorophenyl esters.

As described herein, a guide RNA can comprise a crRNA and a tracrRNA, wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one BNA modification. In some embodiments, the anti -repeat is capable of hybridizing to the crRNA repeat to form a stem loop comprising a first stem and a second stem.

As described herein, the present disclosure also provides a nucleic acid molecule comprising a tracrRNA, wherein the tracrRNA comprises: (a) an anti-repeat; (b) a tail; and (c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one BNA modification. In some embodiments, the antirepeat of the tracrRNA is capable of hybridizing to a crRNA repeat of a crRNA to form a stem loop comprising a first stem and a second stem. In some embodiments, a gRNA comprising the tracrRNA is capable of binding to an RGN.

As described herein, the present disclosure also provides a nucleic acid molecule comprising a crRNA comprising: (a) a spacer; and (b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2', 4'- di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA. In some embodiments, the crRNA repeat is capable of hybridizing to an anti -repeat of a tracrRNA to form a stem loop comprising a first stem and a second stem. In some embodiments, a gRNA comprising the crRNA is capable of binding to an RNA guided nuclease (RGN) that requires a tracrRNA for activity.

As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an antirepeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

As described herein, the present disclosure provides a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an antirepeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

The present invention provides, inter alia, CRISPR RNAs (crRNAs) or polynucleotides encoding CRISPR RNAs that comprise at least one BNA (e.g., LNA and/or cEt) modification. As used herein, the term “crRNA” refers to an RNA molecule or portion thereof that includes a spacer, which is the nucleotide sequence that directly hybridizes with the target strand of a target sequence, and a CRISPR repeat that comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN molecule. As used herein, the term “tracrRNA” or “transactivating crRNA” refers to an RNA molecule that comprises an anti-repeat sequence that has sufficient complementarity to hybridize to at least a portion of the CRISPR repeat of a crRNA to form a structure that is recognized by an RGN molecule. In some embodiments, additional secondary structure(s) (e.g., stem-loops) within the tracrRNA molecule is required for binding to an RGN.

In some embodiments, the crRNAs comprise at least one other chemical modification. In some embodiments, the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O- methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and/or cEt) modification. In certain embodiments, the at least one modification is a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the BNA modification comprises a 2', 4' BNA modification. In certain embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C- ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is an LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification. In some embodiments, the at least one chemical modification is a 2'-0-Me modification. In certain embodiments, the at least one chemical modification is an MS modification.

A crRNA comprises a spacer and a CRISPR repeat. The “spacer” is a nucleotide sequence that directly hybridizes with the target strand of a target sequence (e.g., target DNA sequence) of interest. The spacer is engineered to have full or partial complementarity with the target strand of a target sequence of interest. In some embodiments, the spacer can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the spacer can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the spacer is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the spacer is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In some embodiments, the spacer is about 30 nucleotides in length. In some embodiments, the spacer is 30 nucleotides in length. In some embodiments, the degree of complementarity between a spacer and the target strand of a target sequence (e.g., target DNA sequence), when optimally aligned using a suitable alignment algorithm, is between 50% and 99% or more, including but not limited to about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, the degree of complementarity between a spacer and the target strand of a target sequence (e.g., target DNA sequence), when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the spacer can be identical in sequence to the non-target strand of a target sequence. In some of those embodiments wherein the target sequence is a target DNA sequence, the spacer can be identical in sequence to the non-target strand of the target DNA sequence, with the exception of the thymidines (Ts) in the nontarget strand are replaced by uracils (Us) in the spacer. In embodiments, the spacer is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9: 133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(l):23-24).

In some embodiments, a spacer of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or to the phosphate backbone of the spacer. In certain embodiments, a spacer of the disclosure includes at least one chemical modification. In some embodiments, the at least one modification is selected from the group consisting of: 2'-O- methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O- methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification. In certain embodiments, a spacer of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification. In some embodiments, a spacer of the disclosure includes at least a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, a spacer of the disclosure includes at least one LNA modification. In some embodiments, a spacer of the disclosure includes at least one 2'-0-Me modification. In some embodiments, a spacer of the disclosure includes at least one MS modification.

In some embodiments, a spacer of the disclosure includes at least one 2'-0-Me modification and at least one MS modification. In certain embodiments, a spacer of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification (e.g., 2'-0-Me or MS). In some embodiments, a spacer of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one PS modification. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 14 or that differs from SEQ ID NO: 14 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 14 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 14 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 14.

For clarification, as used herein, when a nucleotide sequence “differs from a SEQ ID NO by a certain number of nucleotides” or “has a certain percentage identity to a SEQ ID NO”, the difference only occurs in the nucleotide sequence, and chemical modifications or lack thereof remain the same.

In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 16 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 16. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 15 or differs from SEQ ID NO: 15 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 15 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 15 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 15.

In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 17 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 17.

In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 89 or that differs from SEQ ID NO: 89 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 89 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 89 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 89.

In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 91 or that differs from SEQ ID NO: 91 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 91 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 91.

In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 90 or that differs from SEQ ID NO: 90 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 90 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 90 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 90.

In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 92 or that differs from SEQ ID NO: 92 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 92 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 92.

In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 111 or that differs from SEQ ID NO: 111 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 111 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 111 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 111.

In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 113 or that differs from SEQ ID NO: 113 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 113 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 113.

In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 112 or that differs from SEQ ID NO: 112 by 1 or 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 112 by 2 nucleotides. In some embodiments, a spacer, absent any chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 112 by 1 nucleotide. In some embodiments, a spacer, absent any chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 112.

In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 114 or that differs from SEQ ID NO: 114 by 1 to 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 5 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 4 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 3 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 2 nucleotides. In some embodiments, a chemically modified spacer has a nucleotide sequence that differs from SEQ ID NO: 114 by 1 nucleotide. In some embodiments, a chemically modified spacer has the nucleotide sequence set forth as SEQ ID NO: 114.

Along with a spacer, a crRNA further comprises a CRISPR RNA (crRNA) repeat. The crRNA repeat comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN molecule. In embodiments, the crRNA repeat can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the crRNA repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In embodiments, the crRNA repeat is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In particular embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

In some embodiments, a crRNA repeat of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or to the phosphate backbone of the crRNA repeat. In certain embodiments, a crRNA repeat of the disclosure includes at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'-O-methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and/or cEt) modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification. In some embodiments, a crRNA repeat of the disclosure includes at least a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4’-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one LNA modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one cEt modification. In some embodiments, a crRNA repeat of the disclosure includes at least one 2’-0-Me modification. In some embodiments, a crRNA repeat of the disclosure includes at least one MS modification. In some embodiments, a crRNA repeat of the disclosure includes at least one 2’-0-Me modification and at least one MS modification. In certain embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification (e.g., 2’- O-Me or MS). In some embodiments, a crRNA repeat of the disclosure includes at least one BNA (e.g., LNA In certain embodiments, a crRNA repeat of the disclosure includes at least one LNA modification.) modification and at least one PS modification.

In some embodiments, the crRNA repeat comprises the nucleotide sequence of any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516, or an active variant or fragment thereof, that when comprised within a guide RNA, is capable of directing the sequence-specific binding of an associated RNA-guided nuclease provided herein to a presently disclosed target sequence. In some embodiments, an active crRNA repeat variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516. In some embodiments, an active crRNA repeat fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 2 or differs from SEQ ID NO: 2 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 2 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 2 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 2. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 70 or differs from SEQ ID NO: 70 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 70 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 70 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 70. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 94 or differs from SEQ ID NO: 94 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 94 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 94 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 94. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 241 or differs from SEQ ID NO: 241 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 241 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 241 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 241. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 253 or differs from SEQ ID NO: 253 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 253 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 253 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 253. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 538 or differs from SEQ ID NO: 538 by 1 or 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 538 by 2 nucleotides. In some embodiments, a crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 538 by 1 nucleotide. In some embodiments, a crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 538.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 384.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 385.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as (c) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 386 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 386 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 386.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 387 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 387 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 387.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 388.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 389 or that differs from SEQ ID NO: 389 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 389.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 390.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 391.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 392.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 393.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 397.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512.

In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide. In some embodiments, a chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516.

In some embodiments, the crRNA is not naturally-occurring. In some embodiments, the specific crRNA repeat sequence is not linked to the engineered spacer sequence in nature and the crRNA repeat sequence is considered heterologous to the spacer sequence. In some embodiments, the spacer sequence is an engineered sequence that is not naturally occurring.

In some embodiments, a crRNA of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or to the phosphate backbone of the crRNA. In certain embodiments, a crRNA of the disclosure includes at least one chemical modification. In some embodiments, the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'- fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3' phosphorothioate (MS) modification; 2'-O-methyl 3' thiophosphonoacetate (MSP) modification; 2'- O-methyl 3' phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA) modification. In certain embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification. In some embodiments, a crRNA of the disclosure includes at least a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'- O,4’-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In certain embodiments, a crRNA of the disclosure includes at least one LNA modification. In some embodiments, a crRNA of the disclosure includes at least one 2'-O-Me modification. In some embodiments, a crRNA of the disclosure includes at least one MS modification. In some embodiments, a crRNA of the disclosure includes at least one 2'-O-Me modification and at least one MS modification. In certain embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification (e.g., 2'-O-Me or MS). In some embodiments, a crRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one PS modification. In some embodiments, a chemically modified crRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337- 339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399-401, 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 18. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 19. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 71. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 71. In some embodiments, a chemically modified crRNA has the nucleotide sequence set forth as SEQ ID NO: 74. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 72. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 95. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 96. In some embodiments, a crRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 96.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 4.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 5.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 6.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 7.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 9.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 708. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 708.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 292. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 292, and wherein with reference to SEQ ID NO: 292.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 293. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 293.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 73. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 73.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 74. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 75. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 75.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 301. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 301.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 302. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 302.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 303. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 303.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 305. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 305.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 306. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 307. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 307.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 309. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 309.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 310. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 310.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 311. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 311.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 313. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 313.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 314. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 315. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 315.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 321. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 321.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 322. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 322.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 323. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 323.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 345. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 345.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 346. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 347. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 347.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 349. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 349.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 350. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 350.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 351. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 351.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 353. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 353.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 354. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 355. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 355.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 357. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 357.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 358. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 358.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 359. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 359.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 361. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 361.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 362. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 363. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 363.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 97. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 97.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 98. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 98.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 99. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 99.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 325. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 325.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 326. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 327. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 327.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 329. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 329.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 330. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 330.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 331. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 331.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 333.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 334. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 335. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 335.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 337. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 337.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 338. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 338.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 339. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 339.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 42.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 43. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 44.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 380. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 380.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 381. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 381.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 382. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 382.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 399. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 399.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 400. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 401. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 401.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 466. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 466.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 467. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 467.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 468. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 468.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 470. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 470.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 471. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 472. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 472.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 474. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 474.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 475. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 475.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 476. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 476.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 478. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 478.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 479. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 480. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 480.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 482. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 482.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 483. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 483.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 484. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 484.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 509. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 509.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 510. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 511. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 511.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 513. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 513.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 514. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 514.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 515. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 515.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 517. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 517.

In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 518. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 519. In some embodiments, a chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 519.

Presently disclosed guide RNAs comprise a crRNA and a trans-activating CRISPR RNA (tracrRNA). A tracrRNA molecule comprises a nucleotide sequence comprising a region, referred to herein as the anti-repeat, that has sufficient complementarity to hybridize to a crRNA repeat. In embodiments, the tracrRNA molecule further comprises a region with secondary structure (e.g., stemloop). In some embodiments, secondary structure includes nucleotides that are in one of two states, paired or unpaired, where nucleotide or base pairing includes base-base hydrogen bonding interactions (e.g., adenine (A) pairs with uracil (U), cytosine (C) pairs with guanine (G)) between two complementary nucleic acid strands to form a helix. In some embodiments, the combination of one or more helical elements interspersed with unpaired, single-stranded nucleotides constitutes an RNA structure.

A “stem loop” as used herein refers to a form of secondary structure comprising at least one “stem” and at least one “loop”, “bulge”, or “bubble” found in polynucleotides. A stem loop can form intramolecularly (within one molecule, e.g., within a tracrRNA or a sgRNA) or intermolecularly (between two distinct nucleic acids, e.g., in a dgRNA by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA). Stem loops are created when there is at least some complementarity between two nucleic acid sequences to form a paired double helix. The paired double helix region with full complementarity or sometimes including a G:U wobble base pair (or I:U, I:A, or I:C, where I refers to inosine) is referred to as a “stem”. The term “loop”, “bulge”, or “bubble” refers to a single stranded region within the “stem loop” structure where there is no complementarity between nucleotides, excluding G:U wobble base pairs (or I:U, I:A, or I:C, where I refers to inosine). Thus, “loops”, “bulges” and “bubbles” include nucleotides that are not paired. In some embodiments, a “loop” is distinguished from a “bulge” or “bubble” by being located at one end of the “stem loop” structure, while a “bulge” or a “bubble” is located between two “stems” in the “stem loop” structure.

In certain embodiments, a stem loop structure comprises a stem and a loop at one end of the stem. In some embodiments, a stem loop structure comprises a first stem and a second stem with a bubble in between the stems. In some embodiments, a stem loop structure comprises a loop, multiple stems and multiple bubbles in between the stems. In this circumstance, the bubbles in the order of closeness to the loop are referred to as a “first bubble”, a “second bubble”, a “third bubble”, etc., and the stems in the order of closeness to the loop are referred to as a “first stem”, a “second stem”, a “third stem”, etc. In embodiments of dgRNA, the stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA does not include a loop, and thus the bubbles in the order of closeness to the 5’ end of the tracrRNA (or 3’ end of the crRNA) are referred to as a “first bubble”, a “second bubble”, a “third bubble”, etc., and the stems in the order of closeness to the 5’ end of the tracrRNA (or 3 ’ end of the crRNA) are referred to as a “first stem”, a “second stem”, a “third stem”, etc.

The term “first stem of a crRNA repeat of a crRNA”, “first stem of a crRNA repeat”, or “first stem of a crRNA” means the region in the crRNA repeat of the crRNA that forms the first stem of a stem loop structure when hybridizing with an anti-repeat of a tracrRNA. The term “second stem of a crRNA repeat of a crRNA”, “second stem of a crRNA repeat”, or “second stem of a crRNA” means the region in the crRNA repeat of the crRNA that forms the second stem of a stem loop structure when hybridizing with an anti-repeat of a tracrRNA. Similarly, the term “first stem of an anti-repeat of a tracrRNA”, “first stem of an anti -repeat”, or “first stem of a tracrRNA” means the region in the anti-repeat of the tracrRNA that forms the first stem of a stem loop structure when hybridizing with a crRNA repeat of a crRNA. The term “second stem of an anti-repeat of a tracrRNA”, “second stem of an anti-repeat”, or “second stem of a tracrRNA” means the region in the anti-repeat of the tracrRNA that forms the second stem of a stem loop structure when hybridizing with a crRNA repeat of a crRNA.

In some embodiments, a stem loop formed intramolecularly is a hairpin stem loop. Base pairings occur in the stem part of a stem loop and typically involve guanine-cytosine base pairing and adenine-uracil(thymidine) base pairing, although guanine -uracil base pairing is possible. Base stacking interactions promote helix formation. The loop part of a stem loop includes bases that are not paired. In some embodiments, a loop is the point at which a nucleic acid strand turns back on itself for nucleotide pairing to create a stem. In some embodiments, loops that are less than three bases long are sterically impossible and do not form. In some embodiments, optimal loop length is about 4-8 bases long. Common loops with four nucleotide sequences such as GAAA, AAAG, ACUU, or UUCG are known as the "tetraloop" and are particularly stable due to the base-stacking interactions of its component nucleotides.

In some embodiments, the region of the tracrRNA that is fully or partially complementary to a crRNA repeat is at the 5' end of the molecule and the 3' end of the tracrRNA comprises secondary structure. This region of secondary structure generally comprises several hairpin structures, including the nexus hairpin, which is found adjacent to the anti-repeat. The nexus forms the core of the interactions between the guide RNA and the RGN, and is at the intersection between the guide RNA, the RGN, and the target sequence. The nexus hairpin often has a conserved nucleotide sequence in the base of the hairpin stem, with the motif UNANNC found in many nexus hairpins in tracrRNAs. In embodiments, guide RNAs or RGN systems of the disclosure use tracrRNAs that comprise non- canonical sequences in the base of the hairpin stem of their nexus hairpins, including UNANNG and CNANNC. In some embodiments, a guide RNA or an RGN system of the disclosure uses a tracrRNA that includes, in the base of the nexus hairpin stem, the non-canonical sequence of UNANNG. In some embodiments, a guide RNA or an RGN system of the disclosure uses a tracrRNA that includes, in the base of the nexus hairpin stem, the non-canonical sequence of CNANNC. There are often terminal hairpins at the 3' end of the tracrRNA that can vary in structure and number, but often comprise a GC-rich Rho-independent transcriptional terminator hairpin followed by a string of U’s at the 3' end. See, for example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc, doi: 10. 1101/pdb.top090902, and U.S. Publication No. 2017/0275648, each of which is herein incorporated by reference in its entirety.

In some embodiments, a tracrRNA of the disclosure includes additional hairpin or stem loop structures in addition to the nexus hairpin. In some embodiments, a tracrRNA includes at least one stem loop. In some embodiments, a tracrRNA includes at least one stem loop proximal to the antirepeat and at least one stem loop proximal to the 3’ end of the tracrRNA. “Proximal” refers to being within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides of a region or an end of a nucleic acid molecule. In certain embodiments, “proximal” refers to being within 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides of a region or an end of a nucleic acid molecule. “Most proximal” refers to being the nearest to a region or to an end of a nucleic acid molecule. For example, a stem loop most proximal to the tail of a tracrRNA is the first stem loop nearest the tail of the tracrRNA. “Distal” refers to being at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more away from a region or an end of a nucleic acid molecule. In some embodiments, “distal” refers to being at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more away from a structure of a nucleic acid molecule (e.g., bubble, loop). For example, the first stem of the anti -repeat of a dual guide RNA lengthened at the end distal to the first bubble of the stem loop is lengthened from the 3 ’ terminal nucleotide of the crRNA and from the 5’ terminal nucleotide of the tracrRNA. A tracrRNA also forms secondary structure upon hybridizing with its corresponding crRNA. The anti-repeat region of a tracrRNA is fully or partially complementary to the crRNA repeat of a crRNA. In some embodiments, a portion of the anti-repeat of a tracrRNA and a portion of a crRNA repeat hybridize and form a stem. In some embodiments, the crRNA:tracrRNA stem includes at least one nucleotide pair (i.e. base pair) because these portions of the anti-repeat and crRNA repeat are complementary. As described elsewhere herein, a portion of the anti-repeat of a tracrRNA forming a first stem is the first stem of the anti-repeat, a portion of the anti-repeat of a tracrRNA forming a second stem is the second stem of the anti-repeat, a portion of the anti-repeat of a tracrRNA forming a third stem is the third stem of the anti-repeat, etc. As described elsewhere herein, a portion of the crRNA repeat of a crRNA forming a first stem is the first stem of the crRNA repeat, a portion of the crRNA repeat of a crRNA forming a second stem is the second stem of the crRNA repeat, a portion of the crRNA repeat of a crRNA forming a third stem is the third stem of the crRNA repeat, etc. In some embodiments, a portion of the anti-repeat of a tracrRNA and a portion of the crRNA repeat are not complementary with each other and thus do not hybridize to form base pairs. In some embodiments, the region of non-complementarity between the anti -repeat and the crRNA repeat forms a bulge or a bubble. In some embodiments, hybridization of the anti-repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one stem. In some embodiments, hybridization of the anti-repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one bubble. In some embodiments, hybridization of the anti -repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes at least one stem and at least one bubble. In some embodiments, hybridization of the anti -repeat of a tracrRNA and the crRNA repeat of a crRNA forms a secondary structure that includes two stems and one bubble in between.

In certain embodiments, a stem loop in a gRNA that is formed solely by portions of a tracrRNA does not include BNA (e.g., LNA and/or cEt) modifications. In some embodiments, a stem loop in a gRNA that is formed solely by portions of a tracrRNA does not include any chemical modifications.

In certain embodiments, the nucleotides in a loop, bulge, or bubble do not include BNA (e.g., LNA) modifications. In some embodiments, the nucleotides in a loop, bulge, or bubble do not include any chemical modifications.

In some embodiments, the anti-repeat of the tracrRNA that is fully or partially complementary to the crRNA repeat comprises from about 8 nucleotides to about 30 nucleotides, or more. For example, the stem formed by the tracrRNA anti-repeat and the crRNA repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the stem formed by the tracrRNA anti-repeat and the crRNA repeat is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, the degree of complementarity between a crRNA repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

In some embodiments, the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides. In some embodiments, the tracrRNA comprises a total length of 60 to 80 nucleotides, 80 to 100 nucleotides, 100 to 120 nucleotides, 120 to 140 nucleotides, 140 to 160 nucleotides, 160 to 180 nucleotides, or more than 180 nucleotides. For example, the tracrRNA can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, or more nucleotides in length. In embodiments, the tracrRNA is 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210 or more nucleotides in length. In some embodiments, the tracrRNA is about 70 to about 105 nucleotides in length, including about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, and about 105 nucleotides in length. In some embodiments, the tracrRNA is 70 to 105 nucleotides in length, including 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105 nucleotides in length. In some embodiments, the tracrRNA is about 90 to about 125 nucleotides in length, including about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, and about 125 nucleotides in length. In some embodiments, the tracrRNA is 90 to 125 nucleotides in length, including 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and 125 nucleotides in length.

In some embodiments, a tracrRNA of the disclosure comprises chemical modifications to at least one nucleotide, at least one sugar, at least one nucleobase, and/or the phosphate backbone of the tracrRNA. In certain embodiments, a tracrRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification. In certain embodiments, a tracrRNA of the disclosure includes at least one BNA (e.g., LNA and/or cEt) modification and at least one other chemical modification. In some embodiments, the at least one other chemical modification is selected from the group consisting of: 2'- O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In certain embodiments, the BNA modification comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the BNA modification is an LNA modification. Thus, in some embodiments, the tracrRNA comprises at least one LNA modification. In some embodiments, the BNA modification is a cEt modification. Thus, in some embodiments, the tracrRNA comprises at least one cEt modification. In certain embodiments, the tracrRNA comprises at least one LNA modification and at least one other chemical modification. In some embodiments, the tracrRNA comprises at least one LNA modification and at least one other chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'- O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments, the tracrRNA comprises at least one LNA modification and at least one PS modification. In certain embodiments, the tracrRNA comprises at least one cEt modification and at least one other chemical modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one other chemical modification selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. In some embodiments, the tracrRNA comprises at least one cEt modification and at least one PS modification.

In some embodiments, the tracrRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713, or an active variant or fragment thereof, that when comprised within a guide RNA is capable of directing the sequence-specific binding of an associated RNA- guided nuclease provided herein to a presently disclosed target sequence. In some embodiments, an active tracrRNA sequence variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 10, 12, 51 - 53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713. In embodiments, an active tracrRNA sequence fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of any one of the nucleotide sequences set forth as SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713.

In some embodiments, a tracrRNA comprises at least one chemical modification at its 5' region or at its 3' region. In some embodiments, a tracrRNA comprises at least one chemical modification at both its 5' region and 3' region. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 3. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 100. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 242. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 254. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 539. In some embodiments, a tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to SEQ ID NO: 539.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity toSEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 10.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 12.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 709. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 709.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 713. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 713.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 294. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 294. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 294. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 294.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 295. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 295. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 295. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 295.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 80. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 80.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 81. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 81.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 364. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 364. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 364. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 364.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 365. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 365. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 365. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 365.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 366. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 366. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 366. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 366.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 367. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 367. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 367. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 367.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 369. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 369. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 369. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 369.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 375. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 375. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 375. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 375.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 376. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 376. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 376.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 377. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 377. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 377. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 377.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 378. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 378. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 378. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 378.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 379. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 379. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 379. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 379.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 102. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 102.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 103. In some embodiments, a chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 103.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 370. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 370. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 370. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 370.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 371. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 371. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 371. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 371.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 372. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 372. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 372. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 372.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 373. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 373. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 373. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 373.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 710. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 710. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 710. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 710.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 711. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 711. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 711. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 711.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 51. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 51.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 52. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 52.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 53. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 53. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 53.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 383. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 383. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 383. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 383.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 499. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 499. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 499. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 499.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 500. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 500. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 500. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 500.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 501. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 501. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 501. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 501.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 504. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 504. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 504. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 504.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 505. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 505. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 505. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 505.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 534. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 534. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 534. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 534.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 535. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 535. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 535. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 535.

In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 537. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 537. In some embodiments, a chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 537. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 537.

The term “derived from” as used herein in the context of a polynucleotide molecule refers to a molecule generated or synthesized using a parent molecule or information from that parent molecule. For example, a tracrRNA, gRNA, or crRNA of the disclosure comprising at least one BNA (e.g., LNA) modification is derived from its respective unmodified tracrRNA, gRNA, or crRNA by having at least one of its nucleotides modified with a BNA (e.g., LNA) modification. In some embodiments, the tracrRNA, gRNA, or crRNA comprising at least one BNA (e.g., LNA) modification derived from its respective unmodified parent tracrRNA, gRNA, or crRNA has the same polynucleotide sequence as the parent molecule. The term “unmodified” in the context of a crRNA, spacer, crRNA repeat, tracrRNA, anti-repeat, or gRNA refers to a conventional crRNA, spacer, crRNA repeat, tracrRNA, anti-repeat, or gRNA that does not include any modified nucleotides, BNA modifications, modified sugars, modified nucleobases, and/or modified phosphate backbones, or any chemical modifications.

Two polynucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. Likewise, an RGN is considered to bind to a particular target sequence within a sequence-specific manner if the guide RNA bound to the RGN binds to a target sequence under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which the two polynucleotide sequences will hybridize to each other to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short sequences (e.g., 10 to 50 nucleotides) and at least about 60°C for long sequences (e.g. , greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and a wash in IX to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched sequence. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley- Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

The term “sequence specific” can also refer to the binding of a RGN polypeptide to a target sequence at a greater affinity than binding to a randomized background sequence.

The guide RNA can be a single guide RNA (sgRNA) or a dual -guide RNA (dgRNA). A sgRNA comprises the crRNA and tracrRNA on a single molecule of RNA, whereas a dgRNA comprises a crRNA and a tracrRNA present on two distinct RNA molecules, hybridized to one another through at least a portion of the crRNA repeat of the crRNA and at least a portion of the antirepeat of the tracrRNA, which may be fully or partially complementary to each other. Hybridization of the anti-repeat of a tracrRNA to the crRNA repeat of a crRNA forms a stem loop comprising the anti-repeat and the crRNA repeat. In some embodiments, the stem loop includes one or more stems formed by the anti-repeat and the crRNA repeat. In some embodiments wherein the guide RNA is a sgRNA, the crRNA and tracrRNA are separated by a linker nucleotide sequence. In general, the linker nucleotide sequence is one that does not include bases complementary within itself or to other parts of the sgRNA in order to avoid the formation of secondary structure within or comprising nucleotides of the linker nucleotide sequence. In certain embodiments, the linker forms a loop at one end of the first stem in the stem loop structure comprising the crRNA repeat and the anti-repeat. In some embodiments, the linker nucleotide sequence between the crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more nucleotides in length. In certain embodiments, the linker nucleotide sequence of a sgRNA is at least 4 nucleotides in length. In certain embodiments, the linker nucleotide sequence includes a nucleotide sequence set forth as any of AAAG, GAAA, ACUU, and CAAAGG.

The total length of a guide RNA can comprise about 100 nt to 120 nt, about 120 nt to 140 nt, about 140 nt to about 160 nt, about 160 nt to about 180 nt, about 180 nt to about 200 nt, or more. In some embodiments, the total length of a guide RNA is 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt, 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt, 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt, 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt, 157 nt, 158 nt, 159 nt, 160 nt. 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt, 171 nt, 172 nt, 173 nt, 174 nt, 175 nt, 176 nt, 177 nt, 178 nt, 179 nt, 180 nt, 181 nt, 182 nt, 183 nt, 184 nt, 185 nt, 186 nt, 187 nt, 188 nt, 189 nt, 190 nt, 191 nt, 192 nt, 193 nt, 194 nt, 195 nt, 196 nt, 197 nt, 198 nt, 199 nt, 200 nt, or more.

In some embodiments, a chemically modified sgRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

The sgRNA or dgRNA can be synthesized chemically or via in vitro transcription. Assays for determining sequence -specific binding between an RGN and a guide RNA are known in the art and include, but are not limited to, in vitro binding assays between an expressed RGN and the guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the guide RNA:RGN complex is captured via the detectable label (e.g., with streptavidin beads). A control guide RNA with an unrelated sequence or structure to the guide RNA can be used as a negative control for non-specific binding of the RGN to RNA.

In some embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule. The guide RNA can be chemically synthesized.

In embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, wherein the guide RNA is bound to an RGN polypeptide.

The guide RNA directs an associated RGN to a particular target nucleotide sequence of interest through hybridization of the guide RNA to the target sequence of interest. The target sequence can be bound (and in some embodiments, cleaved) by an RGN in vitro or in a cell. A target sequence can comprise DNA, RNA, or a combination of both and can be single -stranded or doublestranded. In some embodiments, a target sequence can be genomic DNA (i.e., chromosomal DNA), plasmid DNA, episomal DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). In those embodiments wherein the target sequence is a chromosomal sequence, the chromosomal sequence can be a nuclear, plastid or mitochondrial chromosomal sequence. In the presently disclosed compositions and methods, the target sequence is within a target nucleic acid molecule that is double-stranded (e.g., a target DNA sequence). In some embodiments, the target sequence is unique in the target genome. In some embodiments, the target sequence comprises a target strand and a non-target strand, and the target sequence (i.e., the sequence on the non-target strand) has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

The target sequence is adjacent to a protospacer adjacent motif (PAM) and the non-target strand of the target sequence is the strand that comprises the PAM. The PAM is immediately adjacent to the target sequence and often comprise Ns, which represent any nucleotide. In some embodiments, the PAM comprises about 1 to about 10 Ns, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 Ns. In some embodiments, a PAM comprises 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns. The PAM can be 5' or 3' of the target sequence on its non-target strand. In some embodiments, the PAM is 3' of the target sequence on its non-target strand for the presently disclosed guide RNAs and RGN systems. Generally, the PAM is a consensus sequence of about 3-4 nucleotides, but in some embodiments, it can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length.

In some embodiments, a PAM sequence adjacent to a presently disclosed target sequence on its non-target strand comprises the consensus sequence set forth as any one of the PAM sequences in Table 1. In some embodiments, a PAM sequence adjacent to the presently disclosed target sequence on its non-target strand includes the consensus sequence set forth as any one of NNNNCC, NNGRR, NNRYA, and NGG. In some embodiments, the PAM sequence is 3' of the target sequence on its non- target strand.

It is well-known in the art that PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (see, e.g., Karvelis et al. (2015) Genome Biol 16:253), which may be modified by altering the promoter used to express the RGN, or the amount of ribonucleoprotein complex delivered to the cell, organelle, or embryo.

Upon recognizing its corresponding PAM sequence, the RGN can cleave one or both strands of a target sequence at a specific cleavage site. As used herein, a cleavage site is made up of the two particular nucleotides within a target sequence between which the target strand, non-target strand, or both strands of a target sequence are cleaved by an RGN. The cleavage site can comprise the 1^st and 2^nd, 2^nd and 3^rd, 3^rd and 4^th, 4^th and 5^th, 5^th and 6^th, 7^th and 8^th, or 8^th and 9^th nucleotides from the PAM in either the 5' or 3' direction. In embodiments, the cleavage site may be over 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in either the 5' or 3' direction. As RGNs can cleave a target sequence resulting in staggered ends, in embodiments, the cleavage site is defined based on the distance of the two nucleotides from the PAM on the non-target strand of the target sequence, and for the target strand, the distance of the two nucleotides from the complement of the PAM.

III. Chemical modifications and length modifications to guide RNA

Nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA of the disclosure can in some embodiments comprise at least one BNA (e.g., LNA) modification. In some embodiments, the at least one BNA (e.g., LNA) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA) modification in the first stem of the anti -repeat of the tracrRNA. Nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA of the disclosure can in some embodiments include a modification in the ribose (e.g., sugar) group, phosphate group, nucleobase, or any combination thereof. The term "chemical modification" in the context of an oligonucleotide or polynucleotide includes but is not limited to (a) end modifications, e.g., 5' end modifications or 3' end modifications, (b) nucleobase (or "base") modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2', 3', and/or 4' positions, and (d) backbone modifications, including modification or replacement of the phosphodiester linkages. The term "modified nucleotide" generally refers to a nucleotide having a modification to the chemical structure of one or more of the base, the sugar, and the phosphodiester linkage or backbone portions, including nucleotide phosphates. The terms “modification” and “chemical modification” are used interchangeably herein.

In some embodiments, a modified nucleotide includes a sugar modification. Non-limiting examples of sugar modifications include 2'-deoxy-2'-fluoro-oligoribonucleotide (2'- fluoro-2'- deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5 '-triphosphate), 2'-deoxy-2'-deamine oligoribonucleotide (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'- deoxyuridine-5'- triphosphate), 2'-O-alkyl oligoribonucleotide, 2'-deoxy-2'-C-alkyl oligoribonucleotide (2 '-O- methylcytidine-5'-triphosphate, 2'-methyluridine-5 '-triphosphate), 2'-C-alkyl oligoribonucleotide, and isomers thereof (2'-aracytidine-5'-triphosphate, 2'- arauridine-5'-triphosphate), azidotriphosphate (2'- azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate), and combinations thereof.

In some embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more 2'-fluoro, 2'-amino and/or 2'-thio modifications. In some embodiments, the modification is a 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'- amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, 5 -amino-allyl- uridine, 5- bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl- pyrene -uridine, 5-fluoro-cytidine, and/or 5 -fluoro-uridine. There are more than 96 naturally occurring nucleoside modifications found on mammalian RNA. See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196 (1994). A nucleoside includes a purine or pyrimidine base linked to a sugar (i.e., nucleotides without a phosphate group). The preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art and described in, e.g., U.S. Patent Nos. 4,373,071; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,047,524; 5,132,418; 5,153,319; 5,262,530; and 5,700,642. Numerous modified nucleosides and modified nucleotides that are suitable for use in the present disclosure are commercially available. The nucleoside can be an analogue of a naturally occurring nucleoside. In some embodiments, a nucleoside analogue includes dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxy cytodine, and deoxyuridine.

In some cases, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, atracrRNA, an anti-repeat, or a guide RNA) includes a nucleobase-modified ribonucleotide, i.e., a ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Non-limiting examples of modified nucleobases which can be incorporated into modified nucleosides and modified nucleotides include m5C (5 -methylcytidine), m5U (5 - methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1- methyl adenosine), m2A (2- methyladenosine), Am (2-1-O-methyladenosine), ms2m6A (2- methylthio-N6-methyladenosine), i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio- N6isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2- methylthio- N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycinylcarbamoyladenosine), t6A (N6-threonyl carbamoyladenosine), ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine), m6t6A (N6-methyl- N6-threonylcarbamoyladenosine), hn6A (N6-hydroxynorvalylcarbamoyl adenosine), ms2hn6A (2- methylthio-N6-hydroxynorvalyl carbamoyladenosine), Ar(p) (2'-O-ribosyladenosine(phosphate)), I (inosine), mil (1 -methylinosine), m'lm (l,2'-O-dimethylinosine), m3C (3 -methylcytidine), Cm (2T-O- methylcytidine), s2C (2 -thiocytidine), ac4C (N4-acetylcytidine), f5C (5-fonnylcytidine), m5Cm (5,2- O-dimethylcytidine), ac4Cm (N4acetyl2TOmethylcytidine), k2C (lysidine), mlG (1- methylguanosine), m2G (N2-methylguanosine), m7G (7-methylguanosine), Gm (2'-0- methylguanosine), m22G (N2,N2-dimethylguanosine), m2Gm (N2,2'-O-dimethylguanosine), m22Gm (N2,N2,2'-O-trimethylguanosine), Gr(p) (2'-O-ribosylguanosine(phosphate)), yW (wybutosine), o2yW (peroxywybutosine), OHyW (hydroxywybutosine), OHyW* (undermodified hydroxywybutosine), imG (wyosine), mimG (methylguanosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galtactosyl-queuosine), manQ (mannosyl- queuosine), preQo (7-cyano-7- deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G (archaeosine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2 -thiouridine), s2Um (2-thio- 2'-0-methyluridine), acp3U (3-(3-amino-3- carboxypropyl)uridine), ho5U (5 -hydroxyuridine), mo5U (5 -methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5- (carboxyhydroxymethyl)uridine)), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5 -methoxycarbonyl methyluridine), mcm5Um (S-methoxycarbonyhnethyl-2- O-methyluridine), mcm5s2U (5 -methoxy carbonylmethyl -2 -thiouridine), nm5s2U (5- aminomethyl-2- thiouridine), mnm5U (5 -methylaminomethyluridine), mnm5s2U (5- methylaminomethyl-2- thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5 -carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-0-methyluridine), cmnm5U (5- carboxymethylaminomethyluridine), cnmm5Um (5 -carboxymethylaminomethyl- 2-L- Omethyluridine), cmnm5s2U (5 -carboxymethylaminomethyl -2 -thiouridine), m62A (N6,N6- dimethyladenosine), Tm (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2-O- dimethylcytidine), hm5C (5 -hydroxymethylcytidine), m3U (3 -methyluridine), cm5U (5- carboxymethyluridine), m6Am (N6,T-O-dimethyladenosine), m62Am (N6,N6,0-2- trimethyladenosine), m2'7G (N2,7-dimethylguanosine), m2'2'7G (N2,N2,7- trimethylguanosine), m3Um (3,2T-O-dimethyluridine), m5D (5 -methyldihydrouridine), f5Cm (5 -formyl -2'-0- methylcytidine), mlGm (l,2'-O-dimethylguanosine), m'Am (1,2-0- dimethyl adenosine)irinomethyluridine), tm5s2U (S-taurinomethyl-2-thiouridine)), imG-14 (4-demethyl guanosine), imG2 (isoguanosine), or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo- adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2- thiouracil, 4-thiouracil, 5- aminouracil, 5-(Ci-Cg)-alkyluracil, 5 -methyluracil, 5-(C2-Cg)- alkenyluracil, 5-(C2-Cg)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5- fluorouracil, 5 -bromouracil, 5 -hydroxy cytosine, 5-(Ci- Cg)-alkylcytosine, 5 -methylcytosine, 5-(C2-Cg)-alkenylcytosine, 5-(C2-Cg)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5- bromocytosine, N²-dimethylguanine, 7-deazaguanine, 8- azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-Cg)alkynylguanine, 7-deaza-8-substituted guanine, 8- hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4- diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7- substituted purine, 7-deaza-8-substituted purine, and combinations thereof.

In some embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more modifications in the phosphate backbone. The modification can include one or more of phosphorothioate, phosphorodithioate, phosphoramidate (e.g., N3'- P5'-phosphoramidate (NP)), and/or methylphosphonate linkages. In some embodiments, a backbone modification includes a neutral backbone modification including: phosphorodiamidate morpholino oligomer (PMO) and peptide nucleic acid (PNA) modifications. In some embodiments, all stereoisomers of these backbone modifications are useful in the present disclosure.

In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include modifications at the 2’ position of the ribose sugar. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl (2'-0-Me) modification (“B” indicates “base” in chemical structures herein):

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O- methoxy-ethyl (2'-M0E) modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-fluoro (2'- F) modification:

In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include modifications at the 2' position and 4' position of the ribose sugar. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'F-4'Ca-OMe modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2',4'-di-Ca- OMe modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a phosphorothioate (PS) modification (e.g., in the backbone):

In some embodiments, one or more of the modified nucleotides of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include modifications at the 2' position of the ribose sugar and the phosphate backbone. In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl 3'- phosphorothioate (MS) modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl 3'thiophosphonoacetate (MSP; 2'-O-methyl 3'thioPACE) modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'-O-methyl 3'phosphonoacetate (MP) modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include 2'-O- methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2’-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3 ’thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA (e.g., LNA and/or cEt) modification. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more MS modifications and one or more BNA (e.g., LNA and/or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more BNA (e.g., LNA and/or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA) includes one or more MS modifications and one or more 2’,4’-BNA (e.g., LNA and/or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more 2’,4’-BNA (e.g., LNA and/or cEt) modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more MS modifications and one or more LNA modifications. In some embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more MS modifications and one or more cEt modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more LNA modifications. In certain embodiments, a modified molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) includes one or more PS modifications and one or more cEt modifications.

In some embodiments, a modification includes a bridged nucleic acid (BNA) modification. The term "bridged nucleic acid" refers to a nucleic acid having a structure wherein the degree of freedom of the nucleic acid is restricted through an intramolecular bond or crosslink. In certain embodiments, a BNA modification includes a 2', 4' BNA modification. In some embodiments, the 2' oxygen and 4' carbon of the ribose are linked through a “bridge”.

First generation BNA modifications include locked nucleic acid (LNA) modifications. LNA nucleotides comprise conformationally-restricted RNA nucleotides in which the 2' oxygen in the ribose forms a covalent bond to the 4' carbon, inducing N-type (C3'-endo) sugar puckering and preference for an A-form helix (Y ou et al. (2006) Nucleic Acids Res 34(8):e60), depicted as follows:

LNAs display improved base stacking and thermal stability compared to RNA, resulting in highly efficient binding to complementary nucleic acids and improved mismatch discrimination, as well as nuclease resistance (You et al. (2006) Nucleic Acids Res 34(8):e60; Vester & Wengel (2004) Biochemistry 43(42): 13233-13241). They have been successfully used in numerous applications ranging from SNP detection assays to siRNA (Vester & Wengel (2004) Biochemistry 43(42): 13233- 13241; Elmen et al. (2005) Nucleic Acids Res 33(l):439-447). N-methyl substituted bridged nucleic acids (BNA^NC[N-Me]) have been designed to improve upon the original first generation LNA scaffold by introducing more conformational flexibility for DNA binding, even greater nuclease resistance due to steric bulk, and reduced cellular toxicity (Rahman et al. (2008) J Am Chem Soc 130(14):4886-4896).

A number of bridged nucleic acids are known to those of skill in the art and are available from commercial sources (e.g., Biosynthesis, Inc.). In some embodiments, BNAs include: 2'-O,4'-C- ethylene BNA (2',3'-ENA); 2'-O,4'-C-methylenecytidine; 2'-O,4'-C-methyleneuridine; 2',4'-BNA-l- isoquinolone; 2',4'-BNA-2-pyridone; 2',4'-BNA-TeNA; 2',4'-BNA-TrNA; 2',4'-BNA^C0C; 2', 4'- BNA^NC[NBn]; 2',4'-BNA^NC[NH]; 2',4'-BNA^NC[NMe]; 3'-amino-2',4'-BNA; AmNA; DpNA (3,4- dihydro-2H-pyran bridge moiety; EoNA; GuNA (guanidine BNA); HxNA; PrNA; scpBNA (2'-O,4'- C-spirocycloprepylene BNA); six-membered AmNA; SuNA; urea-BNA; a-L-LNA; 5-methyl-2'-O,4'- C-methyleneuridine; 5-bromo-2'-O,4'-C-methyleneuridine; 3'-O-benzyl-5'-O-mesyl-5-methyl-2'-O,4'- C-methyleneuridine; and benzylidene acetal -type BNA (BA-BNA).

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a BNA^NC[N-Me] modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification:

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a S- constrained ethyl (cEt) modification:

Other BNAs are disclosed in: U.S. Patent Nos. US 6,770,748; US 6,770,748; 8,153,365; 8,080,644; 7,060,809; 7,084,125; 7,060,809; 7,053,207; 6,670,461; 6,436,640; 6,316,198; and 7,427,672, each of which is herein incorporated by reference in its entirety.

In certain embodiments, one or more modifications of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA) include a combination of chemical modifications disclosed herein.

One or more nucleotides of a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA of the disclosure can be a modified nucleotide. In some embodiments, a crRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides up to all nucleotides being chemically modified. In some embodiments, a crRNA repeat of a crRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more chemically modified nucleotides up to all nucleotides being chemically modified. In some embodiments, a spacer of a crRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more chemically modified nucleotides up to all nucleotides being chemically modified. In some embodiments, a tracrRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides up to all nucleotides being chemically modified. In some embodiments, an anti-repeat of a tracrRNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more chemically modified nucleotides up to all nucleotides being chemically modified. In some embodiments, a guide RNA may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chemically modified nucleotides up to all chemically nucleotides being modified. The modified nucleotides can be located at any nucleotide position of the RNA sequence, including the first nucleotide position, the last nucleotide position, and/or any nucleotide position between the first and last nucleotide positions. For example, for a guide RNA that is 20 nucleotides in length, the one or more modified nucleotides can be located at nucleotide position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, and/or position 20 from the 5’ end of the guide RNA. In certain embodiments, from about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 30%, about 20% to about 30%, or about 25% to about 30% of an RNA molecule of the disclosure can comprise modified nucleotides. In other instances, from about 10% to about 30%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% of a RNA molecule or region of the disclosure can comprise modified nucleotides.

In certain embodiments, the chemical modifications are within one or more stems in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are within the one or more stems of one stem loop. In some embodiments, the chemical modifications are within the one or more stems of multiple stem loops. In some embodiments, the chemical modifications are within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are not within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first and the second stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first stem but not within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the first stem and one or more nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the first stem but no chemical modifications are present within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first and the second stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first stem but no chemical modifications are present within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA).

In some embodiments, the chemical modifications are within one stem of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within one stem of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within one stem of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).

In certain embodiments, the chemical modifications are within two stems of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each of the two stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within two stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are within three stems of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each of the three stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within three stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).

In some embodiments, the chemical modifications are present on one or more nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).

In some embodiments, a stem loop comprising chemical modifications in its one or more stems in a guide RNA is formed by hybridization of a crRNA and a tracrRNA. In some embodiments, a stem loop comprising chemical modifications in its one or more stems in a guide RNA is formed by hybridization of crRNA repeat of a crRNA and anti-repeat of a tracrRNA.

In some embodiments, the chemical modifications are within the “tail” in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). The term “tail” as used herein refers to the non-complementary region closest to the 3' end (e.g., within twelve, eleven, ten, nine, eight, seven, six, five nucleotides from the 3' end) of a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, a tail of a tracrRNA includes 1-12, 1-8, or 1-6 nucleotides from the 3' end of the tracrRNA. In some embodiments, a tail of a tracrRNA includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides from the 3' end of the tracrRNA. In some embodiments, the chemical modifications are within one or more stems and within the tail in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first and second stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first stem and no chemical modifications are within the second stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA, and chemical modifications are present within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first, second, and third stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).

In some embodiments, the chemical modifications are not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In certain embodiments, the chemical modifications are within one or more stems but not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within one or more stems and within the tail but not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first and second stems of a stem loop formed by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first, second, and third stems of a stem loop formed by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA and the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first stem and not within any other stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).

In some embodiments, the chemical modifications are present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In certain embodiments, the chemical modifications are within one or more stems and present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within one or more stems and within the tail as well as present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within a first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within a first and second stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within a first, second, and third stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).

In certain embodiments of gRNA, a first stem is formed by hybridization between the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA. In some embodiments, the crRNA and the tracrRNA are two separate molecules, which together form a dgRNA. In embodiments of dgRNA, the first stem is the stem closest to the 3' end of the crRNA and closest to the 5' end of the tracrRNA. In some embodiments, the crRNA and the tracrRNA are within one molecule of a sgRNA. In embodiments of sgRNA, the first stem is the stem immediately adjacent to the loop in a stem loop formed by hybridization between the crRNA repeat of the crRNA and the anti-repeat of the tracrRNA.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within a crRNA repeat. In some embodiments, at least one BNA modification (e.g., LNA and/or cEt) is within the first stem of a crRNA repeat. The first stem of a crRNA repeat includes nucleotides within a crRNA repeat forming the first stem of a stem loop by hybridization with nucleotides of an anti-repeat of a tracrRNA, and as described elsewhere herein. In some embodiments, the terminal 5' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the second stem of a crRNA repeat. The second stem of a crRNA repeat includes nucleotides within a crRNA repeat forming the second stem of a stem loop by hybridization with nucleotides of an antirepeat of a tracrRNA, and as described elsewhere herein. In some embodiments, the terminal 5' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, no chemical modifications are within the second stem of a crRNA repeat.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first and second stems of a crRNA repeat. In some embodiments, the terminal 5' nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 3' nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 5' nucleotides and the terminal 3' nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first and second stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first stem and no chemical modifications are within the second stem of a crRNA repeat.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the third stem of a crRNA repeat. The third stem of a crRNA repeat includes nucleotides within a crRNA repeat forming the third stem of a stem loop by hybridization with nucleotides of an anti-repeat of a tracrRNA, and as described elsewhere herein. In some embodiments, the terminal 5' nucleotide of the third stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the third stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, all stems of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 5' nucleotide of each stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of each stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of each stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of each stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of each stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, only the first stem of a crRNA repeat comprises BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, nucleotides of a crRNA repeat within a loop, bulge, or bubble in a stem loop formed by hybridization of the crRNA repeat and the anti-repeat do not comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, a crRNA repeat does not comprise BNA (e.g., LNA and/or cEt) modifications but hybridizes to the anti-repeat of a tracrRNA comprising at least one BNA (e.g., LNA and/or cEt) modification. In some embodiments, the crRNA repeat lacking BNA (e.g., LNA and/or cEt) modifications hybridizes to the anti-repeat of a tracrRNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the stem loop formed by the hybridization of the crRNA repeat and the anti-repeat.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the anti-repeat of a tracrRNA. In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first stem of the anti-repeat of a tracrRNA. The first stem of an anti-repeat includes nucleotides within an anti-repeat forming the first stem of a stem loop by hybridization with nucleotides of a crRNA repeat of a crRNA, and as described elsewhere herein. In some embodiments, the terminal 5' nucleotide of the first stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the first stem of an anti -repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the first stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the first stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the second stem of the anti-repeat of a tracrRNA. The second stem of an anti-repeat includes nucleotides within an anti-repeat forming the second stem of a stem loop by hybridization with nucleotides of a crRNA repeat of a crRNA, and as described elsewhere herein. In some embodiments, the terminal 5' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the second stem of an antirepeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, no chemical modifications are within the second stem of an anti-repeat.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first and second stems of an anti-repeat of a tracrRNA. In some embodiments, the terminal 5' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 3' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 5' nucleotides and the terminal 3' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first stem and no chemical modifications are within the second stem of an anti-repeat of a tracrRNA.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the third stem of the anti-repeat of a tracrRNA. The third stem of an anti-repeat includes nucleotides within an anti-repeat forming the third stem of a stem loop by hybridization with nucleotides of a crRNA repeat of a crRNA, and as described elsewhere herein. In some embodiments, the terminal 5' nucleotide of the third stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the third stem of an anti -repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the third stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the third stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the third stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, all stems of the anti-repeat of a tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 5' nucleotide of each stem of an antirepeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of each stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of each stem of an antirepeat comprises BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of each stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of each stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, only the first stem of the anti-repeat of a tracrRNA comprises BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, nucleotides of an anti-repeat within a loop, bulge, or bubble in a stem loop formed by hybridization of the anti-repeat and the crRNA repeat do not comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, an anti-repeat of a tracrRNA does not comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, a chemical modification at the fourth nucleotide position from the 5' end, a chemical modification at the fifth nucleotide position from the 5' end, or a combination thereof. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end and a modification at the second nucleotide position from the 5' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, and a chemical modification at the third nucleotide position from the 5' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, and a chemical modification at the fourth nucleotide from the 5' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, a chemical modification at the fourth nucleotide from the 5' end, and a chemical modification at the fifth nucleotide from the 5' end. As used herein, a “5' region” of an RNA molecule disclosed herein includes the first nucleotide, the first 2 nucleotides, the first 3 nucleotides, the first 4 nucleotides, or the first 5 nucleotides of the 5' end of the RNA molecule.

In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, a chemical modification at the fourth nucleotide position from the 3' end, a chemical modification at the fifth nucleotide position from the 3' end, or a combination thereof. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end and a chemical modification at the second nucleotide position from the 3' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3’ end, a chemical modification at the second nucleotide position from the 3' end, and a chemical modification at the third nucleotide position from the 3' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, and a chemical modification at the fourth nucleotide from the 3' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, a chemical modification at the fourth nucleotide from the 3' end, and a chemical modification at the fifth nucleotide from the 3' end. As used herein, a “3' region” of an RNA molecule disclosed herein includes the first nucleotide, the first 2 nucleotides, the first 3 nucleotides, the first 4 nucleotides, or the first 5 nucleotides of the 3' end of the RNA molecule. In some embodiments, a 3' region of a crRNA in the context of a single guide RNA includes the first nucleotide, the first 2 nucleotides, the first 3 nucleotides, the first 4 nucleotides, or the first 5 nucleotides from the tracrRNA or the linker that joins the crRNA and the tracrRNA of the single guide RNA.

In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a tracrRNA of the disclosure comprise MS modifications and the remaining nucleotides in the first stem of the anti -repeat of the tracrRNA comprise 2'-0-Me modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the anti-repeat of the tracrRNA comprise 2'-0-Me modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA modifications and at least one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise MS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise MS modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise BNA+PS modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA+PS modifications and the crRNA does not comprise any further chemical modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt +PS modifications and the crRNA does not comprise any further chemical modifications.

In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise MS modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA modifications and the 3' region of the crRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt modifications and the 3' region of the crRNA comprise LNA modifications.

In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise BNA+PS modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise LNA+PS modifications. In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a crRNA comprise cEt+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise LNA+PS modifications and the 3' region of the crRNA comprise cEt+PS modifications. In some embodiments, the three terminal nucleotides at the 5' region of a crRNA comprise cEt+PS modifications and the 3' region of the crRNA comprise LNA+PS modifications.

In some embodiments, the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA modifications at the three terminal nucleotides in the 5' region and cEt modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt modifications at the three terminal nucleotides in the 5' region and LNA modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.

In some embodiments, the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA+PS modifications at the three terminal nucleotides in the 5' region and cEt+PS modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt+PS modifications at the three terminal nucleotides in the 5' region and LNA+PS modifications at the three terminal nucleotides in the 3' region and further comprises 2'-0-Me modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.

In some embodiments, the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises BNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.

In some embodiments, the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises MS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.

In some embodiments, the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises BNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.

In some embodiments, the crRNA comprises LNA modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA modifications at the three terminal nucleotides in the 5' region and cEt modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt modifications at the three terminal nucleotides in the 5' region and LNA modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA modifications at the three terminal nucleotides in the 5' region and cEt modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt modifications at the three terminal nucleotides in the 5' region and LNA modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.

In some embodiments, the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises BNA+PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt +PS modifications at the three terminal nucleotides in both the 5' and 3' regions and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA+PS modifications at the three terminal nucleotides in the 5' region and cEt+PS modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt+PS modifications at the three terminal nucleotides in the 5' region and LNA+PS modifications at the three terminal nucleotides in the 3' region and further comprises LNA modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises LNA+PS modifications at the three terminal nucleotides in the 5' region and cEt+PS modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat. In some embodiments, the crRNA comprises cEt+PS modifications at the three terminal nucleotides in the 5' region and LNA+PS modifications at the three terminal nucleotides in the 3' region and further comprises cEt modifications at one, two, three, four, five, six, seven, eight, nine, ten or more of the remaining nucleotides in the first stem of the crRNA repeat.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA are chemically modified, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA are chemically modified. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5’ region and the 3’ region of the sgRNA are chemically modified, and wherein all the nucleotides in the first stem of the crRNA repeat and all the nucleotides in the first stem of the anti-repeat of the tracrRNA are chemically modified.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises BNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises cEt modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises BNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises LNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises cEt+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein the three terminal 3 ' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein the three terminal 3 ' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

A chemically modified nucleotide and/or a chemical modification “within” a region of a RNA molecule of the disclosure includes all nucleotides and phosphate backbone in that region, including the first and last nucleotide positions that are considered part of that region.

The chemical modifications may allow use of crRNA, crRNA repeat, spacer, tracrRNA, antirepeat, and/or guide RNA that have been engineered to be shorter. Thus, in some embodiments, a crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, and/or a guide RNA of the present disclosure are truncated or shortened. In some embodiments, a truncated crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, and/or guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification maintains or enhances gene editing efficiency as compared to the truncated crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, and/or guide RNA lacking the at least one BNA (e.g., LNA and/or cEt) modification. “Truncation” and “deletion” in the context of engineering a spacer, crRNA repeat, crRNA, anti-repeat, tracrRNA, backbone, or guide RNA, are used interchangeably herein and refer to removal of at least one nucleotide from a spacer, crRNA repeat, crRNA, anti-repeat, tracrRNA, backbone, or guide RNA.

In some embodiments, an engineered spacer comprises a truncation of 1 nucleotide (nt), 2 nt,

3 nt, 4 nt, or 5 nt, as compared to the same spacer prior to the engineering. In some embodiments, an engineered spacer comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, or 5 nt, as compared to a nucleotide sequence set forth as any one of SEQ ID NOs: 14, 15, 89, 90, 111, and 112.

In some embodiments, an engineered crRNA repeat comprises a truncation of 1 nt, 2 nt, 3 nt,

4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt, as compared to the same crRNA repeat prior to the engineering. In certain embodiments, an engineered crRNA repeat comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt, as compared to a nucleotide sequence set forth as any one of SEQ ID NOs: 2, 70, 94, 241, 253, and 538. In some embodiments, an engineered crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 38 or that differs from SEQ ID NO: 38 by 1 or 2 nucleotides. In some embodiments, an engineered crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 38 by 2 nucleotides. In some embodiments, an engineered crRNA repeat, absent chemical modifications, has a nucleotide sequence that differs from SEQ ID NO: 38 by 1 nucleotide. In some embodiments, an engineered crRNA repeat, absent chemical modifications, has the nucleotide sequence set forth as SEQ ID NO: 38.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide, wherein, with reference to SEQ ID NO: 39. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 39.

In some embodiments, an engineered crRNA comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, or 14 nt, as compared to the same crRNA prior to the engineering. In certain embodiments, an engineered crRNA comprises a truncation of 1 nt, 2 nt, 3 nt,

4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, or 14 nt, compared to a nucleotide sequence set forth as any one of SEQ ID NOs: 18, 19, 71, 72, 95, or 96. In some embodiments, an engineered crRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 40 or 41. In some embodiments, an engineered crRNA, absent chemical modifications, comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 40 or 41. In some embodiments, an engineered crRNA, absent chemical modifications, comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 40 or 41. In some embodiments, an engineered crRNA, absent chemical modifications, comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 40 or 41.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, an engineered chemically modified crRNA has the sequence set forth as SEQ ID NO: 42.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, a chemically modified, truncated crRNA has the sequence set forth as SEQ ID NO: 43.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 44. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, an engineered chemically modified crRNA has the sequence set forth as SEQ ID NO: 44.

In some embodiments, an engineered tracrRNA comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt,

5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt,

38 nt, 39 nt, 40 nt, 41 nt, 42 nt, or 43 nt, as compared to the same tracrRNA prior to the engineering.

In certain embodiments, an engineered tracrRNA comprises a truncation of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt,

23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt,

39 nt, 40 nt, 41 nt, 42 nt, or 43 nt, compared to a nucleotide sequence set forth as any one of SEQ ID NOs: 3, 76, 77, 100, 242, 254, and 539. In some embodiments, an engineered tracrRNA comprises a truncation of 2 nucleotides, 4 nucleotides, 6 nucleotides, 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, or 18 nucleotides, as compared to the same tracrRNA prior to the engineering.

In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 to 12 nucleotides within the first stem of the anti -repeat, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, or 12 nt within the first stem of the anti-repeat, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, or 9 nt within the first stem of the anti-repeat, as compared to the same chemically modified tracrRNA prior to the deletion.

In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of nucleotides from the tail, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 to 6 nucleotides from the tail, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides from the tail, as compared to the same chemically modified tracrRNA prior to the deletion.

In some embodiments, an engineered chemically modified tracrRNA comprises a deletion in a stem loop most proximal to the tail, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 to 4 nucleotide pairs within the first stem of the stem-loop most proximal to the tail of the tracrRNA, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 to 3 nucleotide pairs within the first stem of the stem-loop most proximal to the tail of the tracrRNA, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered chemically modified tracrRNA comprises a deletion of 1 nucleotide pair, 2 nucleotide pairs, or 3 nucleotide pairs within the first stem of the stem-loop most proximal to the tail of the tracrRNA, as compared to the same chemically modified tracrRNA prior to the deletion. In some embodiments, an engineered tracrRNA, absent chemical modifications, comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 45-47. In some embodiments, an engineered tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 45-47. In some embodiments, an engineered tracrRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 45-47. In some embodiments, an engineered tracrRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 45-47.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, an engineered chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 51.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 52. In some embodiments, an engineered chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 52.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 53s. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 53. In some embodiments, an engineered chemically modified tracrRNA has the nucleotide sequence set forth as SEQ ID NO: 53.

In some embodiments, a gRNA of the disclosure is a sgRNA that comprises a backbone, wherein the backbone of the sgRNA comprises a crRNA repeat and a tracrRNA linked by a nucleotide linker. In some embodiments, the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG. In some embodiments, the linker has the nucleotide sequence set forth as AAAG. In some embodiments, a chemically modified sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407. In some embodiments, a gRNA of the disclosure is a dgRNA that comprises a backbone, wherein the backbone of the dgRNA comprises a crRNA repeat and a tracrRNA. In some embodiments, the backbone of an engineered chemically modified sgRNA or dgRNA is 2 to 30 nucleotides shorter, as compared to the same backbone prior to the engineering. In some embodiments, the backbone of an engineered chemically modified sgRNA or dgRNA is 2 to 18 nucleotides shorter, as compared to the same backbone prior to the engineering. In some embodiments, the backbone of an engineered chemically modified sgRNA or dgRNA is 2 nucleotides, 4 nucleotides, 6 nucleotides, 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, or 18 nucleotides shorter, as compared to the same backbone prior to the engineering.

In some embodiments, the backbone of an engineered sgRNA, absent chemical modifications, has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 32-34. In some embodiments, the backbone of an engineered sgRNA, absent chemical modifications, has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 32-34. In some embodiments, the backbone of an engineered sgRNA, absent chemical modifications, has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 32-34. In some embodiments, the backbone of an engineered sgRNA, absent chemical modifications, has a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 32-34.

In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 35. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 35. In some embodiments, an engineered chemically modified backbone of a sgRNA has the nucleotide sequence set forth as SEQ ID NO: 35.

In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 36. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 36.

In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 37. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 37. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 37. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 296. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 296. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 296. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 296.

In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 297. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 297. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 297. In some embodiments, an engineered chemically modified backbone of a sgRNA has a nucleotide sequence set forth as SEQ ID NO: 297.

In some embodiments, a gRNA of the disclosure is a sgRNA that comprises a spacer and a backbone, wherein the backbone of the sgRNA comprises a crRNA repeat and a tracrRNA linked by a nucleotide linker. In some embodiments, an engineered chemically modified sgRNA comprises a truncation in the spacer and/or a truncation in the backbone, as compared to the same chemically modified sgRNA prior to the engineering. In some embodiments, an engineered chemically modified sgRNA comprises a truncation in the spacer. In some embodiments, an engineered chemically modified sgRNA comprises a truncation in the backbone. In some embodiments, an engineered chemically modified sgRNA comprises a truncation in the spacer and a truncation in the backbone. In some embodiments, an engineered chemically modified sgRNA comprises a deletion of 1 to 25 nucleotides, as compared to the same chemically modified sgRNA prior to the engineering. In some embodiments, an engineered chemically modified sgRNA comprises a deletion of 13 to 23 nucleotides, as compared to the same chemically modified sgRNA prior to the engineering. In some embodiments, an engineered chemically modified sgRNA comprises a deletion of 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides, as compared to the same chemically modified sgRNA prior to the engineering. In some embodiments, an engineered chemically modified sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 60-68, 298, and 299.

Gene editing efficiency of a dgRNA can be improved by engineering nucleotide substitutions or additions in the crRNA repeat, crRNA, and/or the tracrRNA and chemical modifications (see FIGs. 20-21). A “substitution” or “replacement” as used herein in the context of engineering a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA, refers to either substituting or replacing a given number of nucleotides with a same number of nucleotides (e.g., replacing 3 nucleotides with 3 nucleotides) or substituting or replacing a given number of nucleotides with a different number of nucleotides. In embodiments where the substituting or replacing a given number of nucleotides is with a different number of nucleotides, the different number of nucleotides can be greater or less than the given number of nucleotides (e.g., replacing 8 nucleotides with 3 nucleotides, or replacing 3 nucleotides with 8 nucleotides). An “addition” as used herein in the context of engineering a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA, refers to adding at least one nucleotide to a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA at any location(s) of a spacer, a crRNA repeat, a crRNA, an anti-repeat, a tracrRNA, a backbone, and/or a guide RNA. In some embodiments, the engineering described herein, with or without chemical modifications, allows use of dgRNA in prime editing. In some embodiments, this engineering, with or without chemical modifications, allows use of dgRNA in prime editing with delivery of an RNA-guided nuclease as an mRNA.

In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a substituted or added nucleotide sequence from a native precursor CRISPR RNA (pre- crRNA). A native pre-crRNA includes a series of alternating crRNA repeats and spacers and is transcribed from a CRISPR array in an organism. The native pre-crRNA is further processed by enzymes to a mature crRNA form that is used for genome modification in the organism. Thus, in some embodiments, the native pre-crRNA contains sequences that are not present in an intermediate processed or mature crRNA. For example, nucleotide sequences that are used to lengthen the first stem of an APG05586 crRNA repeat can be from an Enterococcus sp. APG05586 pre-crRNA.

In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a substituted or added nucleotide sequence that is GC-rich. In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a GC-rich nucleotide sequence, wherein the content of G or C in the 3' region is at least 60%, at least 80%, or 100%. In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a GC-rich nucleotide sequence, wherein the 3' region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs. In some embodiments, the first stem of an engineered crRNA repeat further comprises at least one chemical modification. In some embodiments, the first stem of an engineered crRNA repeat further comprises MS, BNA, or BNA+PS modifications at the 3' region.

In some embodiments, the first stem of an engineered anti-repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA). In some embodiments, the first stem of an engineered anti -repeat comprises at the 5' region a GC-rich nucleotide sequence. In some embodiments, the first stem of an engineered anti -repeat comprises at the 5 ' region a GC-rich nucleotide sequence, wherein the content of G or C in the 5' region is at least 60%, at least 80%, or 100%. In some embodiments, the first stem of an engineered anti-repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5' region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs. In some embodiments, the first stem of an engineered anti-repeat further comprises at least one chemical modification. In some embodiments, the first stem of an engineered anti-repeat further comprises BNA (e.g., LNA and/or cEt) modifications on all nucleotides. A “GC-rich nucleotide sequence” refers to a nucleotide sequence that has at least 51%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% GC content. In some embodiments, a GC-rich nucleotide sequence refers to a nucleotide sequence that comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs when the nucleotide sequence comprises at most 5 nucleotides.

In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 380. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 380. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 380. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 380.

In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 381. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 381. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 381. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 381.

In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 382. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 382. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 382. In some embodiments, an engineered, chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 382.

In some embodiments, an engineered, chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 383. In some embodiments, an engineered, chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 383. In some embodiments, an engineered, chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 383. In some embodiments a chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 383.

Chemical modification (e.g., LNA modification of all nucleotides at the first stem of the antirepeat) of a dgRNA does not improve gene editing for some RGN systems when an RNA-guided nuclease is delivered as an mRNA (see FIGs. 10B and 10C). In some embodiments, gene editing efficiency is improved or gene editing is rescued by adding nucleotides to the first stem of the anti- repeat, adding nucleotides to the first stem of the crRNA repeat, and chemically modifying the lengthened first stems of the crRNA repeat and anti-repeat.

In some embodiments, an engineered crRNA repeat comprises an addition of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition of 2 to 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition of 2 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition of 4 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition of 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering.

In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. In some embodiments, nucleotides in the 3' region of the first stem of a crRNA repeat further comprise MS modifications, BNA modifications, or BNA+PS modifications.

In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise MS modifications. In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise BNA modifications. In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise BNA+PS modifications. In some embodiments, the BNA modifications comprise 2', 4' BNA modifications. In some embodiments, the 2', 4' BNA modifications are selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'- C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA modifications are LNA modifications.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 300.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 304.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 308.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 312.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 320.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 344. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 348.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 352.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 356.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 360.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 324.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 328.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 332.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 336.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 465.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 469.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 473.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 477.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 481.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 508.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 512.

In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide. In some embodiments, an engineered chemically modified crRNA repeat has a nucleotide sequence set forth as SEQ ID NO: 516.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 301. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 301. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 301. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 301.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 302. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 302. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 302. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 302.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 303. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 303. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 303. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 303.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 305. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 305. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 305. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 305.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 306. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 306. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 306. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 306.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 307. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 307. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 307. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 307.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 309. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 309. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 309. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 309.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 310. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 310. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 310. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 310.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 311. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 311. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 311. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 311.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 313. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 313. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 313. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 313.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 314. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 314. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 314. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 314.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 315. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 315. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 315. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 315.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 321. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 321. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 321. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 321.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 322. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 322. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 322. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 322.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 323. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 323. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 323. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 323.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 345. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 345. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 345. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 345.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 346. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 346. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 346. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 346.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 347. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 347. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 347. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 347.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 349. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 349. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 349. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 349.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 350. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 350. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 350. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 350.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 351. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 351. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 351. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 351.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 353. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 353. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 353. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 353.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 354. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 354. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 354. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 354.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 355. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 355. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 355. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 355.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 357. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 357. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 357. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 357.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 358. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 358. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 358. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 358.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 359. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 359. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 359. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 359.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 361. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 361. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 361. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 361.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 362. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 362. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 362. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 362.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 363. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 363. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 363. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 363.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 325. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 325. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 325. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 325.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 326. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 326. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 326. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 326.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 327. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 327. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 327. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 327.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 329. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 329. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 329. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 329.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 330. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 330. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 330. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 330.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 331. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 331. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 331. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 331.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 333. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 333. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 333. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 333.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 334. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 334. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 334. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 334.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 335. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 335. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 335. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 335.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 337. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 337. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 337. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 337.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 338. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 338. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 338. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 338.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 339. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 339. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 339. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 339.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 466. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 466. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 466. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 466.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 467. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 467. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 467. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 467.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 468. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 468. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 468. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 468.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 470. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 470. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 470. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 470.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 471. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 471. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 471. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 471.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 472. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 472. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 472. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 472.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 474. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 474. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 474. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 474.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 475. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 475. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 475. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 475.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 476. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 476. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 476. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 476.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 478. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 478. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 478. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 478.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 479. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 479. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 479. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 479.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 480. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 480. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 480. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 480.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 482. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 482. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 482. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 482.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 483. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 483. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 483. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 483.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 484. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 484. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 484. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 484.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 509. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 509. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 509. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 509.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 510. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 510. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 510. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 510.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 511. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 511. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 511. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 511.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 513. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 513. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 513. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 513.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 514. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 514. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 514. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 514.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 515. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 515. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 515. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 515.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 517. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 517. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 517. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 517.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 518. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 518. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 518. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 518.

In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 519. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 519. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 519. In some embodiments, an engineered chemically modified crRNA has a nucleotide sequence set forth as SEQ ID NO: 519.

In some embodiments, an engineered tracrRNA comprises an addition of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering. In some embodiments, an engineered tracrRNA comprises an addition of 2 to 6 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering. In some embodiments, an engineered tracrRNA comprises an addition of 2 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering. In some embodiments, an engineered tracrRNA is lengthened by addition of 4 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering. In some embodiments, an engineered tracrRNA comprises an addition of 6 nucleotides at the 5' region of the tracrRNA, as compared to the same tracrRNA prior to the engineering.

In some embodiments, the first stem of an engineered anti -repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the first stem of an engineered anti-repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of the engineered anti -repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. In some embodiments, all nucleotides of the first stem of the engineered anti-repeat further comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the BNA modifications comprise 2', 4' BNA modifications. In some embodiments, the 2', 4' BNA modifications are selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA modifications are LNA modifications. In some embodiments, the 2', 4' BNA modifications are cEt modifications.

In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise MS modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise BNA modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise LNA modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise BNA+PS modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise LNA+PS modifications.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 364. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 364. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 364. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 364.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 365. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 365. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 365. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 365.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 366. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 366. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 366. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 366.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 367. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 367. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 367. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 367.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 369. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 369. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 369. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 369.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 375. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 375. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 375. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 375.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 376. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 376. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 376.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 377. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 377. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 377. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 377.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 378. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 378. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 378. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 378.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 379. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 379. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 379. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 379.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 370. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 370. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 370. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 370. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 371. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 371. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 371. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 371.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 372. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 372. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 372. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 372.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 373. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 373. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 373. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 373.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 710. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 710. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 710. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 710.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 711. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 711. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 711. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 711.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 499. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 499. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 499. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 499.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 500. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 500. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 500. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 500.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 501. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 501. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 501. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 501.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 504. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 504. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 504. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 504.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 505. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 505. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 505. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 505.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 534. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 534. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 534. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 534.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 535. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 535. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 535. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 535.

In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 537. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 537. In some embodiments, an engineered chemically modified tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 537. In some embodiments an engineered chemically modified tracrRNA has a nucleotide sequence set forth as SEQ ID NO: 537.

IV. RNA-guided nucleases

Provided herein are RNA-guided nuclease systems comprising the presently disclosed guide RNAs, particularly guide RNAs modified with BNA modifications (e.g., guide RNAs modified with LNA modifications). In some embodiments, the RNA-guided nucleases bind the presently disclosed gRNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti -repeat of the tracrRNA. The term RNA-guided nuclease (RGN) refers to a polypeptide that binds to a particular target sequence (e.g., target DNA sequence) in a sequence -specific manner and is directed to the target sequence by a guide RNA molecule that is complexed with the polypeptide and hybridizes with the target strand of the target sequence (e.g., target DNA sequence). Active fragments or variants thereof of naturally-occurring RGNs maintain binding to a target sequence in an RNA-guided sequence -specific manner. Although an RGN can be capable of cleaving the target strand and/or nontarget strand of a target sequence upon binding, the term RGN also encompasses nuclease-dead RGNs that are capable of binding to, but not cleaving, a target sequence. Cleavage of a target strand and/or non-target strand of a target sequence by an RGN can result in a single- or double -stranded break. RGNs only capable of cleaving a single strand of a double -stranded target nucleic acid molecule are referred to herein as nickases. CRISPR-Cas systems are RGN systems classified into Class 1 or Class 2. The Class 1 and 2 systems are subdivided into types (Types I, II, III, IV, V, VI), with some types further divided into subtypes (e.g., Type II-A, Type II-B, Type II-C, Type V-A, Type V-B). Class 2 systems comprise a single effector nuclease and include Types II, V, and VI.

In certain embodiments, the RGN is a naturally-occurring Type II CRISPR-Cas protein or an active variant or fragment thereof. As used herein, the term “Type II CRISPR-Cas protein,” “Type II CRISPR-Cas effector protein,” or “Type II RNA-guided nuclease” refers to an RGN that requires a trans-activating RNA (tracrRNA) and comprises two nuclease domains (i.e., RuvC and HNH), each of which is responsible for cleaving a single strand of a double -stranded DNA molecule. In some embodiments, a representative type II RGN includes a Cas9 protein, such as Streptococcus pyogenes Cas9 (SpCas9 or SpyCas9) or a SpCas9 nickase, the sequences of which are set forth as SEQ ID NOs: 279 and 280, respectively, and are described in U.S. Pat. Nos. 10,000,772 and 8,697,359, each of which is herein incorporated by reference in its entirety. In some embodiments, a representative type II RGN includes a Streptococcus thermophilus Cas9 (StCas9) or a StCas9 nickase, the sequences of which are set forth as SEQ ID NOs: 281 and 282, respectively, and are disclosed in U.S. Pat. No. 10,113,167, which is herein incorporated by reference in its entirety. In some embodiments, a representative type II RGN includes a Streptococcus aureus Cas9 (SaCas9) or a SaCas9 nickase, the sequences of which are set forth as SEQ ID NOs: 283 and 284, respectively, and are disclosed in U.S. Pat. No. 9,752,132, which is herein incorporated by reference in its entirety.

In some embodiments, the CRISPR-Cas protein is a naturally-occurring Type V CRISPR-Cas protein or an active variant or fragment thereof. As used herein, the term “Type V CRISPR-Cas protein,” “Type V CRISPR-Cas effector protein,” or “Type V RNA-guided nuclease” refers to an RGN that cleaves dsDNA and comprises a single RuvC nuclease domain or a split-RuvC nuclease domain and lacks an HNH domain (Zetsche et al 2015, Cell doi: 10.1016/j .cell.2015.09.038; Shmakov et al 2017, Nat Rev Microbiol doi: 10.1038/nrmicro.2016.184; Yan et al 2018, Science doi: 10.1126/science.aav7271; Harrington et al 2018, Science doi: 10.1126/science.aav4294). In some embodiments, a presently disclosed RGN protein comprises a Casl2 (e.g., Casl2a). It is to be noted that Casl2a is also referred to as Cpfl, and does not require a tracrRNA, although other Type V CRISPR-Cas proteins, such as Casl2b, do require a tracrRNA. Most Type V effectors can also target ssDNA (single-stranded DNA), often without a PAM requirement (Zetsche et al 2015; Yan et al 2018; Harrington et al 2018). The terms “Type V CRISPR-Cas protein” and “Type V RGN” encompasses the unique RGNs comprising split RuvC nuclease domains, such as those disclosed in U.S. Provisional Application Nos. 62/955,014 filed December 30, 2019 and 63/058,169 filed July 29, 2020, and PCT International Application No. PCT/US2020/067138 filed December 28, 2020, the contents of each of which are incorporated herein by reference in its entirety. In some embodiments, the present invention provides a RGN protein comprising Francisella novicida Casl2a (FnCasl2a), the sequence of which is set forth as SEQ ID NO: 408 and is disclosed in U.S. Pat. No. 9,790,490, which is herein incorporated by reference in its entirety, or any of the nuclease -inactivating mutants of FnCasl2a disclosed within U.S. Pat. No. 9,790,490.

In some embodiments, the presently disclosed RGN systems comprise an RGN that binds to a target sequence, e.g., such as those that are disclosed herein. In some embodiments, the RGN recognizes a PAM having a consensus nucleotide sequence including a NNNNCC 3' of the target sequence on its non-target strand (where N is A, C, T, or G; R is G or A), and active fragments or variants thereof. In some embodiments, the RGN recognizes a PAM having a consensus nucleotide sequence including NNGRR at 3' of the target sequence on its non-target strand (where N is A, C, T/U, or G; R is G or A), and active fragments or variants thereof. In some embodiments, the RGN recognizes a PAM having a consensus nucleotide sequence including NNRYA at 3' of the target sequence on its non-target strand (where N is A, C, T/U, or G; R is G or A; Y is C or T/U), and active fragments or variants thereof. In some embodiments, the RGN recognizes a PAM having a consensus nucleotide sequence including NGG at 3' of the target sequence on its non-target strand (where N is A, C, T/U, or G), and active fragments or variants thereof. In embodiments, the active fragment or variant of an RGN recognizing such PAM sequences is capable of binding and in some embodiments, cleaving or nicking a target sequence.

In embodiments, an RGN, or an active variant or fragment thereof, capable of binding a target sequence adjacent to a PAM consensus sequence (i.e., capable of recognizing the PAM consensus sequence) set forth as NNNNCC is used in the presently disclosed compositions and methods. In embodiments, the PAM sequence is at 3' of the target sequence on its non-target strand. In embodiments, the RGN binds to a guide RNA comprising a crRNA repeat set forth in SEQ ID NO: 2 or an active variant or fragment thereof, and a tracrRNA set forth in SEQ ID NO: 3 or an active variant or fragment thereof. Non-limiting examples of RGN systems useful in the presently disclosed compositions and methods along with corresponding crRNA sequences and tracrRNA sequences (if needed), are presented in Table 1 below and described further in Examples 1-18, and FIGs. 2-34 of the present specification.

Table 1. Non-limiting examples of RNA-guided nucleases and corresponding crRNA repeat sequences, tracrRNA sequences, and PAM sequences.

*two tracrRNA sequences for APG01604

ND: not determined

N = A, C, T/U, or G; R = G or A; Y = C or T/U; V = A or G or C; D = A or G or T/U; W = A or T/U Non-limiting examples of RGNs useful in the presently disclosed methods and compositions include APG07433.1, APG01604, APG05586, and APG07991 RNA-guided nucleases, the amino acid sequence of each of which is set forth, respectively, as SEQ ID NO: 1, 69, 93, or 252, and active fragments or variants thereof that retain the ability to bind to a target sequence in an RNA-guided sequence -specific manner. In some embodiments, an active variant of an RGN disclosed herein requires a tracrRNA for activity. In some embodiments, an active variant of an RGN disclosed herein includes a type II RGN. In embodiments, an active variant of an RGN disclosed herein comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, or 252. In some embodiments, an active fragment of the APG07433.1 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,

550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1. In some embodiments, an active fragment of the APG01604 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 69. In some embodiments, an active fragment of the APG05586 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 93. In embodiments, an active fragment of the APG07991 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 252.

The presently disclosed compositions and methods can utilize an RGN comprising at least one nuclease domain (e.g., DNase, RNase domain) and at least one RNA recognition and/or RNA binding domain to interact with guide RNAs. Further domains that can be found in RGNs useful in the presently disclosed compositions and methods include, but are not limited to: DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. In embodiments, the RGNs useful in the presently disclosed compositions and methods comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of a DNA binding domain, helicase domain, protein-protein interaction domain, and dimerization domain.

According to the present invention, the presently disclosed target sequences are bound by an RGN. The target strand of the target sequence hybridizes with the guide RNA associated with the RGN. The target strand and/or the non-target strand of the target sequence (e.g., target DNA sequence) can then be subsequently cleaved by the RGN if the polypeptide possesses nuclease activity. The terms “cleave” or “cleavage” refer to the hydrolysis of at least one phosphodiester bond within the backbone of one or both strands of a double-stranded target sequence (e.g., target DNA sequence) that can result in either single-stranded or double-stranded breaks within the target sequence. The cleavage of a presently disclosed target sequence can result in staggered breaks or blunt ends.

RGNs useful in the presently disclosed compositions and methods can be wild-type sequences derived from bacterial or archaeal species. Alternatively, the RGNs can be variants or fragments of wild-type polypeptides. The wild-type RGN can be modified to alter nuclease activity or alter PAM specificity, for example. In embodiments, the RGN is not naturally-occurring.

An RGN useful in the presently disclosed compositions and methods can function with its own cognate guide RNA or a guide RNA that is heterologous to the RGN. For example, the APG07991 RGN (SEQ ID NO: 252) can bind to engineered and chemically modified APG07991 crRNAs and tracrRNAs disclosed herein to effect cleavage or modification of a target nucleic acid molecule (e.g., a APG07991 crRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, and 482-484; and/or a APG07991 tracrRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 499-501, 504, and 505; see FIGs. 27 and 28). The APG07991 RGN also can bind to engineered and chemically modified SpyCas9 crRNAs and tracrRNAs disclosed herein to effect cleavage or modification of a target nucleic acid molecule (e.g., a SpyCas9 crRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 509-511, 513- 515, and 517-519; and/or a SpyCas9 tracrRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 534, 535, and 537; see FIG. 29).

In some embodiments, an RGN useful in the presently disclosed compositions and methods functions as a nickase, only cleaving a single strand of a double-stranded target sequence (e.g., target DNA sequence). Such RGNs have a single functioning nuclease domain. In some embodiments, the nickase is capable of cleaving the target strand or the non-target strand of the double -stranded target sequence (e.g., target sequence). In some embodiments, additional nuclease domains have been mutated such that the nuclease activity is reduced or eliminated. In embodiments wherein a nickase is used, in order to effect a double-stranded cleavage of a double -stranded target sequence (e.g., target DNA sequence), two nickases are needed, each of which nicks a single strand within the doublestranded target sequence.

In some embodiments, the RGN lacks nuclease activity altogether and is referred to herein as nuclease-dead or nuclease inactive. Any method known in the art for introducing mutations into an amino acid sequence, such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used for generating nickases or nuclease-dead RGNs. See, e.g., U.S. Publ. No. 2014/0068797 and U.S. Pat. No. 9,790,490; each of which is incorporated by reference in its entirety.

RGNs that lack nuclease activity can be used to deliver a fused polypeptide, polynucleotide, or small molecule payload to a particular genomic location. In embodiments, the RGN polypeptide or guide RNA can be fused to a detectable label to allow for detection of a particular sequence. The detectable label or purification tag can be located at the N-terminus, the C-terminus, or an internal location of the RNA-guided nuclease, either directly or indirectly via a linker peptide. In some embodiments, the RGN component of the fusion protein is a nuclease-dead RGN. In some embodiments, the RGN component of the fusion protein is an RGN with nickase activity.

A detectable label is a molecule that can be visualized or otherwise observed. The detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to the RGN polypeptide that can be detected visually or by other means. Detectable labels that can be fused to the presently disclosed RGNs as a fusion protein include any detectable protein domain, including but not limited to, a fluorescent protein or a protein domain that can be detected with a specific antibody. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreenl) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellowl). Non-limiting examples of small molecule detectable labels include radioactive labels, such as ³H and ³⁵ S.

RGN polypeptides can also comprise a purification tag, which is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium). Non-limiting examples of purification tags include biotin, myc, maltose binding protein (MBP), glutathione-S-transferase (GST), and 3X FLAG tag.

Alternatively, nuclease-dead RGNs can be targeted to particular genomic locations to alter the expression of a desired gene (i.e., target gene). In some embodiments, the binding of a nuclease-dead RGN to a target sequence results in the reduction in expression of the target gene by interfering with the binding of RNA polymerase or transcription factors within the targeted genomic region. In embodiments, the RGN (e.g., a nuclease-dead RGN) or its complexed guide RNA further comprises an expression modulator that, upon binding to a target sequence within a target gene, serves to either repress or activate the expression of the target gene.

In some embodiments, the expression modulator of the fusion protein comprises a transcriptional repressor domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to reduce or terminate transcription of at least one gene. Transcriptional repressor domains are known in the art and include, but are not limited to, Spl-like repressors, IKB, and Kriippel associated box (KRAB) domains.

In some embodiments, the expression modulator of the fusion protein comprises a transcriptional activation domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to increase or activate transcription of at least one gene. Transcriptional activation domains are known in the art and include, but are not limited to, a herpes simplex virus VP 16 activation domain and an NFAT activation domain.

In some embodiments, the expression modulator modulates the expression of the target sequence or regulated gene through epigenetic mechanisms. In some embodiments, an epigenetic modulator covalently modifies DNA or histone proteins to alter histone structure and/or chromosomal structure without altering the DNA sequence, leading to changes in gene expression (e.g., upregulation or downregulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation of histone proteins, and methylation and hydroxymethylation of cytosine residues in DNA. Non-limiting examples of epigenetic modulators include histone acetyltransferases, histone deacetylases, histone methyltransferases, histone demethylases, DNA methyltransferases, and DNA demethylases.

In embodiments, the nuclease-dead RGNs or an RGN with nickase activity can be targeted to particular genomic locations to modify the sequence of a target polynucleotide through fusion to a base-editing polypeptide, for example a deaminase polypeptide or active variant or fragment thereof, that directly chemically modifies (e.g., deaminates) a nucleobase, resulting in conversion from one nucleobase to another. The base-editing polypeptide can be fused to the RGN at its amino-terminal (N-terminal) or carboxy-terminal (C-terminal) end. Additionally, the base-editing polypeptide may be fused to the RGN via a peptide linker. “Base editors” are fusion proteins comprising a DNA-targeting polypeptide, such as an RGN, and a base-editing polypeptide, such as a deaminase. Non-limiting examples of deaminases or base editors that are useful for such compositions and methods includes a cytosine deaminase, an adenine deaminase, or a base editor (such as the adenine deaminase or base editor described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, and International Publ. No. WO 2018/027078, or any of the deaminases or base editors disclosed in International Publ. Nos. WO 2020/139783, WO 2022/056254, WO 2022/204093, U.S. provisional application no. 63/382,344 (fded November 4, 2022), and U.S. provisional application no. 63/485,642 (fded February 17, 2023), each of which is herein incorporated by reference in its entirety). In some embodiments, the deaminase that is useful for such presently disclosed compositions and methods is a deaminase disclosed in Table 17 of International Publ. No. WO 2020/139783, which is incorporated herein by reference in its entirety. In some embodiments, a deaminase useful for such presently disclosed compositions and methods includes a deaminase set forth as any one of the polypeptides of SEQ ID NOs: 542, 544, and 592-707. In some embodiments, a deaminase useful for such presently disclosed compositions and methods includes a deaminase having at least 85%, 90%, 95%, or 97% sequence identity to any one of the polypeptides of SEQ ID NOs: 542, 544, and 592-707. In some embodiments, a base editor useful for such presently disclosed compositions and methods includes a base editor set forth as any one of the polypeptides of SEQ ID NOs: 550-591. In some embodiments, a base editor useful for such presently disclosed compositions and methods includes a base editor having at least 85%, 90%, 95%, or 97% sequence identity to any one of the polypeptides of SEQ ID NOs: 550-591. Further, it is known in the art that certain base editor comprising an RGN and a base-editing polypeptide (e.g., cytosine deaminase) may also comprise at least one uracil stabilizing polypeptide that increases the mutation rate of a cytidine, deoxycytidine, or cytosine to a thymidine, deoxythymidine, or thymine in a nucleic acid molecule by a deaminase. Non-limiting examples of uracil stabilizing polypeptides include those disclosed in PCT Publication No. WO 2021/217002 and PCT Publication No. WO 2022/015969, each of which is herein incorporated by reference in its entirety. The disclosed uracil stabilizing polypeptides include USP2, and a uracil glycosylase inhibitor (UGI) domain, which may increase base editing efficiency. Therefore, a base editor may comprise an RGN described herein or variant thereof, a deaminase, and optionally at least one uracil stabilizing polypeptide, such as UGI or USP2. In embodiments, the RGN that is fused to the base-editing polypeptide is a nickase that cleaves the DNA strand that is not acted upon by the base-editing polypeptide (e.g., deaminase).

A gRNA modified with BNA modifications (e.g., gRNAs modified with LNA and/or cEt modifications) as described herein can allow the use of dgRNAs or sgRNAs in a base editing system where the RGN is delivered as mRNA encoding the RGN. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein at least one nucleotide in the first stem of the anti- repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications.

A gRNA modified with BNA modifications (e.g., gRNAs modified with LNA and/or cEt modifications) as described herein can allow the use of dgRNAs or sgRNAs in a base editing system where the RGN is delivered as mRNA encoding the RGN. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5 ' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the crRNA repeat and the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the crRNA repeat and the antirepeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.

A gRNA modified with BNA modifications (e.g., gRNAs modified with LNA and/or cEt modifications) as described herein can allow the use of dgRNAs or sgRNAs in a base editing system where the RGN is delivered as mRNA encoding the RGN. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti -repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti- repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.

A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti -repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered crRNA repear and the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered crRNA repear and the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.

A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered antirepeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 8-13 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. A gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a base editing system includes a sgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered antirepeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracrRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.

In some embodiments, an RGN may be fused to a prime editing polypeptide (e.g., a reverse transcriptase). Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein working in association with a polymerase (described in, e.g., US 11,447,77OB1; WO2021072328; WO2021226558; WO2020156575; W02021042047; US11193123; each incorporated by reference in its entirety herein). The prime editing system uses an RGN that is a nickase, and the system is programmed with a prime editing (PE) guide RNA (“PEgRNA”). The PEgRNA is a guide RNA that both specifies the target sequence and provides the template for polymerization of the replacement strand containing the edit by way of an extension engineered onto the guide RNA (e.g., at the 5' or 3' end, or at an internal portion of the guide RNA). The RGN nickase/prime editing polypeptide fusion is guided to the target sequence by the PEgRNA and nicks the non-target strand upstream of sequence to be edited and upstream of the PAM, creating a 3' flap on the non-target strand. The PEgRNA includes a primer binding site (PBS) that is complementary to the 3' flap of the non-target strand. In some embodiments, a PBS is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In certain embodiments, the PEgRNA comprises a PBS that is at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, or 20) nucleotides in length. In some embodiments, the PEgRNA may comprise a PBS that is at least 8 nucleotides in length. Hybridrization of the PBS and 3' flap of the non-target strand allows polymerization of the replacement strand containing the edit using the extension of the PEgRNA as template. The extension of the PEgRNA can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase.

The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the non-target strand of the target sequence to be edited (with the exception that it includes the desired edit). Through DNA repair and/or replication machinery, the non-target strand of the target sequence is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors not only search and locate the desired target sequence to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding non-target strand of the target sequence. Thus, in some embodiments, a guide RNA of the disclosure comprises an extension comprising an edit template for prime editing. In some embodiments, a prime editing polypeptide that can be fused to an RGN includes a DNA polymerase. In certain embodiments, the DNA polymerase is a reverse transcriptase. In certain embodiments, the RGN is a nickase.

In some embodiments, the presently disclosed guide RNAs are combined with an RGN nickase comprising a mutation in the RuvC domain (e.g., a D10A mutation) which renders the RGN capable of cleaving only the target strand (the strand which does not comprise the PAM) of a nucleic acid duplex. D10A nickases are not able to cleave the non-target strand of the DNA, i.e., the strand where base editing is desired. In these embodiments, the RGN nicks the target strand, while the complementary, non-target strand is modified by the deaminase. Cellular DNA-repair machinery may repair the nicked, target strand using the modified non-target strand as a template, thereby introducing a mutation in the DNA.

Thus, in some embodiments, the nickase comprises an inactive RuvC domain. RuvC domains have an RNase H fold structure (see, e.g., Nishimasu et al. (2014) Cell 156(5):935-949, which is incorporated by reference in its entirety). RuvC domains of RGNs are often split RuvC domains, comprising two or more non-adjacent regions within the linear amino acid sequence. For example, the RuvC domain of Streptococcus pyogenes Cas9 comprises amino acid residues 1-59, 718-769 and 909- 1098 of SEQ ID NO: 279. A non-limiting example of a mutation within a RuvC domain that inactivates its nuclease activity is the D10A mutation that mutates the first aspartic acid residue in the split RuvC nuclease domain. nAPG07433.1 (set forth as SEQ ID NO: 543) is a nickase variant of APG07433.1, which is set forth as SEQ ID NO: 1, and is described in WO 2019/236566 (incorporated by reference in its entirety herein). Other D10A mutant (i.e., inactive RuvC/active HNH domain) nickases include, but are not limited to those set forth as SEQ ID NOs: 546-548.

In some embodiments, the nickase RGN of the fusion protein comprises a mutation (e.g., a H840A mutation, wherein amino acid numbering is based on the Streptococcus pyogenes Cas9 sequence set forth as SEQ ID NO: 279), which renders the RGN capable of cleaving only the nontarget strand (the strand which comprises the PAM) of a nucleic acid duplex. In some of these embodiments, the nickase comprises an inactive HNH nuclease domain. The HNH nuclease domain of RGNs have a PPa-metal fold (see, e.g., Nishimasu et al. 2014). The HNH nuclease domain of the Streptococcus pyogenes Cas9, for example, comprises amino acid residues 775-908 of SEQ ID NO: 279. A non-limiting example of a mutation within a HNH domain that inactivates its nuclease activity is the H840A mutation that mutates the first histidine of the HNH nuclease domain. A non-limiting example of an H840A mutant (i.e., active RuvC/inactive HNH domain) nickase includes that set forth as SEQ ID NO: 549.

Inactive RuvC/active HNH domain (e.g., D10A mutant) nickases are useful for base editing with a fused deaminase and active RuvC/inactive HNH domain (e.g., H840A mutant) nickases are useful for prime editing with a fused reverse transcriptase.

Methods for inactivating a RuvC and/or HNH domain of a RGN are known in the art and generally comprise mutating the first aspartic acid within a RuvC domain and/or the first histidine of the HNH domain. Typically, the aspartic acid residue or histidine residue is mutated to an alanine. Other amino acid residues within the RuvC domain that can be mutated to inactivate nuclease activity of the domain include Glu762, His983, and Asp986 (typically to an alanine), wherein amino acid numbering is based on the Streptococcus pyogenes Cas9 sequence set forth as SEQ ID NO: 279. Other amino acid residues within the HNH domain that can be mutated include D839 and N863 (typically to an alanine), wherein amino acid numbering is based on the Streptococcus pyogenes Cas9 sequence set forth as SEQ ID NO: 279.

In some embodiments, a gRNA modified with BNA modifications (e.g., gRNAs modified with LNA modifications) as described herein allow the use of dgRNAs in a prime editing system where the RGN is delivered as mRNA encoding the RGN. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and atracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.

In some embodiments, a gRNA modified with BNA modifications (e.g., gRNAs modified with LNA and/or cEt modifications) as described herein allow the use of dgRNAs in a prime editing system where the RGN is delivered as mRNA encoding the RGN. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification.

In some embodiments, a gRNA modified with BNA modifications (e.g., gRNAs modified with LNA and/or cEt modifications) as described herein allow the use of dgRNAs in a prime editing system where the RGN is delivered as mRNA encoding the RGN. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification, and wherein the three terminal nucleotides at the tail of the tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein at least one nucleotide in the first stem of the anti-repeat of the tracrRNA comprises a BNA (e.g., LNA and/or cEt) modification.

In some embodiments, a gRNA modified with BNA modifications (e.g., gRNAs modified with LNA and/or cEt modifications) as described herein allow the use of dgRNAs in a prime editing system where the RGN is delivered as mRNA encoding the RGN. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of about 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications.

In some embodiments, a gRNA modified with BNA (e.g., LNA) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein the three terminal nucleotides at the 5' region of the crRNA comprises chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications, wherein the first stem of the crRNA repeat and the first stem of the anti-repeat are engineered to a total length of about 11 nucleotide pairs, wherein all the nucleotides in the first stem of the engineered anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications, and wherein the three terminal nucleotides at the tail of the engineered tracRNA comprise chemical modifications selected from MS, BNA (e.g., LNA and/or cEt), and BNA+PS (e.g., LNA+PS and/or cEt+PS) modifications. In some embodiments, a gRNA modified with BNA (e.g., LNA and/or cEt) modifications useful in a prime editing system includes a dgRNA comprising a crRNA and a tracrRNA, wherein all the nucleotides in the first stem of the anti-repeat of the crRNA comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, the engineered first stem of the crRNA repeat comprises an addition or substitution, at the 3' region, of at least one nucleotide. In some embodiments, the engineered first stem of the crRNA repeat comprises an addition or substitution, at the 3' region, of at least one nucleotide from a native pre-crRNA. In some embodiments, the engineered first stem of the crRNA repeat comprises an addition or substitution, at the 3' region, of at least one nucleotide from a repeat of a native pre-crRNA. In some embodiments, the engineered first stem of the crRNA repeat comprises an addition or substitution, at the 3' region, of a GC-rich sequence. In some embodiments, the engineered first stem of the crRNA repeat comprises an addition or substitution, at the 3' region, of a GC-rich sequence, wherein the content of G or C in the 3' region is at least 60%, at least 80%, or 100%. In some embodiments, the engineered first stem of the crRNA repeat comprises an addition or substitution, at the 3' region, of a GC-rich sequence, wherein the 3’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

In some embodiments, the engineered first stem of the anti-repeat comprises an addition or substitution, at the 5' region, of at least one nucleotide. In some embodiments, the engineered first stem of the anti-repeat comprises an addition or substitution, at the 5' region, of at least one nucleotide from a native pre-crRNA. In some embodiments, the engineered first stem of the anti-repeat comprises an addition or substitution, at the 5' region, of at least one nucleotide from a repeat of a native pre-crRNA. In some embodiments, the engineered first stem of the anti-repeat comprises an addition or substitution, at the 5' region, of a GC-rich sequence. In some embodiments, the engineered first stem of the anti -repeat comprises an addition or substitution, at the 5' region, of a GC-rich sequence, wherein the content of G or C in the 5' region is at least 60%, at least 80%, or 100%. In some embodiments, the engineered first stem of the anti-repeat comprises an addition or substitution, at the 5' region, of a GC-rich sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

In some embodiments, both the engineered first stem of the crRNA repeat and the engineered first stem of the anti -repeat comprise an addition or substitution of at least one nucleotide. In some embodiments, both the engineered first stem of the crRNA repeat and the engineered first stem of the anti-repeat comprise an addition or substitution of at least one nucleotide from a native pre-crRNA. In some embodiments, both the engineered first stem of the crRNA repeat and the engineered first stem of the anti-repeat comprise an addition or substitution of at least one nucleotide from a repeat of a native pre-crRNA. In some embodiments, both the engineered first stem of the crRNA repeat and the engineered first stem of the anti-repeat comprise an addition or substitution of a GC-rich nucleotide sequence. In some embodiments, both the engineered first stem of the crRNA repeat and the engineered first stem of the anti-repeat comprise an addition or substitution of a GC-rich nucleotide sequence, wherein the content of G or C in the 3' region of the first stem of the crRNA repeat and in the 5' region of the first stem of the anti-repeat is at least 60%, at least 80%, or 100%. In some embodiments, both the engineered first stem of the crRNA repeat and the engineered first stem of the anti -repeat comprise an addition or substitution of a GC-rich nucleotide sequence, wherein the 3’ region of the engineered first stem of the crRNA repeat and the 5’ region of the engineered first stem of the anti-repeat comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

RGNs that are fused to a polypeptide or domain can be separated or joined by a linker. The term "linker," as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. In embodiments, a linker joins a gRNA binding domain of an RGN and a base-editing polypeptide, such as a deaminase. In embodiments, a linker joins a nuclease-dead RGN or RGN nickase and a deaminase. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

The presently disclosed compositions and methods can utilize RGNs comprising at least one nuclear localization signal (NLS) to enhance transport of the RGN to the nucleus of a cell. Nuclear localization signals are known in the art and generally comprise a stretch of basic amino acids (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-5105). In embodiments, the RGN comprises 2, 3, 4, 5, 6 or more nuclear localization signals. The nuclear localization signal(s) can be a heterologous NLS. Non-limiting examples of nuclear localization signals useful for the presently disclosed RGNs are the nuclear localization signals of SV40 Large T-antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al. (2015) Bioconjug Chem 26(6): 1004-7). In embodiments, the RGN comprises the NLS sequence set forth as SEQ ID NO: 115 or 116. The RGN can comprise one or more NLS sequences at its N-terminus, C- terminus, or both the N-terminus and C-terminus. For example, the RGN can comprise two NLS sequences at the N-terminal region and four NLS sequences at the C-terminal region.

In embodiments, the presently disclosed RGNs comprise at least one cell-penetrating domain that facilitates cellular uptake of the RGN. Cell-penetrating domains are known in the art and generally comprise stretches of positively charged amino acid residues (i.e., polycationic cellpenetrating domains), alternating polar amino acid residues and non-polar amino acid residues (i.e., amphipathic cell-penetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cellpenetrating domains) (see, e.g., Milletti F. (2012) Drug Discov Today 17:850-860). A non-limiting example of a cell-penetrating domain is the trans-activating transcriptional activator (TAT) from the human immunodeficiency virus 1.

The nuclear localization signal and/or cell-penetrating domain can be located at the N- terminus, the C-terminus, or in an internal location of the RGN.

V. Polynucleotides Encoding RNA-guided nucleases

The present disclosure provides polynucleotides encoding RGNs. In some embodiments, the RGNs bind the presently disclosed gRNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti-repeat of the tracrRNA.

The use of the term "polynucleotide" or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. These include peptide nucleic acids (PNAs), PNA-DNA chimers, locked nucleic acids (LNAs), and phosphothiorate linked sequences. The polynucleotides disclosed herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, DNA-RNA hybrids, triplex structures, stem-and- loop structures, and the like.

In some of those embodiments wherein the presently disclosed compositions and methods comprise a nucleic acid molecule encoding an RGN, the nucleic acid molecule is an mRNA (messenger RNA) molecule. An mRNA refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. In embodiments, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail. In embodiments, an mRNA encoding an RGN useful in the presently disclosed methods and compositions can include one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide. For instance, a useful property of an mRNA includes the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced. A “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in an mRNA without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. Chemical modifications to mRNA can involve inclusion of 5 -methylcytosine, N1 -methyl - pseudouridine, pseudouridine, 2-thiouridine, 4-thiouridine, 5 -methoxyuridine, 2 'Fluoroguanosine, 2 'Fluorouridine, 5 -bromouridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3(l-E-propenylamino)] uridine, a-thiocytidine, N6-methyladenosine, 5 -methylcytidine, N4-acetylcytidine, 5 -formylcytidine, or combinations thereof, in an mRNA.

The nucleic acid molecules encoding RGNs can be codon optimized for expression in an organism of interest (e.g., mammal). A "codon-optimized” coding sequence is a polynucleotide coding sequence having its frequency of codon usage designed to mimic the frequency of preferred codon usage or transcription conditions of a particular host cell. Expression in the particular host cell or organism is enhanced as a result of the alteration of one or more codons at the nucleic acid level such that the translated amino acid sequence is not changed. Nucleic acid molecules can be codon optimized, either wholly or in part. Codon tables and other references providing preference information for a wide range of organisms are available in the art (see, e.g., Gaspar et al. (2012) Bioinformatics 28(20): 2683-2684; Komar et al. (1998) Biol. Chem. 379(10): 1295-1300; Inouye et al. (2015) Protein Expr. Purif. 109: 47-54; and Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of plant-preferred codon usage). Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Non-limiting examples of codon-optimized coding sequences for RGNs useful in the presently disclosed compositions and methods are set forth as any one of SEQ ID NOs: 286-288, and 545.

Polynucleotides encoding the RGNs provided herein can be provided in expression cassettes for in vitro expression or expression in a cell, embryo, or organism of interest. The cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding an RGN provided herein that allows for expression of the polynucleotide. The cassette may additionally contain at least one additional gene or genetic element to be co-transformed into the organism. Where additional genes or elements are included, the components are operably linked. The term “operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a promoter and a coding region of interest (e.g. , region coding for an RGN) is a functional link that allows for expression of the coding region of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene.

The expression cassette will include in the 5'-3' direction of transcription, a transcriptional (and, in some embodiments, translational) initiation region (i.e., a promoter), an RGN-encoding polynucleotide of the disclosure, and a transcriptional (and in some embodiments, translational) termination region (i. e. , termination region) functional in the organism of interest. The promoters of the disclosure are capable of directing or driving expression of a coding sequence in a host cell. The regulatory regions (e.g., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

Convenient termination regions include ones from simian virus (SV40), human growth hormone (hGH), bovine growth hormone (BGH), and rabbit beta-globin (rbGlob). See also Proudfoot (1991) Cell 64:671-674; Munroe et al. (1990) Gene 91: 151-158; Schek et al. (1992) Molecular and Cellular Biology 12(12):5386-5393; Gil and Proudfoot (1987) Cell 49(3):399-406; Goodwin and Rottman (1992) The Journal of Biological Chemistry 267(23): 16330-16334; and Lanoix and Acheson (1988) EMBO J. 7(8): 2515-2522. Additional termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter "Sambrook 11"; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein. In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

A number of promoters can be used in the practice of the disclosure. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, inducible, growth stage-specific, cell type-specific, tissue-preferred, tissue-specific, or other promoters for expression in the organism of interest. See, for example, promoters set forth in WO 99/43838 and in US Patent Nos: 8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; herein incorporated by reference.

Exemplary constitutive promoters for expression in cells of the present disclosure include: an SV40 early promoter; a mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter; a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE); a rous sarcoma virus (RSV) promoter; a human ubiquitin C promoter (UBC); a human U6 small nuclear promoter (U6); an enhanced U6 promoter; a human Hl promoter from RNA polymerase III (Hl); a human elongation factor la promoter (EF1A); a human beta-actin promoter (ACTB); a human or mouse phosphoglycerate kinase 1 promoter (PGK); a chicken -Actin promoter coupled with CMV early enhancer (CAGG); a yeast transcription elongation factor promoter (TEF1); and the like. See, for example, Miyagishi et al. (2002) Nature Biotechnology 20:497-500; Xia et al. (2003) Nucleic Acids Res. 31(17):el00-el00; Pasleau et al. (1985) Gene 38:227-232; Martin-Gallardo et al. (1988) Gene 70: 51-56; Oellig and Seliger (1990) JNeurosci Res 26: 390-396; Manthorpe et al. (1993) Hum Gene Ther 4: 419-431; Yew et al. (1991) Hum Gene Ther 8: 575-584; Xu et al. (2001) Gene 272: 149-156; Nguyen et al. (2008) J Surg Res 148: 60-66; Costa et al. (2005) Nat Meth. 2:259-260; Lam and Truong (2020) ACS Synth. Biol. 9(10):2625-2631.

For expression in plants, constitutive promoters also include CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) The or. Appl. Genet. 81:581-588); and MAS (Velten et al. (198A) EMBO J. 3:2723-2730).

Examples of inducible promoters include: stress-regulated promoters such as Adhl promoter, Hsp70 promoter, and Hsp90 promoter (Wurm et al. (1986) Proc. Natl. Acad. Sci. USA. 83:5414- 5418; Nover L. Heat Shock Response. CRC Press; Boca Raton, FL, USA: 1991); light-inducible promoters such as PPDK promoter and pepcarboxylase promoter; metal-regulated promoters (Mayo et al. (1982) Cell. 29:99-108; Searle et al. (1985) Mol. Cell. Biol. 5: 1480-1489); hormone-responsive promoters including a glucocorticoid-responsive promoter (Hynes et al. (1981) Proc. Natl. Acad. Sci. USA. 78:2038-2042; Klock et al. (1987) Nature. 329:734-736). Chemically regulated promoters include: In2-2 promoter which is safener induced (U.S. Pat. No. 5,364,780); the Axigl promoter which is auxin induced and tapetum specific but also active in callus (PCT US01/22169); the steroidresponsive promoters (see, for example, the ERE promoter which is estrogen induced, and the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257); tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237; Gossen et al. (1993) Trends Biochem Sci. 18:471-475; Gossen and Bujard (1992) Proc. Natl Acad. Sci. USA 89:5547- 5551; Zhou et al. (2006) Gene Ther. 13: 1382-1390; and U.S. Pat. Nos. 5,814,618 and 5,789,156); isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoters; and lactose-regulated promoters. Inducible expression can be obtained using operator systems including AlcR/acetaldehyde, ArgR/L-arginine, BirA/biotinyl-AMP, CymR/cumate, EthR/2-phenylethylbutyrate, HdnoR/6- hydroxynicotine, HucR/uric acid, MphR(A)/macrolides, PIP/Streptogramins, Rex/NADH, RheA/heat, ScbR/SCBl, TraR/3-oxo-C8-HSL, and TtgR/phloretin; see, for example, U.S. Patent No. 8,728,759B2; U.S. Patent No. 7,745,592B2; Weber and Fussenegger (2004) Methods Mol. Biol. 267:451-466; Hartenbach et al. (2007) Nucleic Acids Res. 35:el36; Weber et al. (2009) Metab. Eng. 11: 117-124; Weber et a/. (2008) Proc. Natl. Acad. Sci. USA. 105:9994-9998; Malphettes et al. (2005) Nucleic Acids Res. 33 :e 107; Kemmer et al. (20\0) Nat. Biotechnol. 28:355-360; Weber et a/. (2002) Nat. Biotechnol. 20:901-907; Fussenegger et a/. (2000) Nat. Biotechnol. 18: 1203-1208;

Weber et a/. (2006) Metab. Eng. 8:273-280; Weber et a/. (2003) Nucleic Acids Res. 31:e69; Weber et al. (2003) Nucleic Acids Res. 31 :c71 ; Neddermann et al. (2003) EMBO Rep. 4: 159-165; and Gitzinger et a/. (2009) Proc. Natl. Acad. Sci. USA. 106: 10638-10643. Inducible expression can be obtained using protein-protein interaction systems including: rapamycin-induced interaction between FKBP12 (FK506 binding protein 12) and mTOR (Rivera et al. (1996) Nat. Med. 2: 1028-1032; Belshaw et al. (1996) Proc. Natl. Acad. Sci. USA. 93:4604-46077); abscisic acid (ABA)-regulated interaction between PYL1 (abscisic acid receptor) and ABI1 (protein phosphatase 2C56) (Liang et a/. (2011) Sci. Signal. 4(164):rs2-rs2); and light-induced protein-protein interaction systems (Wang et a/. (20Y2) Nat. Methods. 9:266-269; Yamada et a/. (2018) Cell. Rep. 25:487-500).

Tissue-specific or tissue-preferred promoters can be utilized to target expression of an expression construct within a particular tissue. In embodiments, the tissue-specific or tissue-preferred promoters are active in mammalian tissue. Examples of tissue-specific or tissue-preferred promoters include promoters that initiate transcription preferentially in certain tissues, such as white blood cells (e.g., CD4 T cell), heart, kidney, liver, CNS, eye, pancreas, skeletal muscle, testis. In certain embodiments, the tissue-specific or tissue-preferred promoters are active in plant tissue. Examples of promoters under developmental control in plants include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers. A "tissue specific" promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, promoters from homologous or closely related species can be preferable to use to achieve efficient and reliable expression of transgenes in particular tissues. In some embodiments, the expression comprises a tissue-preferred promoter. A "tissue preferred" promoter is a promoter that initiates transcription preferentially, but not necessarily entirely or solely in certain tissues.

In embodiments, the nucleic acid molecules encoding an RGN comprise a cell type-specific promoter. A "cell type specific" promoter is a promoter that primarily drives expression in certain cell types in one or more organs. Some examples of cells in which cell type specific promoters may be primarily active include, for example, a primary cell, a neuronal cell, a glial cell, an adipocyte, a cardiomyocyte, a smooth muscle cell, a photoreceptor cell, and a retinal ganglia cell. Some examples of plant cells in which cell type specific promoters functional in plants may be primarily active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells. The nucleic acid molecules can also include cell type preferred promoters. A "cell type preferred" promoter is a promoter that primarily drives expression mostly, but not necessarily entirely or solely in certain cell types in one or more organs. Some examples of cells in which cell type preferred promoters may be preferentially active include, for example, a primary cell, a neuron, an adipocyte, a cardiomyocyte, a smooth muscle cell, and a photoreceptor cell. Some examples of plant cells in which cell type preferred promoters functional in plants may be preferentially active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells.

The nucleic acid sequences encoding the RGNs can be operably linked to a promoter sequence that is recognized by a phage RNA polymerase for example, for in vitro mRNA synthesis. In embodiments, the in w/ro-transcribcd RNA can be purified for use in the methods described herein. For example, the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variation of a T7, T3, or SP6 promoter sequence. In embodiments, the expressed protein and/or RNAs can be purified for use in the methods of genome modification described herein.

In embodiments, the polynucleotide encoding the RGN also can be linked to a polyadenylation signal (e.g., SV40 polyA signal and other signals functional in plants) and/or at least one transcriptional termination sequence. Additionally, the sequence encoding the RGN also can be linked to sequence(s) encoding at least one nuclear localization signal, at least one cell-penetrating domain, and/or at least one signal peptide capable of trafficking proteins to particular subcellular locations, as described elsewhere herein.

The polynucleotide encoding the RGN can be present in a vector. A “vector” refers to a polynucleotide composition for transferring, delivering, or introducing a nucleic acid into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vector). The vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Additional information can be found in "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3rd edition, 2001.

The vector can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D). Marker genes can include genes that allow selection for growth on a particular nutrient or substance, such as dihydrofolate reductase (DHFR; Simonsen and Levinson (1983) Proc. Natl. Acad. Sci. U.S.A. 80:2495-2499), histidinol dehydrogenase (hisD; Hartman and Mulligan (1988) Proc. Natl. Acad. Set. U.S.A. 85:8047-8051), puromycin-N-acetyl transferase (PAC or puro; de la Luna etal. (1988) Gene 62 121- 126), thymidine kinase (TK; Littlefield (1964) Science 145:709-710), and xanthine-guanine phosphoribosyltransferase (XGPRT or gpt; Mulligan and Berg (1981) Proc. Natl. Acad. Sci. U.S.A. 78:2072- 2076).

As indicated, expression constructs comprising nucleotide sequences encoding an RGN can be used to transform organisms of interest. Methods for transformation involve introducing a nucleotide construct into an organism of interest. By "introducing" is intended to introduce the nucleotide construct to the host cell in such a manner that the construct gains access to the interior of the host cell. The methods of the disclosure do not require a particular method for introducing a nucleotide construct to a host organism, only that the nucleotide construct gains access to the interior of at least one cell of the host organism. The host cell can be a eukaryotic or prokaryotic cell. In embodiments, the eukaryotic host cell is a plant cell, mammalian cell, an avian cell, or an insect cell. In embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a human cell. In embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a primary cell. The term "primary cell" refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro. In some embodiments, a primary cell is a primary T cell. In embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system) including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a chimeric antigen receptor T (CAR-T) cell, a monocyte, a macrophage, and a dendritic cell. In embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is an ocular cell, a muscle cell (e.g., skeletal muscle cell), an epithelial cell (e.g., lung epithelial cell), or a diseased cell (e.g., tumor cell).

Methods for introducing nucleotide constructs into plants and other host cells are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

The methods result in a transformed organism, such as a plant, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). In some embodiments, the presently disclosed methods can result in a transformed organism or cell line derived from these transformed cells.

"Transgenic organisms" or "transformed organisms" or "stably transformed" organisms or cells or tissues refers to organisms that have incorporated or integrated a polynucleotide encoding an RGN of the disclosure. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments may also be incorporated into the host cell. Agrobacterium-and biolistic -mediated transformation remain the two predominantly employed approaches for transformation of plant cells. However, transformation of a host cell may be performed by infection, conjugation, transfection, microinjection, electroporation, microprojection, biolistics or particle bombardment, electroporation, silica/carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE dextran procedure, and viral mediated, liposome mediated and the like. Viral-mediated introduction of a polynucleotide encoding an RGN includes retroviral, lentiviral, adenoviral, and adeno-associated viral mediated introduction and expression, as well as the use of Caulimoviruses (e.g., cauliflower mosaic virus), Geminiviruses (e.g., bean golden yellow mosaic virus or maize streak virus), and RNA plant viruses (e.g., tobacco mosaic virus).

Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of host cell (e.g., monocot or dicot plant cell) targeted for transformation. Methods for transformation are known in the art and include those set forth in US Patent Nos: 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference. See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4: 1-12; Bates, G.W. (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85: 13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107: 1041-1047; Jones et al. (2005) Plant Methods 1:5.

Transformation may result in stable or transient incorporation of the nucleic acid into the cell. "Stable transformation" is intended to mean that the nucleotide construct introduced into a host cell integrates into the genome of the host cell and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome of the host cell.

Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the polynucleotide encoding an RGN or RGN fusion identified. Two or more generations may be grown to ensure that the polynucleotide encoding an RGN or RGN fusion is stably maintained and inherited and then seeds harvested to ensure the presence of the polynucleotide encoding an RGN or RGN fusion. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

In some embodiments, cells that have been transformed are introduced into an organism. These cells could have originated from the organism, wherein the cells are transformed in an ex vivo approach. These cells can be autologous (originated and returned to the same subject), allogeneic (the donor and recipient subjects are of the same species).

The sequences provided herein may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, com (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. Further provided is a processed plant product or byproduct that retains the sequences disclosed herein, including for example, soymeal.

In some embodiments, the polynucleotides encoding an RGN or RGN fusion are used to transform any eukaryotic species, including but not limited to animals (e.g., mammals, humans, insects, fish, birds, and reptiles), plants, fungi, amoeba, algae, and yeast. In some embodiments, the polynucleotides encoding an RGN or RGN fusion are used to transform any prokaryotic species, including but not limited to, archaea and bacteria (e.g., Bacillus spp., Klebsiella spp. Streptomyces spp., Rhizobium spp., Escherichia spp., Pseudomonas spp., Salmonella spp., Shigella spp., Vibrio spp., Yersinia spp., Mycoplasma spp., Agrobacterium spp., and Lactobacillus spp.).

Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian, plant, insect, or avian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of an RGN system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Non-limiting examples include vectors utilizing Caulimoviruses (e.g., cauliflower mosaic virus), Geminiviruses (e.g., bean golden yellow mosaic virus or maize steak virus), and RNA plant viruses (e.g., tobacco mosaic virus). For a review of gene therapy procedures, see Anderson, Science 256: 808- 813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 ( 1): 31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1: 13-26 (1994). Methods of non-viral delivery of nucleic acids include lipofection, Agrobacterium-mediated transformation, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam ™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291- 297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleic acids takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66: 1635-1640 (1992); Sommnerfelt et al., Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., J. Viral. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno- associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Viral. 63:03822-3828 (1989). Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and \|/J2 cells or PA317 cells, which package retrovirus.

Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.

The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.

In some embodiments, a host cell is transiently or non-transiently transfected with one or more nucleic acid molecules or vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject.

In some embodiments, a cell that is transfected is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell (e.g., mammals, humans, insects, fish, birds, and reptiles). In some embodiments, a cell that is transfected is a human cell. In some embodiments, a cell that is transfected is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system) including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a chimeric antigen receptor T (CAR-T) cell, a monocyte, a macrophage, and a dendritic cell.

In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. In some embodiments, the cell or cell line is prokaryotic. In some embodiments, the cell or cell line is eukaryotic. In further embodiments, the cell or cell line is derived from insect, avian, plant, or fungal species. In some embodiments, the cell or cell line may be mammalian, such as for example human, monkey, mouse, cow, swine, goat, hamster, rat, cat, or dog. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF- CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CVI, RPTE, A1O, T24, 182, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI- 231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4. COS, COS-1, COS-6, C0S-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO- 7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfir-/-, COR-L23, COR-L23/CPR, COR-L235010, CORL23/ R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, IY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR/ 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI- H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/ PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)).

In some embodiments, a cell transfected with one or more nucleic acid molecules or vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In embodiments, a cell transiently transfected with the components of an RGN system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of an RGN system, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.

In some embodiments, one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is an insect. In further embodiments, the insect is an insect pest, such as a mosquito or tick. In some embodiments, the insect is a plant pest, such as a com rootworm or a fall armyworm. In some embodiments, the transgenic animal is a bird, such as a chicken, turkey, goose, or duck. In some embodiments, the transgenic animal is a mammal, such as a human, mouse, rat, hamster, monkey, ape, rabbit, swine, cow, horse, goat, sheep, cat, or dog.

VI. Variants and Fragments of Polypeptides and Polynucleotides

The present disclosure provides active variants and fragments of the presently disclosed crRNAs, tracrRNAs, guide RNAs, and RGNs. In some embodiments, variants of crRNAs, tracrRNAs, and guide RNAs disclosed herein include crRNAs, tracrRNAs, and guide RNAs that include a BNA modification (e.g., an LNA and/or cEt modification). An active variant or fragment of a naturally-occurring (i.e., wild-type) RGN binds to a target sequence described herein in an RNA- guided sequence -specific manner. In embodiments, a target sequence described herein includes the nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712. In some embodiments, an active variant or fragment of an RGN disclosed herein requires atracrRNA for activity. In some embodiments, an active variant or fragment of an RGN disclosed herein includes a type II RGN. In some embodiments, the disclosure provides: active variants and fragments of an RGN having an amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252; active variants and fragments of crRNA repeats, including a sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516; active variants and fragments of crRNAs, including a sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337-339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399- 401, 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, 517-519, and 708; and active variants and fragments of tracrRNAs, including a sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713. In some embodiments, the disclosure provides active variants and fragments of a guide RNA backbone, including a sequence set forth as any one of SEQ ID NOs: 35-37, 296, and 297. In some embodiments, the disclosure provides active variants and fragments of a sgRNA, including a sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

While the activity of a variant or fragment may be altered compared to the polynucleotide or polypeptide of interest, the variant and fragment should retain the functionality of the polynucleotide or polypeptide of interest. For example, a variant or fragment may have increased activity, decreased activity, different spectrum of activity or any other alteration in activity when compared to the polynucleotide or polypeptide of interest. Fragments and variants of naturally-occurring RGN polypeptides, such as those disclosed herein, will retain sequence-specific, RNA-guided DNA-binding activity. In embodiments, fragments and variants of naturally-occurring RGN polypeptides, such as those disclosed herein, retain nuclease activity (single-stranded or double -stranded).

Fragments and variants of crRNA repeats, such as those disclosed herein, will retain the ability, when part of a guide RNA (comprising a tracrRNA), to bind to and guide an RNA-guided nuclease (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner.

Fragments and variants of tracrRNAs, such as those disclosed herein, will retain the ability, when part of a guide RNA (comprising a crRNA), to guide an RNA-guided nuclease (complexed with the guide RNA) to a target sequence in a sequence-specific manner.

Fragments and variants of guide RNAs, such as those disclosed herein, will retain the ability to guide an RNA-guided nuclease (complexed with the guide RNA) to a target sequence in a sequence -specific manner.

The term “fragment” refers to a portion of a polynucleotide or polypeptide sequence of the disclosure. "Fragments" or "biologically active portions" include polynucleotides comprising a sufficient number of contiguous nucleotides to retain the biological activity (i.e., binding to and directing an RGN in a sequence-specific manner to a target sequence when comprised within a guide RNA). "Fragments" or "biologically active portions" include polypeptides comprising a sufficient number of contiguous amino acid residues to retain the biological activity (i.e. , binding to a target sequence in a sequence -specific manner when complexed with a guide RNA). Fragments of the RGN proteins include those that are shorter than the full-length sequences due to the use of an alternate downstream start site. A biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700 or more contiguous amino acid residues of an RGN that binds a target nucleotide sequence disclosed herein or any one of SEQ ID NOs: 1, 69, 93, and 252. Such biologically active portions can be prepared by recombinant techniques and evaluated for sequence-specific, RNA- guided DNA-binding activity. A biologically active fragment of a crRNA repeat can comprise at least 8 contiguous nucleotides of any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516. A biologically active portion of a crRNA repeat can be a polynucleotide that comprises, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516. A biologically active fragment of a tracrRNA can comprise at least 8 contiguous nucleotides of any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713. A biologically active portion of a tracrRNA can be a polynucleotide that comprises, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more contiguous nucleotides of any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713. A biologically active fragment of a crRNA can comprise at least 8 contiguous nucleotides of any one of SEQ ID NOs: 4-9, 42-44, 73- 75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337- 339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399-401, 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, 517-519, and 708. A biologically active portion of a crRNA can be a polynucleotide that comprises, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or more contiguous nucleotides of any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337-339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399-401, 466-468, 470- 472, 474-476, 478-480, 482-484, 509-511, 513-515, 517-519, and 708. A biologically active fragment of a sgRNA can comprise at least 90 contiguous nucleotides of any of SEQ ID NOs: 25-30, 60-68, 86- 88, 108-110, 298, 299, and 405-407. A biologically active portion of a gRNA can be a polynucleotide that comprises, for example, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or more contiguous nucleotides of any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

In general, "variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" or “wild type” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest. Naturally occurring allelic variants such as these can be identified with the use of well- known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode the polypeptide or the polynucleotide of interest. Generally, variants of a particular polynucleotide disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide disclosed herein (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides disclosed herein is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In some embodiments, the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide that requires a tracrRNA for activity. In some embodiments, the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide that is a type II RGN. In particular embodiments, the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to an amino acid sequence encoding an RGN that binds a target sequence disclosed herein or an amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252.

A biologically active variant of an RGN polypeptide of the disclosure may differ by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. In embodiments, the polypeptides can comprise an N-terminal or a C-terminal truncation, which can comprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700 amino acids or more from either the N or C terminus of the polypeptide.

In embodiments, the presently disclosed polynucleotides comprise a crRNA repeat comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516.

In embodiments, the presently disclosed polynucleotides comprise a crRNA comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97- 99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337-339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399-401, 466-468, 470-472, 474-476, 478- 480, 482-484, 509-511, 513-515, 517-519, and 708. The presently disclosed polynucleotides can comprise a tracrRNA comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to any one of the nucleotide sequences set forth as any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713.

The presently disclosed polynucleotides can comprise a sgRNA comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to any one of the nucleotide sequences set forth as any one of SEQ ID NOs: 25-30, 60-68, 86- 88, 108-110, 298, 299, and 405-407.

In some embodiments, variants of crRNAs, tracrRNAs, and guide RNAs disclosed herein include crRNAs, tracrRNAs, and guide RNAs that include modified nucleotides, sugars, phosphate backbone, and/or nucleobases as described herein. Variant crRNAs, tracrRNAs, and guide RNAs can include modifications including 2'-0-Me modification; 2'-F modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; MS modification; MSP modification; MP modification; PS modification; BNA modification (e.g., 2', 4' BNA, LNA, BNA^NC[N-Me], 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA), and S-constrained ethyl (cEt)); or a combination thereof.

Biologically active variants of a crRNA repeat, crRNA, tracrRNA, or guide RNA of the disclosure may differ by as few as about 1-15 nucleotides, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleotide. In embodiments, the polynucleotides can comprise a 5' or 3' truncation, which can comprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110 nucleotides or more from either the 5' or 3' end of the polynucleotide.

It is recognized that modifications may be made to the RGN polypeptides, crRNA repeats, crRNAs, tracrRNAs, and guide RNAs provided herein creating variant proteins and polynucleotides. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques. Alternatively, native, as yet-unknown, or as yet unidentified polynucleotides and/or polypeptides structurally and/or functionally-related to the sequences disclosed herein may also be identified that fall within the scope of the present disclosure. Conservative amino acid substitutions may be made in non-conserved regions that do not alter the function of the RGN proteins. Alternatively, modifications may be made that improve the activity of the RGN.

Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different RGN proteins disclosed herein (e.g., SEQ ID NO: 1) is manipulated to create a new RGN protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the RGN sequences provided herein and other known RGN genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K_m in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458. A "shuffled" nucleic acid is a nucleic acid produced by a shuffling procedure such as any shuffling procedure set forth herein. Shuffled nucleic acids are produced by recombining (physically or virtually) two or more nucleic acids (or character strings), for example in an artificial, and optionally recursive, fashion. Generally, one or more screening steps are used in shuffling processes to identify nucleic acids of interest; this screening step can be performed before or after any recombination step. In some (but not all) shuffling embodiments, it is desirable to perform multiple rounds of recombination prior to selection to increase the diversity of the pool to be screened. The overall process of recombination and selection are optionally repeated recursively. Depending on context, shuffling can refer to an overall process of recombination and selection, or, alternately, can simply refer to the recombinational portions of the overall process.

As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i. e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

Two sequences are "optimally aligned" when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978) "A model of evolutionary change in proteins." In "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. M. O. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10915- 10919. The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website (www.ncbi.nlm.nih.gov). Optimal alignments, including multiple alignments, can be prepared using, e.g., PSI-BLAST, available through www.ncbi.nlm.nih.gov and described by Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds to" the position in the reference sequence with which the residue is paired in the alignment. The "position" is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Owing to deletions, insertion, truncations, fusions, etc., that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence as determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

VII. RGN Systems and Ribonucleoprotein Complexes for Binding a Target Sequence of Interest and Methods of Making the Same

The present disclosure provides an RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) a chemically modified tracrRNA described herein; b) a crRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.. In some embodiments, the chemically modified tracrRNA and the crRNA form a guide RNA.

The present disclosure also provides an RGN system, wherein the RGN system comprises: a) a chemically modified crRNA described herein; b) a tracrRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide. In some comediments, the chemically modified crRNA and the tracrRNA form a guide RNA.

The present disclosure also provides an RGN system, wherein the RGN system comprises: a) a chemically modified guide RNA described herein; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

In some embodiments, the RGN system of the disclosure binds a target sequence in a target nucleic acid molecule. In some embodiments, the gRNA of the RGN system of the disclosure is capable of forming a complex with the Type II RGN polypeptide of the RGN system to direct the Type II RGN polypeptide to bind to the target sequence.

In some embodiments, the RNA-guided nuclease systems comprise the presently disclosed gRNAs comprising at least one bridged nucleic acid (BNA) (e.g., LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti -repeat of the tracrRNA. As used herein, an RGN system comprises at least one RGN polypeptide or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide and one or more guide RNAs capable of forming a complex with the RGN polypeptide (ribonucleoprotein complex). In some embodiments, the one or more guide RNAs includes at least one BNA modification (e.g., an LNA and/or cEt modification). The presently disclosed RGN system comprises: a) one or more guide RNAs capable of targeting a bound RGN polypeptide to a target sequence; and b) an RGN polypeptide or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide, wherein the one or more guide RNAs are capable of forming a complex with the RGN polypeptide to direct said RGN polypeptide to bind to a target sequence. The guide RNA hybridizes to the target strand of the target sequence and also forms a complex with the RGN polypeptide, thereby directing the RGN polypeptide to bind to the target sequence. In some embodiments, the target sequence comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712. In some embodiments, the RGN is capable of recognizing a PAM and binding a target sequence adjacent to the PAM. In some embodiments, the RGN comprises an amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252, or active variants or fragments thereof. In some embodiments, an RGN disclosed herein requires atracrRNA for activity. In some embodiments, an RGN disclosed herein includes a type II RGN. In embodiments, the RGN comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 69, 93, and 252. In some embodiments, the guide RNA comprises a crRNA repeat comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516, or active variants or fragments thereof. In some embodiments, the guide RNA comprises a crRNA comprising the nucleotide sequences set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337-339, 345-347, 349-351, 353-355, 357-359, 361-363, 380- 382, 399-401, 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, 517-519, and 708, or active variants or fragments thereof. In embodiments, the guide RNA comprises a tracrRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713, or active variants or fragments thereof. The guide RNA of the system can be a single guide RNA or a dual-guide RNA. In some embodiments, the guide RNA comprises a sgRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407, or active variants or fragments thereof. In embodiments, the system comprises an RNA- guided nuclease that is heterologous to the guide RNA, wherein the RGN and guide RNA are not found complexed to one another (i.e., bound to one another) in nature. For example, the APG07991 RGN (SEQ ID NO: 252) functions with engineered and chemically modified SpyCas9 crRNAs and tracrRNAs disclosed herein (e.g., a SpyCas9 crRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 509-511, 513-515, and 517-519; and/or a SpyCas9 tracrRNA having a nucleotide sequence set forth as any one of SEQ ID NOs: 534, 535, and 537; see FIG. 29). In certain embodiments, one or more RNA sequences (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, a tracrRNA anti-repeat, a sgRNA, or a dgRNA, etc.) in the systems described include one or more various chemical modifications disclosed herein (e.g., a BNA modification).

In certain embodiments, the chemical modifications are within two stems of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each of the two stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within two stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).

In some embodiments, the chemical modifications are within three stems of multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each of the three stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within three stems of the multiple stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA).

In some embodiments, the chemical modifications are present on one or more nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, a stem loop comprising chemical modifications in its one or more stems in a guide RNA is formed by hybridization of a crRNA and a tracrRNA. In some embodiments, a stem loop comprising chemical modifications in its one or more stems in a guide RNA is formed by hybridization of crRNA repeat of a crRNA and anti-repeat of a tracrRNA.

In some embodiments, the chemical modifications are within the tail in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are within one or more stems and within the tail in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first and second stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first stem and no chemical modifications are within the second stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA, and chemical modifications are present within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first, second, and third stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail of a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).

In some embodiments, the chemical modifications are not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In certain embodiments, the chemical modifications are within one or more stems but not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within one or more stems and within the tail but not present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within a first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within a first and second stem of a stem loop formed by the crRNA repeat of a crRNA and the antirepeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within a first, second, and third stem of a stem loop formed by the crRNA repeat of a crRNA and the anti- repeat of a tracrRNA and the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first stem and not within any other stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail but not present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).

In some embodiments, the chemical modifications are present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA). In certain embodiments, the chemical modifications are within one or more stems and present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within one or more stems and within the tail as well as present in a loop, bulge, or bubble in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first stem of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first and second stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within the first, second, and third stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA). In some embodiments, the chemical modifications are within all the stems of a stem loop formed by the crRNA repeat of a crRNA and the anti-repeat of a tracrRNA and within the tail as well as present in a loop, bulge, or bubble in a gRNA of the disclosure (e.g., a sgRNA or a dgRNA).

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within a crRNA repeat. In some embodiments, at least one BNA modification (e.g., LNA and/or cEt) is within the first stem of a crRNA repeat. In some embodiments, the terminal 5' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the second stem of a crRNA repeat. In some embodiments, the terminal 5' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, no chemical modifications are within the second stem of a crRNA repeat.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the third stem of a crRNA repeat. In some embodiments, the terminal 5' nucleotide of the third stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the third stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the third stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the anti-repeat of a tracrRNA. In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first stem of the anti-repeat of a tracrRNA. In some embodiments, the terminal 5' nucleotide of the first stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the first stem of an anti -repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the first stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the first stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the second stem of the anti -repeat of a tracrRNA. In some embodiments, the terminal 5' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the second stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, no chemical modifications are within the second stem of an anti-repeat. In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the first and second stems of an anti-repeat of atracrRNA. In some embodiments, the terminal 5' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 3' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 5' nucleotides and the terminal 3' nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first and second stems of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the third stem of the anti -repeat of a tracrRNA. In some embodiments, the terminal 5' nucleotide of the third stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the third stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the third stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the third stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the third stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, all stems of the anti-repeat of a tracrRNA comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, the terminal 5' nucleotide of each stem of an antirepeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of each stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of each stem of an antirepeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of each stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of each stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, only the first stem of the anti-repeat of a tracrRNA comprises BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, an anti-repeat of a tracrRNA does not comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, a chemical modification at the fourth nucleotide position from the 5' end, a chemical modification at the fifth nucleotide position from the 5' end, or a combination thereof. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end and a modification at the second nucleotide position from the 5' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, and a chemical modification at the third nucleotide position from the 5' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, and a chemical modification at the fourth nucleotide from the 5' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 5' region includes a chemical modification at the first nucleotide position from the 5' end, a chemical modification at the second nucleotide position from the 5' end, a chemical modification at the third nucleotide position from the 5' end, a chemical modification at the fourth nucleotide from the 5' end, and a chemical modification at the fifth nucleotide from the 5' end.

In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, a chemical modification at the fourth nucleotide position from the 3' end, a chemical modification at the fifth nucleotide position from the 3' end, or a combination thereof. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end and a chemical modification at the second nucleotide position from the 3' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3’ end, a chemical modification at the second nucleotide position from the 3' end, and a chemical modification at the third nucleotide position from the 3' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, and a chemical modification at the fourth nucleotide from the 3' end. In some embodiments, an RNA molecule of the disclosure comprising at least one chemical modification at its 3' region includes a chemical modification at the first nucleotide position from the 3' end, a chemical modification at the second nucleotide position from the 3' end, a chemical modification at the third nucleotide position from the 3' end, a chemical modification at the fourth nucleotide from the 3' end, and a chemical modification at the fifth nucleotide from the 3' end.

In some embodiments, the three terminal nucleotides at both the 5' region and the 3' region of a tracrRNA of the disclosure comprise MS modifications and the remaining nucleotides in the first stem of the anti -repeat of the tracrRNA comprise 2'-0-Me modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise MS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5 ’ region and the 3 ’ region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprises BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprises LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, and wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise LNA+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications.

In embodiments of an RGN system, the first stem of an engineered crRNA repeat can comprise at the 3' region a substituted or added nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA). In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a substituted or added nucleotide sequence that is GC-rich. In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a GC-rich nucleotide sequence, wherein the content of G or C in the 3' region is at least 60%, at least 80%, or 100%. In some embodiments, the first stem of an engineered crRNA repeat comprises at the 3' region a GC-rich nucleotide sequence, wherein the 3’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs. In some embodiments, the first stem of an engineered crRNA repeat further comprises at least one chemical modification. In some embodiments, the first stem of an engineered crRNA repeat further comprises MS, BNA, or BNA+PS modifications at the 3' region.

In embodiments of an RGN system, the first stem of an engineered anti-repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA). In some embodiments, the first stem of an engineered anti -repeat comprises at the 5' region a GC-rich nucleotide sequence. In some embodiments, the first stem of an engineered anti-repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the content of G or C in the 5' region is at least 60%, at least 80%, or 100%. In some embodiments, the first stem of an engineered anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs. In some embodiments, the first stem of an engineered antirepeat further comprises at least one chemical modification. In some embodiments, the first stem of an engineered anti-repeat further comprises BNA (e.g., LNA and/or cEt) modifications on all nucleotides.

In some embodiments, an engineered crRNA repeat of an RGN system comprises an addition or substitution of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat of an RGN system comprises addition or substitution of 2 to 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat of an RGN system comprise an addition or substitution of 2 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat of an RGN system comprises an addition or substitution of 4 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat of an RGN system comprises an addition or substitution of 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, a first stem of an engineered crRNA repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. In some embodiments, nucleotides in the 3' region of the first stem of an engineered crRNA repeat further comprise MS modifications, BNA modifications, or BNA+PS modifications.

In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 5' region of the anti-repeat, as compared to the same anti-repeat prior to the engineering. In some embodiments, an engineered antirepeat of an RGN system comprises an addition or substitution of 2 to 6 nucleotides at the 5' region of the anti -repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 2 nucleotides at the 5' region of the anti -repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 4 nucleotides at the 5' region of the anti -repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 6 nucleotides at the 5' region of the anti -repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, the first stem of an engineered anti -repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of an engineered anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. In some embodiments, all nucleotides of the first stem of the engineered anti-repeat further comprise BNA (e.g., LNA and/or cEt) modifications.

The system for binding a target sequence of interest provided herein can be a ribonucleoprotein complex, which is at least one molecule of an RNA bound to at least one protein. The ribonucleoprotein complexes provided herein comprise at least one guide RNA as the RNA component and an RNA-guided nuclease as the protein component. Such ribonucleoprotein complexes can be purified from a cell or organism that naturally expresses an RGN polypeptide and binds a particular guide RNA that is specific for a target sequence of interest. In some embodiments, the ribonucleoprotein complex is purified from a cell or organism that has been transformed with a polynucleotide (e.g., an mRNA) that encodes an RGN polypeptide and wherein a synthetically derived gRNA has been introduced. Thus, methods are provided for making an RGN polypeptide or an RGN ribonucleoprotein complex. Such methods comprise culturing a cell comprising a nucleotide sequence encoding an RGN polypeptide, under conditions in which the RGN polypeptide is expressed. The RGN polypeptide or RGN ribonucleoprotein can then be purified from a lysate of the cultured cells. In some embodiments, the nucleotide sequence encoding an RGN polypeptide includes a mRNA (messenger RNA).

Methods for purifying an RGN polypeptide or RGN ribonucleoprotein complex from a lysate of a biological sample are known in the art (e.g., size exclusion and/or affinity chromatography, 2D- PAGE, HPLC, reversed-phase chromatography, immunoprecipitation). In particular methods, the RGN polypeptide is recombinantly produced and comprises a purification tag to aid in its purification, including but not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG (e.g., 3X FLAG tag), HA, nus, Softag 1, Softag 3, Strep, SBP, Glu- Glu, HSV, KT3, S, SI, T7, V5, VSV-G, 6xHis, lOxHis, biotin carboxyl carrier protein (BCCP), and calmodulin. Generally, the tagged RGN polypeptide or RGN ribonucleoprotein complex is purified using immobilized metal affinity chromatography. It will be appreciated that other similar methods known in the art may be used, including other forms of chromatography or for example immunoprecipitation, either alone or in combination.

An "isolated" or "purified" polypeptide, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polypeptide as found in its naturally occurring environment. Thus, an isolated or purified polypeptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non- protein-of-interest chemicals. Similarly, an “isolated” polynucleotide or nucleic acid molecule is removed from its naturally occurring environment. An isolated polynucleotide is substantially free of chemical precursors or other chemicals when chemically synthesized or has been removed from a genomic locus via the breaking of phosphodiester bonds. An isolated polynucleotide can be part of a vector, a composition of matter or can be contained within a cell so long as the cell is not the original environment of the polynucleotide.

Particular methods provided herein for binding and/or cleaving a target sequence of interest involve the use of an in vitro assembled RGN ribonucleoprotein complex. In vitro assembly of an RGN ribonucleoprotein complex can be performed using any method known in the art in which an RGN polypeptide is contacted with a guide RNA under conditions to allow for binding of the RGN polypeptide to the guide RNA. As used herein, "contact", contacting", "contacted," refer to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction. The RGN polypeptide can be purified from a biological sample, cell lysate, or culture medium, produced via in vitro translation, or chemically synthesized. The guide RNA can be purified from a biological sample, cell lysate, or culture medium, transcribed in vitro, or chemically synthesized. The RGN polypeptide and guide RNA can be brought into contact in solution (e.g., buffered saline solution) to allow for in vitro assembly of the RGN ribonucleoprotein complex.

Some aspects of this disclosure provide kits comprising one or more elements of an RGN system described herein, including: chemically modified guide RNAs (e.g., chemically modified sgRNAs or dgRNAs, including chemically modified crRNAs and tracrRNAs); RGNs or polynucleotides encoding the same; cells; and complete RGN systems. In embodiments, the kit includes suitable reagents, buffers, and/or instructions for using one or more elements of an RGN system, e.g., for in vitro or in vivo nucleic acid editing. Reagents may be provided in any suitable container, such as a vial, a bottle, or a tube. Reagents may be used in a process utilizing one or more of the elements of an RGN system. For example, restriction enzymes may be included for cloning of a polynucleotide encoding an RGN into a vector. In embodiments, the kit includes instructions regarding the design and use of suitable chemically modified guide RNAs (e.g., chemically modified sgRNAs or dgRNAs, including chemically modified crRNAs and tracrRNAs) for targeted editing of a target sequence. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10.

A kit including one or more elements of an RGN system of the disclosure has utility in a wide variety of applications including modifying (e.g., deleting, inserting, translocating, inactivating, activating, base editing, prime editing) a target sequence in a multiplicity of cell types. As such, kits including one or more elements of an RGN system of the disclosure may be useful in, for example, gene therapy, drug screening, disease diagnosis, and prognosis.

In certain embodiments, a kit of the disclosure includes a pharmaceutical kit including a pharmaceutical composition described herein. In embodiments, a pharmaceutical kit may include: (a) a container containing a composition of the disclosure in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized compound of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

VIII. Methods of Binding, Cleaving, or Modifying a Target Nucleic Acid Molecule

The present disclosure provides methods for binding, cleaving, and/or modifying a target nucleic acid molecule of interest comprising a target sequence (e.g., T cell receptor alpha constant (TRAC)). The methods include delivering a system comprising at least one guide RNA, and at least one RGN polypeptide or a polynucleotide encoding the same to the target sequence or a cell, organelle, or embryo comprising the target sequence. In some embodiments, the guide RNA comprises one or more various chemical modifications disclosed herein. In particular embodiments, the guide RNA comprises at least one BNA (e.g., an LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the antirepeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti -repeat of the tracrRNA.

In some embodiments, the target sequence includes a nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712. In embodiments, the RGN is capable of recognizing a PAM and binding a target sequence adjacent to the PAM. In some embodiments, the RGN requires a tracrRNA for activity. In some embodiments, the RGN includes a type II RGN. In some embodiments, the RGN comprises an amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252, or active variants or fragments thereof. In some embodiments, the RGN comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 69, 93, and 252. In embodiments, the guide RNA comprises a crRNA repeat comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516, or active variants or fragments thereof. In some embodiments, the guide RNA comprises a crRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301-303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337-339, 345-347, 349-351, 353-355, 357-359, 361-363, 380- 382, 399-401, 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, 517-519, and 708, or active variants or fragments thereof. In some embodiments, the guide RNA comprises a tracrRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713, or active variants or fragments thereof. The guide RNA of the system can be a single guide RNA or a dual-guide RNA. In some embodiments, the guide RNA comprises a sgRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407, or active variants or fragments thereof. In certain embodiments, one or more RNA sequences (e.g., a crRNA, a crRNA repeat, a spacer, a tracrRNA, a tracrRNA anti-repeat, a sgRNA, or a dgRNA, etc.) used in the methods described include one or more various chemical modifications disclosed herein.

The RGN of the system may be a nuclease dead RGN, have nickase activity, or may be a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a base-editing polypeptide, for example, a cytosine deaminase or an adenine deaminase. In some embodiments, a deaminase useful for such presently disclosed compositions and methods includes a deaminase set forth as any one of the polypeptides of SEQ ID NOs: 542, 544, and 592-707. In some embodiments, a deaminase useful for such presently disclosed compositions and methods includes a deaminase having at least 85%, 90%, 95%, or 97% sequence identity to any one of the polypeptides of SEQ ID NOs: 542, 544, and 592-707. In some embodiments, the fusion polypeptide comprises a base editor set forth as any one of the polypeptides of SEQ ID NOs: 550-591. In some embodiments, the fusion polypeptide comprises a base editor having at least 85%, 90%, 95%, or 97% sequence identity to any one of the polypeptides of SEQ ID NOs: 550-591. Insome embodiments, the RGN fusion protein comprises a prime editing polypeptide, for example, a reverse transcriptase. In some embodiments, the RGN fusion protein comprises a polypeptide that recruits members of a functional nucleic acid repair complex, such as a member of the nucleotide excision repair (NER) or transcription coupled- nucleotide excision repair (TC-NER) pathway (Wei et al., 2015, PNAS USA 112(27):E3495-504 ; Troelstra et al., 1992, Cell 71:939-953; Mamef et a/., 2017, JMol Biol 429(9): 1277-1288), as described in U.S. Provisional Application No. 62/966,203, which was filed on January 27, 2020, and is incorporated by reference in its entirety. In embodiments, the RGN fusion protein comprises CSB (van den Boom et al., 2004, J Cell Biol 166(l):27-36; van Gool et al., 1997, EMBO J 16(19):5955-65; an example of which is set forth as SEQ ID NO: 285), which is a member of the TC-NER (nucleotide excision repair) pathway and functions in the recruitment of other members. In further embodiments, the RGN fusion protein comprises an active domain of CSB, such as the acidic domain of CSB which comprises amino acid residues 356-394 of SEQ ID NO: 285 (Teng et al., 2018, Nat Commun 9(1):4115).

In particular embodiments, the RGN and/or guide RNA is heterologous to the cell or embryo to which the RGN and/or guide RNA (or polynucleotide(s) encoding at least one of the RGN) are introduced.

In some embodiments wherein the method comprises delivering a polynucleotide encoding an RGN polypeptide, the cell or embryo can then be cultured under conditions in which the RGN polypeptide is expressed. In embodiments, the method comprises contacting a target nucleic acid molecule with an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex may comprise an RGN that is nuclease dead or has nickase activity. In embodiments, the RGN of the ribonucleoprotein complex is a fusion polypeptide comprising a base-editing polypeptide. In embodiments, the RGN of the ribonucleoprotein complex is a fusion polypeptide comprising a prime editing polypeptide. In embodiments, the method comprises introducing into a cell or embryo comprising a target nucleic acid molecule an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex can be one that has been purified from a biological sample, recombinantly produced and subsequently purified, or in w/ro-asscmblcd as described herein. In embodiments wherein the RGN ribonucleoprotein complex that is contacted with the target nucleic acid molecule, cell, organelle, or embryo, has been assembled in vitro, the method can further comprise the in vitro assembly of the complex prior to contact with the target nucleic acid molecule, cell, or embryo. A purified or in vitro assembled RGN ribonucleoprotein complex can be introduced into a cell, organelle, or embryo using any method known in the art (e.g., electroporation).

Upon delivery to or contact with the target nucleic acid moleculeor cell, organelle, or embryo comprising the target nucleic acid molecule, the guide RNA directs the RGN to bind to the target sequence within the target nucleic acid molecule in a sequence-specific manner. In those embodiments wherein the RGN has nuclease activity, the RGN polypeptide cleaves the target sequence upon binding. The target sequence can subsequently be modified via endogenous repair mechanisms, such as non-homologous end joining, or homology-directed repair with a provided donor polynucleotide.

Methods to measure binding of an RGN polypeptide to a target sequence are known in the art and include chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter assays, microplate capture and detection assays. Likewise, methods to measure cleavage or modification of a target nucleic acid molecule comprising a target sequence are known in the art and include in vitro or in vivo cleavage assays wherein cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without the attachment of an appropriate label (e.g., radioisotope, fluorescent substance) to the target sequence to facilitate detection of degradation products. Alternatively, the nicking triggered exponential amplification reaction (NTEXPAR) assay can be used (see, e.g., Zhang et al. (2016) Chem. Set. 7:4951-4957). In vivo cleavage can be evaluated using the Surveyor assay (Guschin et al. (2010) Methods Mol Biol 649:247-256).

In some embodiments, the methods involve the use of a single type of RGN complexed with more than one guide RNA. The more than one guide RNA can target different regions of a single gene or can target multiple genes.

In those embodiments wherein a donor polynucleotide is not provided, a double-stranded break introduced by an RGN polypeptide can be repaired by a non-homologous end-joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, repair of the double-stranded break can result in a mutation to the target sequence. In certain embodiments, a “mutation” in reference to a target nucleic acid molecule refers to a change in the nucleotide sequence of the nucleic acid molecule, which can be a deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. Mutation of the target nucleic acid molecule comprising a target sequence can result in the expression of an altered protein product or inactivation of a coding sequence.

In those embodiments wherein a donor polynucleotide is present, the donor sequence in the donor polynucleotide can be integrated into or exchanged with the target nucleotide sequence during the course of repair of the introduced double-stranded break, resulting in the introduction of the exogenous donor sequence. A donor polynucleotide thus comprises a donor sequence that is desired to be introduced into a target sequence of interest. In embodiments, the donor sequence alters the original target nucleotide sequence such that the newly integrated donor sequence will not be recognized and cleaved by the RGN. Integration of the donor sequence can be enhanced by the inclusion within the donor polynucleotide of flanking sequences, referred to herein as “homology arms” that have substantial sequence identity with the sequences flanking the target nucleotide sequence, allowing for a homology-directed repair process. In embodiments, homology arms have a length of at least 50 base pairs, at least 100 base pairs, and up to 2000 base pairs or more, and have at least 90%, at least 95%, or more, sequence homology to their corresponding sequence within the target nucleotide sequence.

In those embodiments wherein the RGN polypeptide introduces double-stranded staggered breaks, the donor polynucleotide can comprise a donor sequence flanked by compatible overhangs, allowing for direct ligation of the donor sequence to the cleaved target nucleotide sequence comprising overhangs by a non-homologous repair process during repair of the double-stranded break. In those embodiments wherein the method involves the use of an RGN that is a nickase (i.e. , is only able to cleave a single strand of a double -stranded polynucleotide), the method can comprise introducing two RGN nickases that target identical or overlapping target sequences and cleave different strands of the polynucleotide. For example, an RGN nickase that only cleaves the positive (+) strand of a double-stranded polynucleotide can be introduced along with a second RGN nickase that only cleaves the negative (-) strand of a double-stranded polynucleotide.

In some embodiments, a method is provided for binding a target nucleotide sequence and detecting the target sequence, wherein the method comprises introducing into a cell or embryo at least one guide RNA useful in the presently disclosed compositions and methods, and at least one RGN polypeptide or a polynucleotide encoding the same, expressing the RGN polypeptide (if coding sequences are introduced), wherein the RGN polypeptide is a nuclease-dead RGN and further comprises a detectable label, and the method further comprises detecting the detectable label. The detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to or incorporated within the RGN polypeptide that can be detected visually or by other means.

Also provided herein are methods for modulating the expression of a target gene of interest comprising a target sequence or a gene under the regulation of a target sequence. The methods comprise introducing into a cell, organelle, or embryo at least one guide RNA useful in the presently disclosed compositions and methods, and at least one RGN polypeptide or a polynucleotide encoding the same, expressing the RGN polypeptide (if coding sequences are introduced), wherein the RGN polypeptide is a nuclease-dead RGN. In embodiments, the nuclease-dead RGN is a fusion protein comprising an expression modulator domain (i.e., epigenetic modification domain, transcriptional activation domain or a transcriptional repressor domain) as described herein.

The present disclosure also provides methods for binding and/or modifying a target nucleic acid molecule of interest comprising a target sequence. The methods include delivering a system comprising at least one guide RNA, and at least one fusion polypeptide comprising an RGN of the disclosure and a base-editing polypeptide, for example a cytosine deaminase or an adenine deaminase, or a polynucleotide encoding the fusion polypeptide, to the target sequence or a cell, organelle, or embryo comprising the target sequence.

In some embodiments wherein a fusion polypeptide comprising an RGN and a base-editing polypeptide is utilized, the binding of the fusion protein to a target sequence results in the modification of nucleotide(s) adjacent to the target sequence. The nucleobase adjacent to the target sequence that is modified by the deaminase may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5' or 3' end of the target sequence.

One of ordinary skill in the art will appreciate that any of the presently disclosed methods can be used to target a single target sequence or multiple target sequences. Thus, methods comprise the use of a single RGN polypeptide in combination with multiple, distinct guide RNAs useful in the presently disclosed compositions and methods, which can target multiple, distinct sequences within a single gene and/or multiple genes. For example, a APG07433.1 RGN is used with an appropriate guide RNA comprising a crRNA selected from SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, and 399- 401 and atracrRNA selected from SEQ ID NOs: 10, 12, 51-53, 294, 295, and 383 to modify a TRAC locus. Also encompassed herein are methods wherein multiple, distinct guide RNAs are introduced in combination with multiple, distinct RGN polypeptides. These guide RNAs and guide RNA/RGN polypeptide systems can target multiple, distinct sequences within a single gene and/or multiple genes.

Also provided herein is a method of increasing efficiency of cleaving and/or modifying a nucleic acid molecule comprising a target sequence. In some embodiments, the method comprises delivering an RGN system, or an RNP complex described herein to a target sequence or to a cell comprising the target sequence. The RGN system or the RNP complex comprises a crRNA, a tracrRNA, and/or a gRNA comprising chemical modifications described herein such that cleavage or modification of the target nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the target nucleic acid molecule by a method comprising delivering to the same target nucleic acid molecule a reference RGN system or RNP complex. In some embodiments, the reference RGN system or RNP complex comprises a tracrRNA, a gRNA, or a crRNA that does not comprise any BNA (e.g., LNA and/or cEt) modifications described herein. In some embodiments, the increased efficiency includes increased efficiency in base editing or prime editing.

In some embodiments, the efficiency of cleaving and/or modifying a target sequence is increased by 2-fold to 100-fold, or 2-fold to 80-fold, or 2-fold to 5-fold. In some embodiments, the efficiency of cleaving and/or modifying a target sequence is increased by 15-fold to 30-fold. In some embodiments, the efficiency of cleaving and/or modifying a target sequence is increased by 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more. In some embodiments, the efficiency of cleaving and/or modifying the target sequence is assessed by measuring the percentage of a target sequence or cells comprising the target sequence that comprise altered expression of the target sequence or of a polypeptide encoded by the target sequence. In some embodiments, the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof. In some embodiments, the target sequence encodes a cell surface expressed protein, and the efficiency of cleaving and/or modifying the target sequence is assessed by measuring the percentage of cells comprising a reduction of the cell surface expressed protein as measured by flow cytometry. In some embodiments, the cell surface expressed protein is T cell receptor alpha (TRAC). In some embodiments, the cell surface expressed protein is beta 2-microglobulin (B2M). In certain embodiments of various methods described herein, the chemical modifications are within one or more stems in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the one or more stems of one stem loop. In some embodiments, the chemical modifications are within the one or more stems of multiple stem loops. In some embodiments, the chemical modifications are within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are not within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first and the second stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are within the first stem but not within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the first stem and one or more nucleotides within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within the first stem but no chemical modifications are present within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first and the second stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within the first stem but no chemical modifications are present within the second stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an antirepeat, or a guide RNA).

In some embodiments, the chemical modifications are present on one or more nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on one or more nucleotides within each stem of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti-repeat, or a guide RNA). In some embodiments, the chemical modifications are present on all nucleotides within all stems of a stem loop in a molecule or region of the disclosure (e.g., crRNA, a crRNA repeat, a spacer, a tracrRNA, an anti -repeat, or a guide RNA).

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within a crRNA repeat. In some embodiments, at least one BNA modification (e.g., LNA and/or cEt) is within the first stem of a crRNA repeat. In some embodiments, the terminal 5' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the first stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the first stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the second stem of a crRNA repeat. In some embodiments, the terminal 5' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the second stem of a crRNA repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two consecutive nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the second stem of a crRNA repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, no chemical modifications are within the second stem of a crRNA repeat.

In some embodiments, at least one BNA (e.g., LNA and/or cEt) modification is within the second stem of the anti -repeat of a tracrRNA. In some embodiments, the terminal 5' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 3' nucleotide of the second stem of an anti-repeat comprises a BNA (e.g., LNA and/or cEt) modification. In some embodiments, the terminal 5' nucleotide and the terminal 3' nucleotide of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine consecutive nucleotides of the second stem of an anti-repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, all nucleotides of the second stem of an anti -repeat comprise BNA (e.g., LNA and/or cEt) modifications. In some embodiments, no chemical modifications are within the second stem of an anti-repeat.

In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise BNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise cEt modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise cEt+PS modifications and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise LNA modifications. In some embodiments, the three terminal nucleotides at the 3' region of a tracrRNA of the disclosure comprise LNA+PS modifications and all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise BNA+PS modifications, wherein all the nucleotides in the first stem of the crRNA repeat of the crRNA comprise 2’-0-Me modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise BNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti-repeat of the tracrRNA comprise LNA modifications. In some embodiments, the gRNA is a sgRNA comprising a crRNA and a tracrRNA connected by a multiple nucleotide (e.g., a four nucleotide) linker, wherein the three terminal nucleotides at both the 5' region and the 3' region of the sgRNA comprise cEt+PS modifications, wherein the three terminal 3' nucleotides in the first stem of the crRNA repeat of the crRNA comprise MS modifications, and wherein all the nucleotides in the first stem of the anti -repeat of the tracrRNA comprise cEt modifications.

In some embodiments, methods of the disclosure use an engineered crRNA repeat, wherein the first stem of the engineered crRNA repeat can comprise at the 3' region a substituted or added nucleotide sequence from a native pre-crRNA. In some embodiments, the first stem of the engineered crRNA repeat comprises at the 3' region a substituted or added nucleotide sequence that is GC-rich. In some embodiments, the first stem of the engineered crRNA repeat comprises at the 3' region a GC- rich nucleotide sequence, wherein the content of G or C in the 3' region is at least 60%, at least 80%, or 100%. In some embodiments, the first stem of the engineered crRNA repeat comprises at the 3' region a GC-rich nucleotide sequence, wherein the 3’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs. In some embodiments, the first stem of the engineered crRNA repeat further comprises MS, BNA, or BNA+PS modifications at the 3' region.

In some embodiments, methods of the disclosure use an engineered anti-repeat, wherein the first stem of the engineered anti -repeat comprises at the 5' region a nucleotide sequence from a native precursor pre-crRNA. In some embodiments, the first stem of the engineered anti-repeat comprises at the 5' region a nucleotide sequence that is GC-rich. In some embodiments, the first stem of the engineered anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the content of G or C in the 5' region is at least 60%, at least 80%, or 100%. In some embodiments, the first stem of the engineered anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs. In some embodiments, the first stem of the engineered anti-repeat further comprises BNA (e.g., LNA and/or cEt) modifications at all nucleotides.

In some embodiments, methods of the disclosure use an engineered crRNA repeat comprising an addition or substitution of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises addition or substitution of 2 to 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprise an addition or substitution of 2 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition or substitution of 4 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, an engineered crRNA repeat comprises an addition or substitution of 6 nucleotides at the 3' region of the crRNA repeat, as compared to the same crRNA repeat prior to the engineering. In some embodiments, a first stem of an engineered crRNA repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of an engineered crRNA repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. In some embodiments, nucleotides in the 3' region of the first stem of an engineered crRNA repeat further comprise MS modifications, BNA modifications, or BNA+PS modifications.

In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise MS modifications. In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise BNA modifications. In some embodiments, three terminal nucleotides at both the 5' region and the 3' region of an engineered crRNA comprise BNA+PS modifications. In some embodiments, the BNA modifications comprise 2', 4' BNA modifications. In some embodiments, the 2', 4' BNA modifications are selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'- C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA modifications are LNA modifications. In some embodiments, the 2', 4' BNA modifications are cEt modifications.

In some embodiments, methods of the disclosure use an engineered anti-repeat comprising an addition or substitution of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or more at the 5' region of the anti -repeat, as compared to the same anti-repeat prior to the engineering. In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 2 to 6 nucleotides at the 5' region of the anti -repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 2 nucleotides at the 5' region of the anti -repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 4 nucleotides at the 5' region of the anti -repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, an engineered anti-repeat of an RGN system comprises an addition or substitution of 6 nucleotides at the 5' region of the anti-repeat, as compared to the same anti -repeat prior to the engineering. In some embodiments, the first stem of an engineered anti-repeat comprises a total length of about 11 nucleotides. In some embodiments, the first stem of an engineered anti -repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. In some embodiments, all nucleotides of the first stem of the engineered antirepeat further comprise BNA (e.g., LNA and/or cEt) modifications.

In some embodiments, all nucleotides in the first stem of the anti-repeat of an engineered tracrRNA comprise BNA modifications. In some embodiments, the BNA modifications comprise 2', 4' BNA modifications. In some embodiments, the 2', 4' BNA modifications are selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C- ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA modifications are LNA modifications. In some embodiments, the 2', 4' BNA modifications are cEt modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise MS modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise BNA modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise LNA modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise cEt modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise BNA+PS modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise LNA+PS modifications. In some embodiments, the 3' three terminal nucleotides at the tail of an engineered tracrRNA comprise cEt+PS modifications. IX. Methods of Engineering a guide RNA

The present disclosure provides methods for engineering a gRNA, comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat and the tracrRNA comprises an anti-repeat; and b) adding or substituting one or more nucleotides in the crRNA repeat and one or more nucleotides in the anti-repeat, wherein the one or more nucleotides added or substituted in the repeat and the one or more nucleotides added or substituted in the antirepeat are capable of hybridizing to each other, wherein the added or substituted one or more nucleotides comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a). In some embodiments, the guide RNA comprises at least one BNA (e.g., an LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti-repeat of the tracrRNA.

In some embodiments, the one or more nucleotides include 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In some embodiments, the gRNA is a dgRNA. In some embodiments, the gRNA is a sgRNA.

A nucleotide sequence added or substituted in a crRNA repeat and/or anti-repeat can be from a native pre-crRNA or a GC-rich sequence. In some embodiments, the content of G or C of the added or substituted one or more nucleotides is at least 51%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

In some embodiments, the added or substituted nucleotides are in the 3' region of the crRNA repeat and in the 5' region of the anti-repeat. The 3’ region of the crRNA repeat and/or the 5’ region of the anti -repeat of an engineered gRNA can comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

The method can include substituting or replacing 1, 2, 3, 4, or 5 nucleotides at the 3' region of the first stem of the crRNA repeat with another shorter or longer nucleotide sequence to form an engineered crRNA repeat. In some embodiments, the method includes adding or substituting 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at the 3' region of the crRNA repeat.

The method can include substituting or replacing 1, 2, 3, 4, or 5 nucleotides at the 5' region of the first stem of the anti-repeat with another shorter or longer nucleotide sequence to form an engineered tracrRNA. In some embodiments, the method includes adding or substituting 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at the 5' region of the anti -repeat.

The method can further comprise modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA modification. The types and the locations of chemical modifications disclosed in previous sections of this application can all be used in various methods described herein.

In some embodiments, the at least one chemical modification is a BNA modification, the BNA modification is a 2', 4' BNA modification. The 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'- C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification.

The at least one chemical modification can be in the crRNA, the tracrRNA, or both. In some embodiments, the at least one chemical modification is in the crRNA repeat. In some embodiments, the at least one chemical modification is in a first stem of the crRNA repeat. In some embodiments, the at least one chemical modification is in a tail of the tracrRNA.

The at least one chemical modification can be in the anti-repeat. In some embodiments, the at least one chemical modification is in a first stem of the anti-repeat. In certain embodiments, the at least one chemical modification is on 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in the first stem of the antirepeat. The at least one chemical modification can be on consecutive nucleotides in the first stem of the anti-repeat. The at least one chemical modification can be on alternate nucleotides in the first stem of the anti-repeat. “Alternate nucleotides” refers to every other nucleotide in a nucleotide sequence having at least one chemical modification. In some embodiments, the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat.

In some embodiments, the at least one chemical modification is in the crRNA repeat and the anti-repeat. In some embodiments, the at least one chemical modification is in the first stem of the crRNA repeat and the first stem of the anti-repeat. In some embodiments, the at least one chemical modification is in the crRNA repeat, the anti-repeat, and the tail of the tracrRNA. In some embodiments, the at least one chemical modification is in the first stem of the crRNA repeat, the first stem of the anti-repeat, and the tail of the tracrRNA.

In some embodiments, the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on three terminal nucleotides at the 3’ region of the tail of the tracrRNA. The at least one chemical modification can be on all nucleotides in the first stem of the anti-repeat and on at least one nucleotide in the first stem of the crRNA repeat. The at least one chemical modification is on all nucleotides in the first stem of the anti-repeat, on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA, and on at least one nucleotide at the 3 ’ region of the first stem of the crRNA repeat.

In some embodiments, the at least one chemical modification comprises MS modifications at both the 5' region and the 3' region of the engineered crRNA. In some embodiments, the at least one chemical modification comprises BNA modifications at both the 5' region and the 3' region of the engineered crRNA. In some embodiments, the at least one chemical modification comprises BNA+PS modifications at both the 5' region and the 3' region of the engineered crRNA.

In some embodiments, the at least one chemical modification comprises BNA modifications on all nucleotides in the first stem of the anti-repeat of an engineered tracrRNA. In some embodiments, the at least one chemical modification comprises BNA modifications on all nucleotides in the first stem of the anti -repeat of an engineered tracrRNA and MS modifications at the 3 ’ region of the first stem of the crRNA repeat. In some embodiments, the at least one chemical modification comprises MS modifications on the tail of an engineered tracrRNA. In some embodiments, the at least one chemical modification comprises BNA modifications on the tail of an engineered tracrRNA. In some embodiments, the at least one chemical modification comprises LNA modifications on the tail of an engineered tracrRNA. In some embodiments, the at least one chemical modification comprises cEt modifications on the tail of an engineered tracrRNA. In some embodiments, the at least one chemical modification comprises BNA+PS modifications on the tail of an engineered tracrRNA. In some embodiments, the at least one chemical modification comprises LNA+PS modifications on the tail of an engineered tracrRNA. In some embodiments, the at least one chemical modification comprises cEt+PS modifications on the tail of an engineered tracrRNA.

The engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a) that has not been engineered. In some embodiments, the editing efficiency of the engineered gRNA is increased at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, or more, as compared to the gRNA that has not been engineered. In some embodiments, the editing efficiency of the engineered gRNA is increased at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, or more, as compared to the gRNA that has not been engineered. In some embodiments, the increased efficiency includes increased efficiency in base editing or prime editing. The editing efficiency can be determined using methods known to a person of ordinary skill in the art, including but not limited to those described in section VIII.

The engineered gRNA can exhibit more stability as compared to the gRNA that has not been engineered. In some embodiments, stability of an engineered gRNA can be measured by measuring the melting temperature (Tm) of a heteroduplex formed between the crRNA repeat and the anti-repeat of the engineered gRNA. In some embodiments, Tm of a nucleic acid molecule is dependent upon GC content, length of the nucleic acid molecule, and sequence of the nucleic acid molecule. Determination of Tm of a nucleic acid molecule can be performed as described in Cromwell et al. (2018) Nature Communications 9: 1448.

The disclosure also provides for an engineered gRNA produced by a method described herein, an RGN system, an RNP complex, or a pharmaceutical composition comprising such an engineered gRNA, and methods of using these in binding, cleaving, and/or modifying a target polynucleotide.

X. Target Polynucleotides

The disclosure provides for methods of modifying a target polynucleotide comprising a target sequence or modifying the expression of a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. The methods comprise using guide RNAs comprising at least one BNA (e.g., an LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including microalgae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the human, non-human animal or plant (including micro-algae).

Using natural variability, plant breeders combine most useful genes for desirable qualities, such as yield, quality, uniformity, hardiness, and resistance against pests. These desirable qualities also include growth, day length preferences, temperature requirements, initiation date of floral or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, tolerance to various environmental factors including drought, heat, wet, cold, wind, and adverse soil conditions including high salinity. The sources of these useful genes include native or foreign varieties, heirloom varieties, wild plant relatives, and induced mutations, e.g., treating plant material with mutagenic agents. Using the present disclosure, plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can employ the present disclosure to induce the rise of useful genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs.

The target polynucleotide of an RGN system of the disclosure can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. In some embodiments, the target polynucleotide is a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). Without wishing to be bound by theory, the non-target strand of the target sequence should be adjacent to a PAM (protospacer adjacent motif), that is, a short sequence recognized by the RGN. The precise sequence and length requirements for the PAM differ depending on the RGN used, but PAMs are typically 2-5 base pair sequences adjacent to the protospacer (that is, the target sequence). The target polynucleotide of an RGN system of the disclosure may include a number of disease-associated genes and polynucleotides as well as signaling biochemical pathway-associated genes and polynucleotides. Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissue compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease (e.g., a causal mutation). The transcribed or translated products may be known or unknown, and further may be at a normal or abnormal level.

Non-limiting examples of disease-associated genes that can be targeted using the presently disclosed methods and compositions are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web.

In some embodiments, the methods comprise contacting a target nucleic acid molecule comprising a target sequence with an RGN of the disclosure, wherein the target nucleic acid molecule is contacted with the RGN in an amount effective and under conditions suitable for editing of the target sequence (e.g., cleaving, modifying expression, base editing, prime editing). In certain embodiments, the methods comprise contacting a target nucleic acid molecule comprising a target sequence with (a) an RGN of the disclosure; and (b) a gRNA targeting the RGN of (a) to the target sequence; wherein the target nucleic acid molecule is contacted with the RGN and the gRNA in an amount effective and under conditions suitable for editing of the target sequence. In some embodiments, the target sequence comprises a sequence associated with a disease or disorder, and wherein the editing of the target sequence results in a sequence that is not associated with a disease or disorder. In some embodiments, the target sequence resides in an allele of a crop plant, wherein the particular allele is associated with a trait that results in a plant of lesser agronomic value. The editing of the target sequence results in an allele that is associated with a trait that increases the agronomic value of the plant.

In some embodiments, the target sequence associated with the disease or disorder encodes a protein, and the editing of the target sequenceintroduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein. In some embodiments, the contacting is performed in vivo in a subject susceptible to having, having, or diagnosed with the disease or disorder. In some embodiments, the disease or disorder is a disease associated with a point mutation, or a single-base mutation, in the genome. In some embodiments, the disease is a genetic disease, a cancer, a metabolic disease, or a lysosomal storage disease.

XI. Cells Comprising a Polynucleotide Genetic Modification

Provided herein are cells and organisms comprising a target nucleic acid molecule that has been modified using a process mediated by an RGN, crRNA, tracrRNA, and/or guide RNA as described herein, e.g., that has been modified to comprise at least one BNA (e.g., LNA and/or cEt) modification. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti-repeat of the tracrRNA. In some embodiments, an RGN disclosed herein requires a tracrRNA for activity. In some embodiments, an RGN disclosed herein includes a type II RGN. In some embodiments, the RGN comprises an amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252, or active variants or fragments thereof. In some embodiments, the RGN comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 69, 93, and 252. In some embodiments, the guide RNA comprises a crRNA repeat comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 39, 300, 304, 308, 312, 320, 324, 328, 332, 336, 344, 348, 352, 356, 360, 384-393, 397, 465, 469, 473, 477, 481, 508, 512, and 516, or active variants or fragments thereof. In some embodiments, the guide RNA comprises a crRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 73-75, 97-99, 292, 293, 301- 303, 305-307, 309-311, 313-315, 321-323, 325-327, 329-331, 333-335, 337-339, 345-347, 349-351, 353-355, 357-359, 361-363, 380-382, 399-401, 466-468, 470-472, 474-476, 478-480, 482-484, 509- 511, 513-515, 517-519, and 708, or active variants or fragments thereof. In some embodiments, the guide RNA comprises a tracrRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 80, 81, 102, 103, 294, 295, 364-367, 369-373, 375-379, 383, 499-501, 504, 505, 534, 535, 537, 709-711, and 713, or active variants or fragments thereof. The guide RNA of the system can be a single guide RNA or a dual-guide RNA. In embodiments, the guide RNA comprises a sgRNA comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407, or active variants or fragments thereof.

The modified cells can be eukaryotic (e.g., mammalian, plant, insect, avian cell) or prokaryotic. Prokaryotic cells can be from species, including but not limited to, archaea and bacteria (e.g., Bacillus sp., Klebsiella sp. Streptomyces sp., Rhizobium sp., Escherichia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Vibrio sp., Yersinia sp., Mycoplasma sp., Agrobacterium, Lactobacillus sp.). Eukaryotic cells can include cells from animals (e.g., mammals, humans, insects, fish, birds, and reptiles), plants, fungi, amoeba, algae, and yeast. In some embodiments, the cell that is modified by the presently disclosed methods include cells of hematopoietic origin, such as cells of the immune system including but not limited to B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. In some embodiments, the cell that is modified by the presently disclosed methods include primary cells. In certain embodiments, the primary cells include primary T cells.

Also provided are embryos comprising at least one target sequence that has been modified by a process utilizing an RGN, crRNA, tracrRNA, and/or guide RNA as described herein. The genetically modified cells, organisms, organelles, and embryos can be heterozygous or homozygous for the modified target sequence.

In some embodiments, the chromosomal modification of the cell, organism, organelle, or embryo can result in altered expression (up-regulation or down-regulation), inactivation, or the expression of an altered protein product or an integrated sequence. In those instances wherein the mutation(s) results in either the inactivation of a gene or the expression of a non-functional protein product, the genetically modified cell, organism, organelle, or embryo is referred to as a “knock out”. The knock-out phenotype can be the result of a deletion mutation (i.e., deletion of at least one nucleotide), an insertion mutation (i.e., insertion of at least one nucleotide), or a nonsense mutation (i.e., substitution of at least one nucleotide such that a stop codon is introduced). In some embodiments, chromosomal modification results in upregulation of expression of a protein product that had been lacking or reduced due to mutation(s) in a target sequence.

In some embodiments, an RGN system of the disclosure includes a fusion of an RGN and a base editing polypeptide (e.g., a deaminase) or a fusion of an RGN and a prime editing polypeptide (e.g., a reverse transcriptase) and the mutation(s) introduced as a result of these RGN systems yields production of a variant protein product. The expressed variant protein product can have at least one amino acid substitution and/or the addition or deletion of at least one amino acid. The variant protein product can exhibit modified characteristics or activities when compared to the wild-type protein, including but not limited to altered enzymatic activity or substrate specificity. In some embodiments, the mutation(s) introduced as a result of these RGN systems yields altered expression pattern of a protein. As a non-limiting example, mutation(s) in the regulatory regions controlling the expression of a protein product can result in the overexpression or downregulation of the protein product or an altered tissue or temporal expression pattern. In some embodiments, the mutation(s) introduced as a result of these RGN systems yields a reduction or elimination in expression of a gene.

Cells that have been modified may be introduced into an organism. These cells could have originated from the same organism (e.g., person) in the case of autologous cellular transplants, wherein the cells are modified in an ex vivo approach. Alternatively, the cells originated from another organism within the same species (e.g., another person) in the case of allogeneic cellular transplants.

XII. Pharmaceutical Compositions

Pharmaceutical compositions comprising: crRNAs comprising at least one BNA modification

(e.g., LNA and/or cEt) and active variants and fragments thereof; tracrRNAs comprising at least one BNA modification (e.g., LNA and/or cEt) and active variants and fragments thereof; gRNAs comprising at least one BNA modification (e.g., LNA and/or cEt) and active variants and fragments thereof; RGN polypeptides comprising at least one BNA modification (e.g., LNA and/or cEt) and active variants and fragments thereof, or polynucleotides encoding the same; RGN systems described herein comprising crRNAs, tracrRNAs, and/or gRNAs comprising at least one BNA modification (e.g., LNA and/or cEt); or cells comprising any of the crRNA, tracrRNA, gRNA, RGN polypeptides or RGN-encoding polynucleotides, or RGN systems; and a pharmaceutically acceptable carrier are provided. In some embodiments, the gRNAs comprising at least one BNA modification (e.g., LNA and/or cEt) are single guide RNAs. In some embodiments, the gRNAs comprising at least one BNA modification (e.g., LNA and/or cEt) are dual guide RNAs. In some embodiments, the at least one BNA (e.g., LNA and/or cEt) modification is in the first stem of the anti-repeat of the tracrRNA. In some embodiments, the guide RNA is an engineered guide RNA comprising at least one BNA (e.g., LNA and/or cEt) modification in the first stem of the anti-repeat of the tracrRNA.

A pharmaceutical composition is a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease that comprises an active ingredient (i.e., crRNA, tracrRNA, gRNA, RGN polypeptides, RGN-encoding polynucleotides, RGN systems, or cells comprising any one of these) and a pharmaceutically acceptable carrier.

As used herein, a “pharmaceutically acceptable carrier” refers to a material that does not cause significant irritation to an organism and does not abrogate the activity and properties of the active ingredient (i.e., crRNA, tracrRNA, gRNA, RGN polypeptides, RGN-encoding polynucleotides, RGN systems, or cells comprising any one of these). Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to a subject being treated. The carrier can be inert, or it can possess pharmaceutical benefits. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. In some embodiments, the pharmaceutically acceptable carrier is not naturally-occurring. In some embodiments, the pharmaceutically acceptable carrier and the active ingredient are not found together in nature.

Pharmaceutical compositions used in the presently disclosed methods can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations are known to those skilled in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st ed. 2005). Suitable formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN vesicles), lipid nanoparticles, DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Pharmaceutical compositions for oral or parenteral use may be prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

In embodiments wherein cells comprising or modified with a crRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a tracrRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a gRNA comprising at least one BNA modification (e.g., LNA and/or cEt), RGN polypeptides, and/or RGN-encoding polynucleotides are administered to a subject, the cells are administered as a suspension with a pharmaceutically acceptable carrier. One of skill in the art will recognize that a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject. A formulation comprising cells can include, e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and/or can be adapted for use with the cells described herein using routine experimentation.

A cell composition can also be emulsified or presented as a liposome composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable and pharmaceutically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active compound used in the cell compositions that is effective in the treatment of a particular disorder or condition can depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. A crRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a tracrRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a gRNA comprising at least one BNA modification (e.g., LNA and/or cEt), RGN polypeptides, and/or RGN-encoding polynucleotides can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form. In some embodiments, these pharmaceutical compositions are formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH can be adjusted to a range from about pH 5.0 to about pH 8. In some embodiments, the compositions can comprise a therapeutically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients. In some embodiments, the compositions comprise a combination of the compounds described herein, or include a second active ingredient useful in the treatment or prevention of bacterial growth (for example and without limitation, antibacterial or anti -microbial agents), or include a combination of reagents of the present disclosure.

Suitable excipients include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients can include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.

In some embodiments, the formulations are provided in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring the addition of the sterile liquid carrier, for example, saline, water-for-injection, a semiliquid foam, or gel, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. In some embodiments, the active ingredient is dissolved in a buffered liquid solution that is frozen in a unitdose or multi-dose container and later thawed for injection or kept/stabilized under refrigeration until use.

The therapeutic agent(s) may be contained in controlled release systems. In order to prolong the effect of a drug, it often is desirable to slow the absorption of the drug from subcutaneous, intrathecal, or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. In some embodiments, the use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term sustained release implants are well-known to those of ordinary skill in the art.

In some embodiments, a composition of the disclosure includes: a) a tracrRNA comprising at least one BNA (e.g., LNA and/or cEt) modification as described herein; b) a crRNA capable of binding to the tracrRNA to form a guide RNA (gRNA); and c) an RGN or a nucleic acid molecule encoding the RGN, wherein the gRNA is capable of forming a complex with said RGN. In some embodiments, a composition of the disclosure includes: a) a gRNA comprising at least one BNA (e.g., LNA and/or cEt) modification as described herein; and b) an RGN or a nucleic acid molecule encoding the RGN, wherein the gRNA is capable of forming a complex with the RGN. In some embodiments, a composition of the disclosure includes: a) a crRNA comprising at least one chemical modification as described herein; b) a tracrRNA capable of binding to the crRNA to form a gRNA; and c) an RGN or a nucleic acid molecule encoding the RGN, wherein the gRNA is capable of forming a complex with said RGN.

XIII. Methods of Treatment

Methods of treating a disease in a subject in need thereof are provided herein. The methods comprise administering to a subject in need thereof an effective amount of a tracrRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a gRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a crRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a presently disclosed RGN or a polynucleotide encoding the same, a presently disclosed RGN system, or a cell modified by or comprising any one of these compositions.

In some embodiments, the treatment comprises in vivo gene editing by administering to a subject in need thereof a tracrRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a gRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a crRNA comprising at least one BNA modification (e.g., LNA and/or cEt), RGN or polynucleotide(s) encoding the same, and/or an RGN system. In some embodiments, the treatment comprises ex vivo gene editing wherein cells are genetically modified ex vivo with a tracrRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a gRNA comprising at least one BNA modification (e.g., LNA and/or cEt), a crRNA comprising at least one BNA modification (e.g., LNA and/or cEt), RGN or polynucleotide(s) encoding the same, and/or an RGN system and then the modified cells are administered to a subject. In some embodiments, the genetically modified cells originate from the subject that is then administered the modified cells, and the transplanted cells are referred to herein as autologous. In some embodiments, the genetically modified cells originate from a different subject (i.e., donor) within the same species as the subject that is administered the modified cells (i.e., recipient), and the transplanted cells are referred to herein as allogeneic. In some examples described herein, the cells can be expanded in culture prior to administration to a subject in need thereof. In some embodiments, the disease to be treated with the presently disclosed compositions is one that can be treated with immunotherapy, such as with a chimeric antigen receptor (CAR) T cell. Such diseases include but are not limited to cancer.

In some embodiments, the disease to be treated with the presently disclosed compositions is associated with a genetic defect, e.g, a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes. Thus, in some embodiments, compositions of the disclosure are used to edit a genomic sequence causal for the disease or disorder or symptoms of such disease or disorder. For example, the deamination of a target nucleobase results in the correction of a genetic defect such that loss of function of a gene product is restored.

In some embodiments, the disease to be treated with the presently disclosed compositions is associated with a causal mutation. As used herein, a “causal mutation” refers to a particular nucleotide, nucleotides, or nucleotide sequence in the genome that contributes to the severity or presence of a disease or disorder in a subject. The correction of the causal mutation leads to the improvement of at least one symptom resulting from a disease or disorder. In some embodiments, the correction of the causal mutation leads to the improvement of at least one symptom resulting from a disease or disorder. In some embodiments, the causal mutation is adjacent to a PAM site recognized by an RGN disclosed herein. Non-limiting examples of diseases associated with a causal mutation include cystic fibrosis, Niemann-Pick disease, and diseases caused by splice site disruptions. Additional non-limiting examples of disease-associated genes and mutations are available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web.

In some embodiments, the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease or disorder. For example, in some embodiments, methods are provided herein that employ an RGN fusion protein to introduce a deactivating point mutation into an oncogene (e.g., in the treatment of a proliferative disease). A deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g. , a truncated protein lacking the function of the full-length protein. In some embodiments, the purpose of the methods provided herein is to restore the function of a dysfunctional gene via genome editing. The RGN fusion proteins can be validated for gene editing -based human therapeutics in vitro, e.g., by correcting a disease associated mutation in human cell culture. It will be understood by the skilled artisan that RGN fusion proteins comprising an RGN and base editing polypeptide or comprising an RGN and prime editing polypeptide can be used to correct any single point mutation.

As used herein, "treatment" or "treating," or "palliating" or "ameliorating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested. In embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their prevention or recurrence.

The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the delivery system in which it is carried.

The term "administering" refers to the placement of an active ingredient into a subject, by a method or route that results in at least partial localization of the introduced active ingredient at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced. In embodiments, the disclosure provides methods comprising delivering any of the RGN polypeptides, nucleic acid molecules, ribonucleoprotein complexes, vectors, pharmaceutical compositions and/or gRNAs described herein. In embodiments, the disclosure further provides cells produced by such methods, and organisms (such as animals or plants) comprising or produced from such cells. In embodiments, a RGN polypeptide and/or nucleic acid molecules as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell.

In embodiments wherein cells are administered, the cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the patient, i.e., long-term engraftment.

In embodiments, the administering comprises administering by viral delivery. Viral vectors comprising a nucleic acid encoding the RGN polypeptides, ribonucleoprotein complexes, or vectors disclosed herein may be administered directly to patients (i.e., in vivo) or they may be used to treat cells in vitro, and the modified cells may optionally be administered to patients (i.e., ex vivo). Conventional viral based systems may include, without limitation, retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division.

In embodiments, the administering comprises administering by other non-viral delivery of nucleic acids. Exemplary non-viral delivery methods, without limitation, include RNP complexes, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO1991/17424; WO 1991/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).

Suitable routes of administering the pharmaceutical compositions described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

In embodiments, the pharmaceutical composition described herein is administered to a subject by injection, inhalation (e.g., of an aerosol), by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. In embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing.

In embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

In embodiments, the pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts.

As used herein, the term "subject" refers to any individual for whom diagnosis, treatment or therapy is desired. In embodiments, the subject is an animal. In embodiments, the subject is a mammal. In embodiments, the subject is a human being.

The efficacy of a treatment can be determined by the skilled clinician. However, a treatment is considered an "effective treatment," if any one or all of the signs or symptoms of a disease or disorder are altered in a beneficial manner (e.g., decreased by at least 10%), or other clinically accepted symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art. Treatment includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

The article “a” and “an” are used herein to refer to one or more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, “a polypeptide” means one or more polypeptides.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended embodiments.

Non-limiting embodiments include:

1. A nucleic acid molecule comprising a transactivating CRISPR RNA (tracrRNA), wherein the tracrRNA comprises:

(a) an anti-repeat; (b) a tail; and

(c) a stem loop most proximal to the tail, wherein the anti -repeat of the tracrRNA is capable of hybridizing to a CRISPR RNA (crRNA) repeat of a crRNA to form a guide RNA (gRNA) comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.

2. The nucleic acid molecule of embodiment 1, wherein the at least one BNA modification is within the anti-repeat.

3. The nucleic acid molecule of embodiment 1 or 2, wherein the at least one BNA modification is within the first stem of the anti-repeat.

4. The nucleic acid molecule of embodiment 3, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen consecutive BNA modifications on consecutive mucleotides, at least three consecutive BNA modifications, at least four consecutive BNA modifications, at least five consecutive BNA modifications, at least six consecutive BNA modifications, at least seven consecutive BNA modifications, at least eight consecutive BNA modifications, or at least nine consecutive BNA modifications, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat.

5. The nucleic acid molecule of embodiment 3 or 4, wherein all nucleotides within the first stem of the anti -repeat comprise BNA modifications.

6. The nucleic acid molecule of embodiment 1 or 2, wherein the at least one BNA modification is not within the second stem of the anti-repeat.

7. The nucleic acid molecule of any one of embodiments 1-6, wherein the at least one BNA modification is not within a bulge of the tracrRNA.

8. The nucleic acid molecule of any one of embodiments 1-7, wherein three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications.

9. The nucleic acid molecule of any one of embodiments 1-7, wherein three terminal nucleotides of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

10. The nucleic acid molecule of any one of embodiments 1-7, wherein three terminal nucleotides of the tail of the tracrRNA comprise 2'-O-methyl 3' phosphorothioate (MS) modifications.

11. The nucleic acid molecule of any one of embodiments 1-10, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

12. The nucleic acid molecule of embodiment 11, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. 13. The nucleic acid molecule of embodiment 11 or 12, wherein the 2', 4' BNA is a LNA modification.

14. The nucleic acid molecule of embodiment 11 or 12, wherein the 2', 4' BNA is a cEt modification.

15. The nucleic acid molecule of any one of embodiments 1-14, wherein the tracrRNA further comprises at least one other chemical modification.

16. The nucleic acid molecule of embodiment 15, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

17. The nucleic acid molecule of embodiment 15 or 16, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

18. The nucleic acid molecule of embodiment 15, wherein the at least one other chemical modification is within the tail of the tracrRNA.

19. The nucleic acid molecule of any one of embodiments 15-18, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.

20. The nucleic acid molecule of embodiment 19, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications.

21. The nucleic acid molecule of embodiment 20, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications and all nucleotides of the first stem of the antirepeat comprise BNA modifications.

22. The nucleic acid molecule of embodiment 21, wherein the BNA modifications comprise LNA modifications.

23. The nucleic acid molecule of embodiment 21, wherein the BNA modifications comprise cEt modifications.

24. The nucleic acid molecule of any one of embodiments 1-23, wherein the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

25. The nucleic acid molecule of any one of embodiments 1-23, wherein the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

26. The nucleic acid molecule of any one of embodiments 1-23, wherein the first stem of the anti -repeat comprises a total length of about 11 nucleotides.

27. The nucleic acid molecule of any one of embodiments 1-23, wherein the first stem of the anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides. 28. The nucleic acid molecule of any one of embodiments 1-27, wherein the first stem of the anti -repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

29. The nucleic acid molecule of embodiment 28, wherein the first stem of the anti-repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

30. The nucleic acid molecule of any one of embodiments 1-29, wherein the tracrRNA comprises atotal length of 60-80 nt, 80-100 nt, 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, or more than 180 nt.

31. The nucleic acid molecule of any one of embodiments 1-30, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

32. The nucleic acid molecule of embodiment 31, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

33. The nucleic acid molecule of embodiment 31 or 32, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

34. The nucleic acid molecule of any one of embodiments 31-33, wherein the tracrRNA has a nucleotide sequence having the sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

35. The nucleic acid molecule of any one of embodiments 1-30, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

36. The nucleic acid molecule of embodiment 35, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

37. The nucleic acid molecule of embodiment 35 or 36, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 80, 81, 364- 367, 369, and 375-379.

38. The nucleic acid molecule of any one of embodiments 35-37, wherein the tracrRNA has a nucleotide sequence having the sequence set forth as any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

39. The nucleic acid molecule of any one of embodiments 1-30, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711. 40. The nucleic acid molecule of embodiment 39, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

41. The nucleic acid molecule of embodiment 39 or 40, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 102, 103, 370- 373, 710, and 711.

42. The nucleic acid molecule of any one of embodiments 39-41, wherein the tracrRNA has a nucleotide sequence having the sequence set forth as any one of SEQ ID NOs: 102, 103, 370- 373, 710, and 711.

43. The nucleic acid molecule of any one of embodiments 1-30, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 499-501,

504, 505, 534, 535, and 537.

44. The nucleic acid molecule of embodiment 43, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

45. The nucleic acid molecule of embodiment 43 or 44, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 499-501, 504,

505, 534, 535, and 537.

46. The nucleic acid molecule of any one of embodiments 43-45, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

47. The nucleic acid molecule of any one of embodiments 1-46, wherein the tracrRNA is part of a gRNA that is capable of binding to an RGN.

48. The nucleic acid molecule of embodiment 47, wherein the RGN is a Type II RGN.

49. The nucleic acid molecule of embodiment 47 or 48, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

50. The nucleic acid molecule of embodiment 49, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

51. The nucleic acid molecule of embodiment 49 or 50, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

52. The nucleic acid molecule of any one of embodiments 49-51, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 1.

53. The nucleic acid molecule of embodiment 47 or 48, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

54. The nucleic acid molecule of embodiment 53, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 69.

55. The nucleic acid molecule of embodiment 53 or 54, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69. 56. The nucleic acid molecule of any one of embodiments 53-55, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 69.

57. The nucleic acid molecule of embodiment 47 or 48, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 93.

58. The nucleic acid molecule of embodiment 57, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93.

59. The nucleic acid molecule of embodiment 57 or 58, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 93.

60. The nucleic acid molecule of any one of embodiments 57-59, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 93.

61. The nucleic acid molecule of embodiment 47 or 48, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 252.

62. The nucleic acid molecule of embodiment 61, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 252.

63. The nucleic acid molecule of embodiment 61 or 62, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 252.

64. The nucleic acid molecule of any one of embodiments 61-63, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 252.

65. A nucleic acid molecule comprising a transactivating CRISPR RNA (tracrRNA), wherein the tracrRNA comprises:

(a) an anti-repeat;

(b) a tail; and

(c) a stem loop most proximal to the tail, wherein the anti-repeat of the tracrRNA comprises a first stem and a second stem, and wherein the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.

66. The nucleic acid molecule of embodiment 65, wherein the at least one BNA modification is within the anti-repeat.

67. The nucleic acid molecule of embodiment 65 or 66, wherein the at least one BNA modification is within the first stem of the anti-repeat.

68. The nucleic acid molecule of embodiment 67, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen consecutive BNA modifications on consecutive mucleotides, at least three consecutive BNA modifications, at least four consecutive BNA modifications, at least five consecutive BNA modifications, at least six consecutive BNA modifications, at least seven consecutive BNA modifications, at least eight consecutive BNA modifications, or at least nine consecutive BNA modifications, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat. 69. The nucleic acid molecule of embodiment 67 or 68, wherein all nucleotides within the first stem of the anti -repeat comprise BNA modifications.

70. The nucleic acid molecule of embodiment 65 or 66, wherein the at least one BNA modification is not within the second stem of the anti-repeat.

71. The nucleic acid molecule of any one of embodiments 65-70, wherein the at least one BNA modification is not within a bulge of the tracrRNA.

72. The nucleic acid molecule of any one of embodiments 65-71, wherein three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications.

73. The nucleic acid molecule of any one of embodiments 65-71, wherein three terminal nucleotides of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

74. The nucleic acid molecule of any one of embodiments 65-71, wherein three terminal nucleotides of the tail of the tracrRNA comprise 2'-O-methyl 3' phosphorothioate (MS) modifications.

75. The nucleic acid molecule of any one of embodiments 65-74, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

76. The nucleic acid molecule of embodiment 75, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

77. The nucleic acid molecule of embodiment 75 or 76, wherein the 2', 4' BNA is a LNA modification.

78. The nucleic acid molecule of embodiment 75 or 76, wherein the 2', 4' BNA is a cEt modification.

79. The nucleic acid molecule of any one of embodiments 65-78, wherein the tracrRNA further comprises at least one other chemical modification.

80. The nucleic acid molecule of embodiment 79, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

81. The nucleic acid molecule of embodiment 79 or 80, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

82. The nucleic acid molecule of embodiment 79, wherein the at least one other chemical modification is within the tail of the tracrRNA.

83. The nucleic acid molecule of any one of embodiments 79-82, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3 'phosphorothioate (MS) modification; 2'- O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification. 84. The nucleic acid molecule of embodiment 83, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications.

85. The nucleic acid molecule of embodiment 83, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications and all nucleotides of the first stem of the antirepeat comprise BNA modifications.

86. The nucleic acid molecule of embodiment 85, wherein the BNA modifications comprise LNA modifications.

87. The nucleic acid molecule of embodiment 85, wherein the BNA modifications comprise cEt modifications.

88. The nucleic acid molecule of any one of embodiments 65-87, wherein the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

89. The nucleic acid molecule of any one of embodiments 65-87, wherein the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

90. The nucleic acid molecule of any one of embodiments 65-87, wherein the first stem of the anti -repeat comprises a total length of about 11 nucleotides.

91. The nucleic acid molecule of any one of embodiments 65-87, wherein the first stem of the anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

92. The nucleic acid molecule of any one of embodiments 65-91, wherein the first stem of the anti -repeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

93. The nucleic acid molecule of embodiment 92, wherein the first stem of the anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

94. The nucleic acid molecule of any one of embodiments 65-93, wherein the tracrRNA comprises atotal length of 60-80 nt, 80-100 nt, 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, or more than 180 nt.

95. The nucleic acid molecule of any one of embodiments 65-94, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, and 383, 709, and 713.

96. The nucleic acid molecule of embodiment 95, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, and 383, 709, and 713.

97. The nucleic acid molecule of embodiment 95 or 96, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, and 383, 709, and 713. 98. The nucleic acid molecule of any one of embodiments 95-97, wherein the tracrRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, and 383,

709, and 713.

99. The nucleic acid molecule of any one of embodiments 65-94, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

100. The nucleic acid molecule of embodiment 99, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

101. The nucleic acid molecule of embodiment 99 or 100, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 80, 81, 364- 367, 369, and 375-379.

102. The nucleic acid molecule of any one of embodiments 99-101, wherein the tracrRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

103. The nucleic acid molecule of any one of embodiments 65-94, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 102, 103, and 370-373, 710, and 711.

104. The nucleic acid molecule of embodiment 103, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 102, 103, and 370-373, 710, and 711.

105. The nucleic acid molecule of embodiment 103 or 104, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 102, 103, and 370-373, 710, and 711.

106. The nucleic acid molecule of any one of embodiments 103-105, wherein the tracrRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 102, 103, and 370-373,

710, and 711.

107. The nucleic acid molecule of any one of embodiments 65-94, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 499-501,

504, 505, 534, 535, and 537.

108. The nucleic acid molecule of embodiment 107, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 499-501, 504,

505, 534, 535, and 537.

109. The nucleic acid molecule of embodiment 107 or 108, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537. 110. The nucleic acid molecule of any one of embodiments 107-109, wherein the tracrRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

111. The nucleic acid molecule of any one of embodiments 65-110, wherein a gRNA comprising the tracrRNA is capable of binding to an RGN.

112. The nucleic acid molecule of embodiment 111, wherein the RGN is a Type II RGN.

113. The nucleic acid molecule of embodiment 111 or 112, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

114. The nucleic acid molecule of embodiment 113, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

115. The nucleic acid molecule of embodiment 113 or 114, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

116. The nucleic acid molecule of any one of embodiments 113-115, wherein the RGN has the amino acid sequence set forth as SEQ ID NO: 1.

117. The nucleic acid molecule of embodiment 111 or 112, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

118. The nucleic acid molecule of embodiment 117, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 69.

119. The nucleic acid molecule of embodiment 117 or 118, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69.

120. The nucleic acid molecule of any one of embodiments 117-119, wherein the RGN has the amino acid sequence set forth as SEQ ID NO: 69.

121. The nucleic acid molecule of embodiment 111 or 112, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 93.

122. The nucleic acid molecule of embodiment 121, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93.

123. The nucleic acid molecule of embodiment 121 or 122, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 93.

124. The nucleic acid molecule of any one of embodiments 121-123, wherein the RGN has the amino acid sequence set forth as SEQ ID NO: 93.

125. The nucleic acid molecule of embodiment 111 or 112, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 252.

126. The nucleic acid molecule of embodiment 125, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 252.

127. The nucleic acid molecule of embodiment 125 or 126, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 252. 128. The nucleic acid molecule of any one of embodiments 125-127, wherein the RGN has the amino acid sequence set forth as SEQ ID NO: 252.

129. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a spacer and a crRNA repeat, wherein the tracrRNA comprises a tail and an anti-repeat, wherein the anti -repeat is capable of hybridizing to the crRNA repeat to form the gRNA comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BN A) modification.

130. The gRNA of embodiment 129, wherein the gRNA is a single guide RNA (sgRNA).

131. The gRNA of embodiment 130, wherein the sgRNA comprises a total length of 100- 120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

132. The gRNA of embodiment 129, wherein the gRNA is a dual guide RNA (dgRNA).

133. The gRNA of any one of embodiments 129-132, wherein the at least one BNA modification is within the crRNA repeat.

134. The gRNA of any one of embodiments 129-133, wherein the at least one BNA modification is within the first stem of the crRNA repeat.

135. The gRNA of any one of embodiments 129-134, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications.

136. The gRNA of any one of embodiments 129-135, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications and phosphorothioate (PS) modifications.

137. The gRNA of any one of embodiments 129-134, wherein the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat.

138. The gRNA of any one of embodiments 129-133, wherein the at least one BNA modification is not within the second stem of the crRNA repeat.

139. The gRNA of any one of embodiments 129-138, wherein the at least one BNA modification is within the anti-repeat.

140. The gRNA of embodiment 139, wherein the at least one BNA modification is within the first stem of the anti-repeat.

141. The gRNA of embodiment 140, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat. 142. The gRNA of embodiment 140 or 141, wherein all nucleotides within the first stem of the anti-repeat comprises BNA modifications.

143. The gRNA of embodiment 139, wherein the at least one BNA modification is not within the second stem of the anti-repeat.

144. The gRNA of any one of embodiments 129-143, wherein the at least one BNA modification is not within a bulge of the gRNA.

145. The gRNA of any one of embodiments 129-144, wherein the at least one BNA modification is within the tail of the tracrRNA.

146. The gRNA of embodiment 145, wherein the three terminal nucleotides at the 3’ region of the tail of the tracrRNA comprise BNA modifications.

147. The gRNA of embodiment 145, wherein the three terminal nucleotides at the 3’ region of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

148. The gRNA of any one of embodiments 129-147, wherein at least 3 terminal nucleotides in the 3' region of the first stem of the crRNA repeat and all nucleotides in the first stem of the anti -repeat comprise BNA modifications.

149. The gRNA of any one of embodiments 129-147, wherein all nucleotides in the first stem of the crRNA repeat lack chemical modifications and all nucleotides in the first stem of the antirepeat comprise BNA modifications.

150. The gRNA of any one of embodiments 129-149, wherein the at least one BNA modification is within the spacer.

151. The gRNA of embodiment 150, wherein three terminal nucleotides at the 5 ' region of the spacer comprise BNA modifications.

152. The gRNA of embodiment 150, wherein the three terminal nucleotides at the 5' region of the spacer comprise both BNA modifications and phosphorothioate (PS) modifications.

153. The gRNA of any one of embodiments 129-152, wherein the spacer is 18-30 nucleotides in length.

154. The gRNA of any one of embodiments 129-153, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

155. The gRNA of embodiment 154, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

156. The gRNA of embodiment 154 or 155, wherein the 2', 4' BNA is a LNA modification.

157. The gRNA of embodiment 154 or 155, wherein the 2', 4' BNA is a cEt modification.

158. The gRNA of any one of embodiments 129-157, wherein the gRNA further comprises at least one other modification. 159. The gRNA of embodiment 158, wherein the at least one other modification is within the crRNA.

160. The gRNA of embodiment 158 or 159, wherein the at least one other modification is within the 5' region or the 3' region of the crRNA.

161. The gRNA of embodiment 158 or 159, wherein the at least one other modification is within the 5' region and the 3' region of the crRNA.

162. The gRNA of any one of embodiments 158-161, wherein the at least one other chemical modification is within the crRNA repeat of the crRNA.

163. The gRNA of any one of embodiments 158-162, wherein the at least one other chemical modification is within the first stem of the crRNA repeat.

164. The gRNA of any one of embodiments 158-163, wherein the at least one other chemical modification is within the spacer of the crRNA.

165. The gRNA of embodiment 158, wherein the at least one other chemical modification is within the tracrRNA.

166. The gRNA of embodiment 165, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

167. The gRNA of embodiment 165 or 166, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

168. The gRNA of embodiment 158, wherein the at least one other chemical modification is within the tail of the tracrRNA.

169. The gRNA of any one of embodiments 158-168, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.

170. The gRNA of embodiment 169, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications.

171. The gRNA of embodiment 169 or 170, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-O-Me modifications.

172. The gRNA of any one of embodiments 129-171, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

173. The gRNA of any one of embodiments 129-171, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. 174. The gRNA of any one of embodiments 129-171, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of about 11 nucleotides.

175. The gRNA of any one of embodiments 129-171, wherein the first stem of the crRNA repeat or the first stem of the anti -repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

176. The gRNA of any one of embodiments 129-171, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti-repeat at the 5’ region comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

177. The gRNA of embodiment 176, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises a GC-rich nucleotide sequence, wherein the the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

178. The gRNA of embodiment 169, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

179. The gRNA of any one of embodiments 129-178, wherein the crRNA repeat has a nucleotide sequence set forth as:

(a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides;

(b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides;

(c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides;

(d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides;

(e) SEQ ID NO: 387 or that differs from SEQ ID NO: 387 by 1 or 2 nucleotides; or

(f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides.

180. The gRNA of embodiment 179, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 2 nucleotides; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides.

181. The gRNA of embodiment 179, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 1 nucleotide; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide. 182. The gRNA of embodiment 179, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 384-387, and 397.

183. The gRNA of any one of embodiments 179-182, wherein the spacer has the sequence set forth as:

(a) SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1 to 5 nucleotides; or

(b) SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1 to 5 nucleotides.

184. The gRNA of embodiment 183, wherein the spacer has the sequence set forth as SEQ ID NO: 16 or 17.

185. The gRNA of any one of embodiments 179-184, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

186. The gRNA of embodiment 185, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

187. The gRNA of embodiment 185 or 186, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382,399- 401, and 708.

188. The gRNA of any one of embodiments 185-187, wherein the crRNA has the sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

189. The gRNA of any one of embodiments 179-188, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

190. The gRNA of embodiment 189, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

191. The gRNA of embodiment 189 or 190, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

192. The gRNA of any one of embodiments 189-191, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

193. The gRNA of any one of embodiments 129-178, wherein the crRNA repeat has a nucleotide sequence set forth as

(a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides;

(b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides;

(c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides;

(d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides;

(e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides; (f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides;

(g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides;

(h) SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides;

(i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides;

(j) SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides;

(k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides;

(l) SEQ ID NO: 389 or that differs from SEQ ID NO: 389 by 1 or 2 nucleotides; or

(m) SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides.

194. The gRNA of embodiment 193, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides.

195. The gRNA of embodiment 193, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide. 196. The gRNA of embodiment 193, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 300, 304, 308, 312, 320, 344, 348, 352, 356, 360, and 388-390.

197. The gRNA of any one of embodiments 193-196, wherein the spacer has:

(a) a nucleotide sequence set forth as SEQ ID NO: 91 or that differs from SEQ ID NO: 91 by 1 to 5 nucleotides; or

(b) a nucleotide sequence set forth as SEQ ID NO: 92 or that differs from SEQ ID NO: 92 by 1 to 5 nucleotides.

198. The gRNA of embodiment 197, wherein the spacer has the sequence set forth as SEQ ID NO: 91 or 92.

199. The gRNA of any one of embodiments 193-198, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

200. The gRNA of embodiment 199, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, S ISS IS, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

201. The gRNA of embodiment 199 or 200, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

202. The gRNA of any one of embodiments 199-201, wherein the crRNA has the sequence set forth as any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

203. The gRNA of any one of embodiments 193-202, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 80, 81, 364- 367, 369, and 375-379.

204. The gRNA of embodiment 203, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

205. The gRNA of embodiment 203 or 204, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

206. The gRNA of any one of embodiments 203-205, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

207. The gRNA of any one of embodiments 129-178, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides;

(b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides;

(c) SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides;

(d) SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides; (e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides;

(f) SEQ ID NO: 392 or that differs from SEQ ID NO: 392 by 1 or 2 nucleotides; and

(g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides.

208. The gRNA of embodiment 207, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides.

209. The gRNA of embodiment 207, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide.

210. The gRNA of embodiment 207, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 324, 328, 332, 336, and 391-393.

211. The gRNA of any one of embodiments 207-210, wherein the spacer has:

(a) a nucleotide sequence set forth as SEQ ID NO: 113 or that differs from SEQ ID NO: 113 by 1 to 5 nucleotides; or

(b) a nucleotide sequence set forth as SEQ ID NO: 114 or that differs from SEQ ID NO: 114 by 1 to 5 nucleotides, wherein, with reference to SEQ ID NO: 114.

212. The gRNA of embodiment 211, wherein the spacer has the sequence set forth as SEQ ID NO: 113 or 114.

213. The gRNA of any one of embodiments 207-212, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

214. The gRNA of embodiment 213, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

215. The gRNA of embodiment 213 or 214, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339. 216. The gRNA of any one of embodiments 213-215, wherein the crRNA has the sequence set forth as any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

217. The gRNA of any one of embodiments 213-216, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 102, 103, 370- 373, 710, and 711.

218. The gRNA of embodiment 217, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

219. The gRNA of embodiment 217 or 218, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

220. The gRNA of any one of embodiments 217-219, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

221. The gRNA of any one of embodiments 129-178, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides;

(b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides;

(c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides;

(d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides;

(e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides;

(f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides;

(g) SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides; and

(h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides.

222. The gRNA of embodiment 221, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides;

(g) a nucleotide sequence that differs from SEQ ID NO: 512 by 2 nucleotides; and

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides.

223. The gRNA of embodiment 221, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide; (e) a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide; and

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide.

224. The gRNA of embodiment 221, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 465, 469, 473, 477, 481, 508, 512, and 516.

225. The gRNA of any one of embodiments 221-224, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474- 476, 478-480, 482-484, 509-511, 513-515, and 517-519.

226. The gRNA of embodiment 225, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482- 484, 509-511, 513-515, and 517-519.

227. The gRNA of embodiment 225 or 226, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478- 480, 482-484, 509-511, 513-515, and 517-519.

228. The gRNA of any one of embodiments 225-227, wherein the crRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509- 511, 513-515, and 517-519.

229. The gRNA of any one of embodiments 225-228, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

230. The gRNA of embodiment 229, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

231. The gRNA of embodiment 229 or 230, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

232. The gRNA of any one of embodiments 229-231, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

233. The gRNA of any one of embodiments 129-232, wherein the crRNA and the tracrRNA are linked by a linker between 3’ terminal nucleotide of the crRNA repeat and 5’ terminal nucleotide of the anti-repeat.

234. The gRNA of embodiment 233, wherein the linker comprises an azide functional group or an alkyne functional group.

235. The gRNA of embodiment 233, wherein the linker is a polynucleotide.

236. The gRNA of embodiment 235, wherein the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG. 237. The gRNA of embodiment 235 or 236, wherein the linker has a nucleotide sequence set forth as AAAG.

238. The gRNA of any one of embodiments 235-237, wherein the gRNA is a sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and the spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat, the linker, and the tracrRNA.

239. The gRNA of embodiment 238, wherein the backbone of the sgRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

240. The gRNA of embodiment 239, wherein the backbone of the sgRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

241. The gRNA of embodiment 239 or 240, wherein the backbone of the sgRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

242. The gRNA of any one of embodiments 239-241, wherein the backbone of the sgRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 35-37, 296, and 297.

243. The gRNA of embodiment 238, wherein the sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

244. The gRNA of any one of embodiments 129-243, wherein the gRNA is capable of binding to an RGN.

245. The gRNA of embodiment 244, wherein the RGN is a Type II RGN.

246. The gRNA of embodiment 244 or 245, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

247. The gRNA of embodiment 246, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

248. The gRNA of embodiment 246 or 247, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

249. The gRNA of any one of embodiments 246-248, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 1.

250. The gRNA of embodiment 244 or 245, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

251. The gRNA of embodiment 250, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 69.

252. The gRNA of embodiment 250 or 251, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69.

253. The gRNA of any one of embodiments 250-252, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 69.

254. The gRNA of embodiment 244 or 245, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 93. 255. The gRNA of embodiment 254, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93.

256. The gRNA of embodiment 254 or 255, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 93.

257. The gRNA of any one of embodiments 254-256, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 93.

258. The gRNA of embodiment 244 or 245, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 252.

259. The gRNA of embodiment 258, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 252.

260. The gRNA of embodiment 258 or 259, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 252.

261. The gRNA of any one of embodiments 258-260, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 252.

262. The gRNA of any one of embodiments 129-261, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

263. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BN A) modification.

264. The gRNA of embodiment 263, wherein the gRNA is a single guide RNA (sgRNA).

265. The gRNA of embodiment 264, wherein the sgRNA comprises a total length of 100- 120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

266. The gRNA of embodiment 263, wherein the gRNA is a dual guide RNA (dgRNA).

267. The gRNA of any one of embodiments 263-266, wherein the at least one BNA modification is within the crRNA repeat.

268. The gRNA of any one of embodiments 263-267, wherein the at least one BNA modification is within the first stem of the crRNA repeat.

269. The gRNA of any one of embodiments 263-268, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications.

270. The gRNA of any one of embodiments 263-268, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications and phosphorothioate (PS) modifications. 271. The gRNA of any one of embodiments 263-270, wherein the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat.

272. The gRNA of any one of embodiments 263-271, wherein the at least one BNA modification is not within the second stem of the crRNA repeat.

273. The gRNA of any one of embodiments 263-272, wherein the at least one BNA modification is within the anti-repeat.

274. The gRNA of embodiment 273, wherein the at least one BNA modification is within the first stem of the anti-repeat.

275. The gRNA of embodiment 274, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat.

276. The gRNA of embodiment 274 or 275, wherein all nucleotides within the first stem of the anti-repeat comprises BNA modifications.

277. The gRNA of embodiment 273, wherein the at least one BNA modification is not within the second stem of the anti-repeat.

278. The gRNA of any one of embodiments 263-277, wherein the at least one BNA modification is not within a bulge of the gRNA.

279. The gRNA of any one of embodiments 263-278, wherein the at least one BNA modification is within the tail of the tracrRNA.

280. The gRNA of embodiment 279, wherein the three terminal nucleotides at the 3’ region of the tail of the tracrRNA comprise BNA modifications.

281. The gRNA of embodiment 279, wherein the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

282. The gRNA of any one of embodiments 263-281, wherein at least 3 terminal nucleotides in the 3' region of the first stem of the crRNA repeat and all nucleotides in the first stem of the anti -repeat comprise BNA modifications.

283. The gRNA of any one of embodiments 263-281, wherein all nucleotides in the first stem of the crRNA repeat lack chemical modifications and all nucleotides in the first stem of the antirepeat comprise BNA modifications.

284. The gRNA of any one of embodiments 263-283, wherein the at least one BNA modification is within the spacer.

285. The gRNA of embodiment 284, wherein three terminal nucleotides at the 5' region of the spacer comprise BNA modifications. 286. The gRNA of embodiment 284, wherein the three terminal nucleotides at the 5' region of the spacer comprise both BNA modifications and phosphorothioate (PS) modifications.

287. The gRNA of any one of embodiments 263-286, wherein the spacer is 18-30 nucleotides in length.

288. The gRNA of any one of embodiments 263-287, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

289. The gRNA of embodiment 288, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

290. The gRNA of embodiment 288 or 289, wherein the 2', 4' BNA is a LNA modification.

291. The gRNA of embodiment 288 or 289, wherein the 2', 4' BNA is a cEt modification.

292. The gRNA of any one of embodiments 263-291, wherein the gRNA further comprises at least one other modification.

293. The gRNA of embodiment 292, wherein the at least one other modification is within the crRNA.

294. The gRNA of embodiment 292 or 293, wherein the at least one other modification is within the 5' region or the 3' region of the crRNA.

295. The gRNA of embodiment 292 or 293, wherein the at least one other modification is within the 5' region and the 3' region of the crRNA.

296. The gRNA of any one of embodiments 292-295, wherein the at least one other chemical modification is within the crRNA repeat of the crRNA.

297. The gRNA of any one of embodiments 292-296, wherein the at least one other chemical modification is within the first stem of the crRNA repeat.

298. The gRNA of any one of embodiments 292-297, wherein the at least one other chemical modification is within the spacer of the crRNA.

299. The gRNA of embodiment 292, wherein the at least one other chemical modification is within the tracrRNA.

300. The gRNA of embodiment 299, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

301. The gRNA of embodiment 299 or 300, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

302. The gRNA of embodiment 292, wherein the at least one other chemical modification is within the tail of the tracrRNA.

303. The gRNA of any one of embodiments 292-302, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.

304. The gRNA of embodiment 303, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications.

305. The gRNA of embodiment 303 or 304, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-O-Me modifications.

306. The gRNA of any one of embodiments 263-305, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

307. The gRNA of any one of embodiments 263-305, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

308. The gRNA of any one of embodiments 263-305, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of about 11 nucleotides.

309. The gRNA of any one of embodiments 263-305, wherein the first stem of the crRNA repeat or the first stem of the anti -repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

310. The gRNA of any one of embodiments 263-305, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti-repeat at the 5’ region comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

311. The gRNA of embodiment 310, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises a GC-rich nucleotide sequence, wherein the the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

312. The gRNA of embodiment 303, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

313. The gRNA of any one of embodiments 263-312, wherein the crRNA repeat has a nucleotide sequence set forth as:

(a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides;

(b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides;

(c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides;

(d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides;

(f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides. 314. The gRNA of embodiment 313, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 2 nucleotides; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides.

315. The gRNA of embodiment 313, wherein the crRNA repeat has :

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 1 nucleotide; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide.

316. The gRNA of embodiment 313, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 39, 384-387, and 397.

317. The gRNA of any one of embodiments 313-316, wherein the spacer has the sequence set forth as:

(a) SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1 to 5 nucleotides; or

(b) SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1 to 5 nucleotides.

318. The gRNA of embodiment 317, wherein the spacer has the sequence set forth as SEQ ID NO: 16 or 17.

319. The gRNA of any one of embodiments 313-318, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

320. The gRNA of embodiment 319, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

321. The gRNA of embodiment 319 or 320, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382,399- 401, and 708.

322. The gRNA of any one of embodiments 319-321, wherein the crRNA has the sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

323. The gRNA of any one of embodiments 313-322, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713. 324. The gRNA of embodiment 323, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

325. The gRNA of embodiment 323 or 324, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

326. The gRNA of any one of embodiments 323-325, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

327. The gRNA of any one of embodiments 263-312, wherein the crRNA repeat has a nucleotide sequence set forth as

(a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides;

(b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides;

(c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides;

(d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides;

(e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides;

(f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides;

(g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides;

(h) SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides;

(i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides;

(j) SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides;

(k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides;

(m) SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides.

328. The gRNA of embodiment 327, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides.

329. The gRNA of embodiment 327, wherein the crRNA repeat has: (a) a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide.

330. The gRNA of embodiment 327, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 300, 304, 308, 312, 320, 344, 348, 352, 356, 360, and 388-390.

331. The gRNA of any one of embodiments 327-330, wherein the spacer has:

332. The gRNA of embodiment 331, wherein the spacer has the sequence set forth as SEQ ID NO: 91 or 92.

333. The gRNA of any one of embodiments 327-332, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

334. The gRNA of embodiment 333, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, S ISS IS, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

335. The gRNA of embodiment 333 or 334, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

336. The gRNA of any one of embodiments 333-335, wherein the crRNA has the sequence set forth as any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

337. The gRNA of any one of embodiments 327-336, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 80, 81, 364- 367, 369, and 375-379. 338. The gRNA of embodiment 337, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

339. The gRNA of embodiment 337 or 338, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

340. The gRNA of any one of embodiments 337-339, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

341. The gRNA of any one of embodiments 263-312, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides;

(b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides;

(c) SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides;

(d) SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides;

(e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides;

(g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides.

342. The gRNA of embodiment 341, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides.

343. The gRNA of embodiment 341, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide.

344. The gRNA of embodiment 341, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 324, 328, 332, 336, and 391-393.

345. The gRNA of any one of embodiments 341-344, wherein the spacer has:

(a) a nucleotide sequence set forth as SEQ ID NO: 113 or that differs from SEQ ID NO: 113 by 1 to 5 nucleotides; or (b) a nucleotide sequence set forth as SEQ ID NO: 114 or that differs from SEQ ID NO: 114 by 1 to 5 nucleotides, wherein, with reference to SEQ ID NO: 114.

346. The gRNA of embodiment 345, wherein the spacer has the sequence set forth as SEQ ID NO: 113 or 114.

347. The gRNA of any one of embodiments 341-346, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

348. The gRNA of embodiment 347, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

349. The gRNA of embodiment 347 or 348, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

350. The gRNA of any one of embodiments 347-349, wherein the crRNA has the sequence set forth as any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

351. The gRNA of any one of embodiments 347-350, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 102, 103, 370- 373, 710, and 711.

352. The gRNA of embodiment 351, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

353. The gRNA of embodiment 351 or 352, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

354. The gRNA of any one of embodiments 351-353, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

355. The gRNA of any one of embodiments 263-312, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides;

(b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides;

(c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides;

(d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides;

(e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides;

(f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides;

(h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides.

356. The gRNA of embodiment 355, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides; (b) a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides;

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides.

357. The gRNA of embodiment 355, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide; and

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide.

358. The gRNA of embodiment 355, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 465, 469, 473, 477, 481, 508, 512, and 516.

359. The gRNA of any one of embodiments 355-358, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474- 476, 478-480, 482-484, 509-511, 513-515, and 517-519.

360. The gRNA of embodiment 359, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482- 484, 509-511, 513-515, and 517-519.

361. The gRNA of embodiment 359 or 360, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478- 480, 482-484, 509-511, 513-515, and 517-519.

362. The gRNA of any one of embodiments 359-361, wherein the crRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509- 511, 513-515, and 517-519.

363. The gRNA of any one of embodiments 359-362, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

364. The gRNA of embodiment 363, wherein the tracrRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537. 365. The gRNA of embodiment 363 or 364, wherein the tracrRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

366. The gRNA of any one of embodiments 363-365, wherein the tracrRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

367. The gRNA of any one of claims 263-366, wherein the crRNA and the tracrRNA are linked by a linker between 3 ’ terminal nucleotide of the crRNA repeat and 5 ’ terminal nucleotide of the anti-repeat.

368. The gRNA of claim 367, wherein the linker comprises an azide functional group or an alkyne functional group.

369. The gRNA of claim 367, wherein the linker is a polynucleotide.

370. The gRNA of embodiment 369, wherein the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG.

371. The gRNA of embodiment 369 or 370, wherein the linker has a nucleotide sequence set forth as AAAG.

372. The gRNA of any one of embodiments 369-371, wherein the gRNA is a sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and the spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat, the linker, and the tracrRNA.

373. The gRNA of embodiment 372, wherein the backbone of the sgRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

374. The gRNA of embodiment 373, wherein the backbone of the sgRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

375. The gRNA of embodiment 373 or 374, wherein the backbone of the sgRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

376. The gRNA of any one of embodiments 373-375, wherein the backbone of the sgRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 35-37, 296, and 297.

377. The gRNA of embodiment 372, wherein the sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

378. The gRNA of any one of embodiments 263-377, wherein the gRNA is capable of binding to an RGN.

379. The gRNA of embodiment 378, wherein the RGN is a Type II RGN.

380. The gRNA of embodiment 378 or 379, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

381. The gRNA of embodiment 380, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. 382. The gRNA of embodiment 380 or 381, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

383. The gRNA of any one of embodiments 380-382, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 1.

384. The gRNA of embodiment 378 or 379, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

385. The gRNA of embodiment 384, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 69.

386. The gRNA of embodiment 384 or 385, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69.

387. The gRNA of any one of embodiments 384-386, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 69.

388. The gRNA of embodiment 378 or 379, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 93.

389. The gRNA of embodiment 388, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93.

390. The gRNA of embodiment 388 or 389, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 93.

391. The gRNA of any one of embodiments 388-390, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 93.

392. The gRNA of embodiment 378 or 379, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 252.

393. The gRNA of embodiment 392, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 252.

394. The gRNA of embodiment 392 or 393, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 252.

395. The gRNA of any one of embodiments 392-394, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 252.

396. The gRNA of any one of embodiments 263-395, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

397. A nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising:

(a) a spacer; and

(b) a crRNA repeat, wherein the crRNA repeat is capable of hybridizing to an anti -repeat of a tracrRNA to form a guide RNA (gRNA) comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-0-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'- fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'- O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA.

398. The nucleic acid molecule of embodiment 397, wherein the at least one chemical modification is a BNA modification.

399. The nucleic acid molecule of embodiment 398, wherein the BNA modification is a 2', 4' BNA modification.

400. The nucleic acid molecule of embodiment 399, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

401. The nucleic acid molecule of embodiment 399 or 400, wherein the 2', 4' BNA is a LNA modification.

402. The nucleic acid molecule of embodiment 399 or 400, wherein the 2', 4' BNA is a cEt modification.

403. The nucleic acid molecule of any one of embodiments 397-402, wherein the three terminal nucleotides at the 3' region of the crRNA comprise BNA modifications.

404. The nucleic acid molecule of any one of embodiments 397-402, wherein the three terminal nucleotides at the 3' region of the crRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

405. The nucleic acid molecule of any one of embodiments 397-402, wherein the three terminal nucleotides at the 5' region of the crRNA comprise BNA modifications.

406. The nucleic acid molecule of any one of embodiments 397-402, wherein the three terminal nucleotides at the 5' region of the crRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

407. The nucleic acid molecule of any one of embodiments 397-402, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

408. The nucleic acid molecule of embodiment 397, wherein the at least one chemical modification is a MS modification.

409. The nucleic acid molecule of embodiment 408, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications. 410. The nucleic acid molecule of embodiment 408 or 409, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-O-Me modifications.

411. The nucleic acid molecule of any one of embodiments 397-410, wherein the spacer is 18-30 nucleotides in length.

412. The nucleic acid molecule of any one of embodiments 397-411, wherein the first stem of the crRNA repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

413. The nucleic acid molecule of any one of embodiments 397-411, wherein the first stem of the crRNA repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

414. The nucleic acid molecule of any one of embodiments 397-411, wherein the first stem of the crRNA repeat comprises a total length of about 11 nucleotides.

415. The nucleic acid molecule of any one of embodiments 397-411, wherein the first stem of the crRNA repeat comprises atotal length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

416. The nucleic acid molecule of any one of embodiments 397-411, wherein the 3' region of the crRNA comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

417. The nucleic acid molecule of embodiment 416 wherein the 3' region of the crRNA comprises a GC-rich nucleotide sequence, and wherein the 3' region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

418. The nucleic acid molecule of any one of embodiments 397-417, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides;

(b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides;

(c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides;

(d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides;

(f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides.

419. The nucleic acid molecule of embodiment 418, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 2 nucleotides; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides. 420. The nucleic acid molecule of embodiment 418, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 1 nucleotide; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide.

421. The nucleic acid molecule of embodiment 418, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 39, and 384-387.

422. The nucleic acid molecule of any one of embodiments 418-421, wherein the spacer has:

(a) SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1 to 5 nucleotides; or

(b) SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1 to 5 nucleotides.

423. The nucleic acid molecule of embodiment 422, wherein the spacer has the sequence set forth as SEQ ID NO: 16 or 17.

424. The nucleic acid molecule of any one of embodiments 418-423, wherein the crRNA comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4- 9, 42-44, 292, 293, 380-382, 399-401, and 708.

425. The nucleic acid molecule of embodiment 424, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

426. The nucleic acid molecule of embodiment 424 or 425, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

427. The nucleic acid molecule of any one of embodiments 424-426, wherein the crRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399- 401, and 708.

428. The nucleic acid molecule of any one of embodiments 397-417, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides;

(b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides;

(c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides;

(d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides;

(e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides;

(f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides;

(g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides; (h) SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides;

(i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides;

(j) SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides;

(k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides;

(m) SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides.

429. The nucleic acid molecule of embodiment 428, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides.

430. The nucleic acid molecule of embodiment 428, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide.

431. The nucleic acid molecule of embodiment 428, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 300, 304, 308, 312, 320, 344, 348, 352, 356, 360, and 388-390. 432. The nucleic acid molecule of any one of embodiments 428-431, wherein the spacer has:

(b) a nucleotide sequence set forth as SEQ ID NO: 92 or that differs from SEQ ID NO: 92 by 1 to 5 nucleotides, wherein, with reference to SEQ ID NO: 92.

433. The nucleic acid molecule of embodiment 432, wherein the spacer has the sequence set forth as SEQ ID NO: 91 or 92.

434. The nucleic acid molecule of any one of embodiments 428-433, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

435. The nucleic acid molecule of embodiment 434, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

436. The nucleic acid molecule of embodiment 434 or 435, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 73-75, 301- 303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

437. The nucleic acid molecule of any one of embodiments 428-436, wherein the crRNA has the sequence set forth as any of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321- 323, 345-347, 349-351, 353-355, 357-359, and 361-363.

438. The nucleic acid molecule of any one of embodiments 397-417, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides;

(b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides;

(c) SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides;

(d) SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides;

(e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides;

(g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides.

439. The nucleic acid molecule of embodiment 438, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides. 440. The nucleic acid molecule of embodiment 438, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide.

441. The nucleic acid molecule of embodiment 438, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 324, 328, 332, 336, and 391-393.

442. The nucleic acid molecule of any one of embodiments 438-441, wherein the spacer has:

(b) a nucleotide sequence set forth as SEQ ID NO: 114 or that differs from SEQ ID NO: 114 by 1 to 5 nucleotides.

443. The nucleic acid molecule of embodiment 442, wherein the spacer has the sequence set forth as SEQ ID NO: 113 or 114.

444. The nucleic acid molecule of any one of embodiments 438-443, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

445. The nucleic acid molecule of embodiment 444, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

446. The nucleic acid molecule of embodiment 444 or 445, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 97-99, 325- 327, 329-331, 333-335, and 337-339.

447. The nucleic acid molecule of any one of embodiments 438-446, wherein the crRNA has the sequence set forth as SEQ ID NO: 97-99, 325-327, 329-331, 333-335, and 337-339.

448. The nucleic acid molecule of any one of embodiments 397-417, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides;

(b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides;

(c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides;

(d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides;

(e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides;

(f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides; (g) SEQ ID NO: 512 or that differs from SEQ ID NO: 512 by 1 or 2 nucleotides; and

(h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides.

449. The nucleic acid molecule of embodiment 448, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides;

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides.

450. The nucleic acid molecule of embodiment 448, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide; and

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide.

451. The nucleic acid molecule of embodiment 448, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 465, 469, 473, 477, 481, 508, 512, and 516.

452. The nucleic acid molecule of any one of embodiments 448-451, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.

453. The nucleic acid molecule of embodiment 452, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474- 476, 478-480, 482-484, 509-511, 513-515, and 517-519.

454. The nucleic acid molecule of embodiment 452 or 453, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 466-468, 470- 472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.

455. The nucleic acid molecule of any one of embodiments 452-454, wherein the crRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.

456. The nucleic acid molecule of any one of embodiments 397-455, wherein the gRNA is capable of binding to an RNA guided nuclease (RGN). 457. The nucleic acid molecule of embodiment 456, wherein the RGN is a Type II RGN.

458. The nucleic acid molecule of embodiment 456 or 457, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

459. The nucleic acid molecule of embodiment 458, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

460. The nucleic acid molecule of embodiment 458 or 459, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

461. The nucleic acid molecule of any one of embodiments 458-460, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 1.

462. The nucleic acid molecule of embodiment 456 or 457, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

463. The nucleic acid molecule of embodiment 462, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 69.

464. The nucleic acid molecule of embodiment 462 or 463, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69.

465. The nucleic acid molecule of any one of embodiments 462-464, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 69.

466. The nucleic acid molecule of embodiment 456 or 457, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 93.

467. The nucleic acid molecule of embodiment 466, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93.

468. The nucleic acid molecule of embodiment 466 or 467, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 93.

469. The nucleic acid molecule of any one of embodiments 466-468, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 93.

470. The nucleic acid molecule of embodiment 456 or 457, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 252.

471. The nucleic acid molecule of embodiment 470, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 252.

472. The nucleic acid molecule of embodiment 470 or 471, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 252.

473. The nucleic acid molecule of any one of embodiments 470-472, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 252.

474. A nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising:

(a) a spacer; and

(b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2', 4'- di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA.

475. The nucleic acid molecule of embodiment 474, wherein the at least one chemical modification is a BNA modification.

476. The nucleic acid molecule of embodiment 475, wherein the BNA modification is a 2', 4' BNA modification.

477. The nucleic acid molecule of embodiment 476, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

478. The nucleic acid molecule of embodiment 476 or 477, wherein the 2', 4' BNA is a LNA modification.

479. The nucleic acid molecule of embodiment 476 or 477, wherein the 2', 4' BNA is a cEt modification.

480. The nucleic acid molecule of any one of embodiments 474-479, wherein the three terminal nucleotides at the 3' region of the crRNA comprise BNA modifications.

481. The nucleic acid molecule of any one of embodiments 474-479, wherein the three terminal nucleotides at the 3' region of the crRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

482. The nucleic acid molecule of any one of embodiments 474-479, wherein the three terminal nucleotides at the 5' region of the crRNA comprise BNA modifications.

483. The nucleic acid molecule of any one of embodiments 474-479, wherein the three terminal nucleotides at the 5' region of the crRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

484. The nucleic acid molecule of any one of embodiments 474-479, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

485. The nucleic acid molecule of embodiment 474, wherein the at least one chemical modification is a MS modification.

486. The nucleic acid molecule of embodiment 485, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications. 487. The nucleic acid molecule of embodiment 485 or 486, wherein the three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-0-Me modifications.

488. The nucleic acid molecule of any one of embodiments 474-487, wherein the spacer is 18-30 nucleotides in length.

489. The nucleic acid molecule of any one of embodiments 474-488, wherein the first stem of the crRNA repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

490. The nucleic acid molecule of any one of embodiments 474-488, wherein the first stem of the crRNA repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

491. The nucleic acid molecule of any one of embodiments 474-488, wherein the first stem of the crRNA repeat comprises a total length of about 11 nucleotides.

492. The nucleic acid molecule of any one of embodiments 474-488, wherein the first stem of the crRNA repeat comprises atotal length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

493. The nucleic acid molecule of any one of embodiments 474-488, wherein the 3' region of the crRNA comprises a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

494. The nucleic acid molecule of embodiment 493 wherein the 3' region of the crRNA comprises a GC-rich nucleotide sequence, and wherein the 3' region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

495. The nucleic acid molecule of any one of embodiments 474-494, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides;

(b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides;

(c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides;

(d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides;

(f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides.

496. The nucleic acid molecule of embodiment 495, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 2 nucleotides; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 2 nucleotides. 497. The nucleic acid molecule of embodiment 495, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 39 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 384 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 385 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 386 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 387 by 1 nucleotide; or

(f) a nucleotide sequence that differs from SEQ ID NO: 397 by 1 nucleotide.

498. The nucleic acid molecule of embodiment 495, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 39, and 384-387.

499. The nucleic acid molecule of any one of embodiments 495-498, wherein the spacer has:

(a) SEQ ID NO: 16 or that differs from SEQ ID NO: 16 by 1 to 5 nucleotides; or

(b) SEQ ID NO: 17 or that differs from SEQ ID NO: 17 by 1 to 5 nucleotides.

500. The nucleic acid molecule of embodiment 499, wherein the spacer has the sequence set forth as SEQ ID NO: 16 or 17.

501. The nucleic acid molecule of any one of embodiments 495-500, wherein the crRNA comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4- 9, 42-44, 292, 293, 380-382, 399-401, and 708.

502. The nucleic acid molecule of embodiment 501, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

503. The nucleic acid molecule of embodiment 501 or 502, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

504. The nucleic acid molecule of any one of embodiments 501-503, wherein the crRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399- 401, and 708.

505. The nucleic acid molecule of any one of embodiments 474-494, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides;

(b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides;

(c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides;

(d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides;

(e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides;

(f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides;

(i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides;

(j) SEQ ID NO: 360 or that differs from SEQ ID NO: 360 by 1 or 2 nucleotides;

(k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides;

(m) SEQ ID NO: 390 or that differs from SEQ ID NO: 390 by 1 or 2 nucleotides.

506. The nucleic acid molecule of embodiment 505, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 300 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 2 nucleotides;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 2 nucleotides;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 2 nucleotides;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 2 nucleotides;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 2 nucleotides;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 2 nucleotides;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 2 nucleotides;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 2 nucleotides; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 2 nucleotides.

507. The nucleic acid molecule of embodiment 505, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 300 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 304 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 308 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 312 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 320 by 1 nucleotide;

(f ) a nucleotide sequence that differs from SEQ ID NO: 344 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 348 by 1 nucleotide;

(h) a nucleotide sequence that differs from SEQ ID NO: 352 by 1 nucleotide;

(i) a nucleotide sequence or that differs from SEQ ID NO: 356 by 1 nucleotide;

(j) a nucleotide sequence or that differs from SEQ ID NO: 360 by 1 nucleotide;

(k) a nucleotide sequence that differs from SEQ ID NO: 388 by 1 nucleotide;

(l) a nucleotide sequence that differs from SEQ ID NO: 389 by 1 nucleotide; or

(m) a nucleotide sequence that differs from SEQ ID NO: 390 by 1 nucleotide.

508. The nucleic acid molecule of embodiment 505, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 300, 304, 308, 312, 320, 344, 348, 352, 356, 360, and 388-390. 509. The nucleic acid molecule of any one of embodiments 505-508, wherein the spacer has:

510. The nucleic acid molecule of embodiment 509, wherein the spacer has the sequence set forth as SEQ ID NO: 91 or 92.

511. The nucleic acid molecule of any one of embodiments 505-510, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

512. The nucleic acid molecule of embodiment 511, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

513. The nucleic acid molecule of embodiment 511 or 512, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 73-75, 301- 303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353-355, 357-359, and 361-363.

514. The nucleic acid molecule of any one of embodiments 505-513, wherein the crRNA has the sequence set forth as any of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321- 323, 345-347, 349-351, 353-355, 357-359, and 361-363.

515. The nucleic acid molecule of any one of embodiments 474-494, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 324 or that differs from SEQ ID NO: 324 by 1 or 2 nucleotides;

(b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides;

(c) SEQ ID NO: 332 or that differs from SEQ ID NO: 332 by 1 or 2 nucleotides;

(d) SEQ ID NO: 336 or that differs from SEQ ID NO: 336 by 1 or 2 nucleotides;

(e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides;

(g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides.

516. The nucleic acid molecule of embodiment 515, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 2 nucleotides; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 2 nucleotides. 517. The nucleic acid molecule of embodiment 515, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 324 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 328 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 332 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 336 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 391 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 392 by 1 nucleotide; or

(g) a nucleotide sequence that differs from SEQ ID NO: 393 by 1 nucleotide.

518. The nucleic acid molecule of embodiment 515, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 324, 328, 332, 336, and 391-393.

519. The nucleic acid molecule of any one of embodiments 515-518, wherein the spacer has:

520. The nucleic acid molecule of embodiment 519, wherein the spacer has the sequence set forth as SEQ ID NO: 113 or 114.

521. The nucleic acid molecule of any one of embodiments 515-520, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

522. The nucleic acid molecule of embodiment 521, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

523. The nucleic acid molecule of embodiment 521 or 522, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 97-99, 325- 327, 329-331, 333-335, and 337-339.

524. The nucleic acid molecule of any one of embodiments 515-523, wherein the crRNA has the sequence set forth as SEQ ID NO: 97-99, 325-327, 329-331, 333-335, and 337-339.

525. The nucleic acid molecule of any one of embodiments 474-494, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides;

(b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides;

(c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides;

(d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides;

(e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides;

(h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides.

526. The nucleic acid molecule of embodiment 525, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 2 nucleotides;

(b) a nucleotide sequence that differs from SEQ ID NO: 469 by 2 nucleotides;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 2 nucleotides;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 2 nucleotides;

(e) a nucleotide sequence that differs from SEQ ID NO: 481 by 2 nucleotides;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 2 nucleotides;

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 2 nucleotides.

527. The nucleic acid molecule of embodiment 525, wherein the crRNA repeat has:

(a) a nucleotide sequence that differs from SEQ ID NO: 465 by 1 nucleotide;

(b) a nucleotide sequence that differs from SEQ ID NO: 469 by 1 nucleotide;

(c) a nucleotide sequence that differs from SEQ ID NO: 473 by 1 nucleotide;

(d) a nucleotide sequence that differs from SEQ ID NO: 477 by 1 nucleotide;

(e) a nucleotide sequence that differs from SEQ ID NO: 481 by 1 nucleotide;

(f) a nucleotide sequence that differs from SEQ ID NO: 508 by 1 nucleotide;

(g) a nucleotide sequence that differs from SEQ ID NO: 512 by 1 nucleotide; and

(h) a nucleotide sequence that differs from SEQ ID NO: 516 by 1 nucleotide.

528. The nucleic acid molecule of embodiment 525, wherein the crRNA repeat has a nucleotide sequence set forth as any one of SEQ ID NOs: 465, 469, 473, 477, 481, 508, 512, and 516.

529. The nucleic acid molecule of any one of embodiments 525-528, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.

530. The nucleic acid molecule of embodiment 529, wherein the crRNA has a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 466-468, 470-472, 474- 476, 478-480, 482-484, 509-511, 513-515, and 517-519.

531. The nucleic acid molecule of embodiment 529 or 530, wherein the crRNA has a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 466-468, 470- 472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.

532. The nucleic acid molecule of any one of embodiments 529-531, wherein the crRNA has a nucleotide sequence set forth as any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519. 533. The nucleic acid molecule of any one of embodiments 474-532, wherein a gRNA comprising the crRNA is capable of binding to an RNA guided nuclease (RGN) that requires a tracrRNA for activity.

534. The nucleic acid molecule of embodiment 533, wherein the RGN is a Type II RGN.

535. The nucleic acid molecule of embodiment 533 or 534, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

536. The nucleic acid molecule of embodiment 535, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1.

537. The nucleic acid molecule of embodiment 535 or 536, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

538. The nucleic acid molecule of any one of embodiments 535-537, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 1.

539. The nucleic acid molecule of embodiment 533 or 534, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 69.

540. The nucleic acid molecule of embodiment 539, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 69.

541. The nucleic acid molecule of embodiment 539 or 540, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69.

542. The nucleic acid molecule of any one of embodiments 539-541, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 69.

543. The nucleic acid molecule of embodiment 533 or 534, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 93.

544. The nucleic acid molecule of embodiment 543, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 93.

545. The nucleic acid molecule of embodiment 543 or 544, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 93.

546. The nucleic acid molecule of any one of embodiments 543-545, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 93.

547. The nucleic acid molecule of embodiment 533 or 534, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 252.

548. The nucleic acid molecule of embodiment 547, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 252.

549. The nucleic acid molecule of embodiment 547 or 548, wherein the RGN comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 252.

550. The nucleic acid molecule of any one of embodiments 547-549, wherein the RGN comprises an amino acid sequence set forth as SEQ ID NO: 252. 551. An RNA-guided nuclease (RGN) system for binding a target sequence in a target nucleic acid molecule, wherein the RGN system comprises: a) the transactivating crRNA (tracrRNA) of any one of embodiments 1-128; b) a crRNA capable of hybridizing to the tracrRNA to form a guide RNA (gRNA); and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide; wherein the gRNA is capable of forming a complex with the RGN polypeptide to direct the RGN polypeptide to bind to the target sequence.

552. An RNA-guided nuclease (RGN) system for binding a target sequence in a target nucleic acid molecule, wherein the RGN system comprises: a) the gRNA of any one of embodiments 129-396; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide; wherein the gRNA is capable of forming a complex with the RGN polypeptide to direct the RGN polypeptide to bind to the target sequence.

553. An RNA-guided nuclease (RGN) system for binding a target sequence in a target nucleic acid molecule, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) of any one of embodiments 397-550; b) a tracrRNA capable of binding to the crRNA to form a guide RNA (gRNA); and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide; wherein the gRNA is capable of forming a complex with the RGN polypeptide to direct the RGN polypeptide to bind to the target sequence.

554. The RGN system of any one of embodiments 551-553, wherein the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence set forth as NNNNCC, NNGRR, NNRYA, or NGG.

555. The RGN system of any one of embodiments 551-554, wherein the gRNA is a sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

556. The RGN system of embodiment 555, wherein the RGN polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 69, 93, and 252.

557. The RGN system of embodiment 556, wherein the RGN polypeptide comprises an amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252.

558. The RGN system of any one of embodiments 551-557, wherein the RGN polypeptide and the gRNA are not found complexed to one another in nature. 559. The RGN system of any one of embodiments 551-558, wherein the target sequence is a eukaryotic target sequence.

560. The RGN system of any one of embodiments 551-559, wherein the target sequence has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

561. The RGN system of any one of embodiments 551-560, wherein the target sequence is within a cell.

562. The RGN system of any one of embodiments 551-561, wherein a complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence.

563. The RGN system of embodiment 562, wherein the cleavage generates a doublestranded break.

564. The RGN system of embodiment 562, wherein the cleavage generates a singlestranded break.

565. The RGN system of any one of embodiments 551-561, wherein the RGN polypeptide is nuclease inactive.

566. The RGN system of any one of embodiments 551-561, wherein the RGN polypeptide is a nickase.

567. The RGN system of any one of embodiments 551-561, wherein the RGN polypeptide is fused to a base-editing polypeptide.

568. The RGN system of embodiment 567, wherein the base-editing polypeptide comprises a deaminase.

569. The RGN system of any one of embodiments 551-561, wherein the RGN polypeptide is fused to a prime editing polypeptide.

570. The RGN system of embodiment 569, wherein the prime editing polypeptide comprises a DNA polymerase.

571. The RGN system of embodiment 570, wherein the DNA polymerase comprises a reverse transcriptase.

572. The RGN system of any one of embodiments 569-571, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

573. The RGN system of any one of embodiments 551-572, wherein the RGN polypeptide is fused to a detectable label.

574. The RGN system of any one of embodiments 551-573, wherein the RGN system further comprises a donor polynucleotide.

575. The RGN system of any one of embodiments 551-574, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN is an mRNA.

576. The RGN system of any one of embodiments 551-575, wherein the nucleotide sequence encoding the RGN polypeptide is operably linked to a heterologous promoter. 577. The RGN system of any one of embodiments 551-576, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is within a vector.

578. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the transactivating crRNA (tracrRNA) of any one of embodiments 1-128; b) a crRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

579. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) of any one of embodiments 397-550; b) a tracrRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

580. The RGN system of embodiment 578 or 579, wherein the crRNA and the tracrRNA form a guide RNA.

581. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the gRNA of any one of embodiments 129-396; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

582. The RGN system of any one of embodiments 578-581, wherein the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence set forth as NNNNCC, NNGRR, NNRYA, or NGG.

583. The RGN system of any one of embodiments 578-582, wherein the gRNA is a sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

584. The RGN system of embodiment 583, wherein the RGN polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 69, 93, and 252.

585. The RGN system of embodiment 584, wherein the RGN polypeptide comprises an amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252.

586. The RGN system of any one of embodiments 578-585, wherein the RGN polypeptide and the gRNA are not found complexed to one another in nature.

587. The RGN system of any one of embodiments 578-586, wherein the RGN system binds a target sequence in a target nucleic acid molecule.

588. The RGN system of any one of embodiments 578-587, wherein the target sequence is a eukaryotic target sequence.

589. The RGN system of any one of embodiments 578-588, wherein the target sequence has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712. 590. The RGN system of any one of embodiments 578-589, wherein the target sequence is within a cell.

591. The RGN system of any one of embodiments 578-590, wherein a complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence.

592. The RGN system of embodiment 591, wherein the cleavage generates a doublestranded break.

593. The RGN system of embodiment 591, wherein the cleavage generates a singlestranded break.

594. The RGN system of any one of embodiments 578-590, wherein the RGN polypeptide is nuclease inactive.

595. The RGN system of any one of embodiments 578-590, wherein the RGN polypeptide is a nickase.

596. The RGN system of any one of embodiments 578-590, wherein the RGN polypeptide is fused to a base-editing polypeptide.

597. The RGN system of embodiment 596, wherein the base-editing polypeptide comprises a deaminase.

598. The RGN system of any one of embodiments 578-590, wherein the RGN polypeptide is fused to a prime editing polypeptide.

599. The RGN system of embodiment 598, wherein the prime editing polypeptide comprises a DNA polymerase.

600. The RGN system of embodiment 599, wherein the DNA polymerase comprises a reverse transcriptase.

601. The RGN system of any one of embodiments 598-600, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

602. The RGN system of any one of embodiments 578-601, wherein the RGN polypeptide is fused to a detectable label.

603. The RGN system of any one of embodiments 578-593, wherein the RGN system further comprises a donor polynucleotide.

604. The RGN system of any one of embodiments 578-603, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN is an mRNA.

605. The RGN system of any one of embodiments 578-603, wherein the nucleotide sequence encoding the RGN polypeptide is operably linked to a heterologous promoter.

606. The RGN system of any one of embodiments 578-603, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is within a vector.

607. A ribonucleoprotein (RNP) complex comprising the RGN system of any one of embodiments 551-606. 608. A cell comprising the nucleic acid molecule comprising a tracrRNA of any one of embodiments 1-128, the gRNA of any one of embodiments 129-396, the crRNA of any one of embodiments 397-550, the RGN system of any one of embodiments 551-606, or the RNP complex of embodiment 607.

609. The cell of embodiment 608, wherein the cell comprises a target sequence capable of being bound by a formed crRNA/tracrRNA/RGN polypeptide or gRNA/RGN polypeptide complex of the RGN system of any one of embodiments 551-606, or by the RNP complex of embodiment 607.

610. The cell of embodiment 608 or 609, wherein the target sequence comprises a nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

611. The cell of any one of embodiments 608-610, wherein the cell is a prokaryotic cell.

612. The cell of any one of embodiments 608-610, wherein the cell is a eukaryotic cell.

613. The cell of embodiment 612, wherein the eukaryotic cell is a primary cell.

614. The cell of embodiment 613, wherein the primary cell is a T cell.

615. The cell of embodiment 612, wherein the eukaryotic cell is a plant cell.

616. A plant comprising the cell of embodiment 615.

617. A seed comprising the cell of embodiment 615.

618. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the tracrRNA of any one of embodiments 1-128, the gRNA of any one of embodiments 129-396, the crRNA of any one of embodiments 397-550, the RGN system of any one of embodiments 551-606, the RNP complex of embodiment 607, or the cell of any one of embodiments 608-614.

619. The pharmaceutical composition of embodiment 618, wherein the pharmaceutically acceptable carrier is not naturally-occurring.

620. A method for binding a target sequence in a target nucleic acid molecule comprising delivering the RGN system of any one of embodiments 551-606, or the RNP complex of embodiment 607 to the target sequence or to a cell comprising the target sequence.

621. The method of embodiment 620, wherein the RGN polypeptide or the gRNA further comprises a detectable label, thereby allowing for detection of the target sequence.

622. The method of embodiment 620 or 621, wherein the RGN polypeptide or the gRNA further comprises an expression modulator, thereby modulating expression of a target gene comprising the target sequence.

623. A method for cleaving and/or modifying a target nucleic acid molecule that comprises a target sequence comprising delivering the RGN system of any one of embodiments 551-606, or the RNP complex of embodiment 607 to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

624. A method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) of any one of embodiments 1-128 and a CRISPR RNA (crRNA); and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

625. The method of embodiment 624, wherein the assembled RNP complex directs cleavage of the target sequence.

626. The method of any one of embodiments 620-624, wherein the RGN is fused to a prime editing polypeptide.

627. The method of embodiment 626, wherein the prime editing polypeptide comprises a DNA polymerase.

628. The method of embodiment 627, wherein the DNA polymerase comprises a reverse transcriptase.

629. The method of any one of embodiments 626-628, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

630. The method of any one of embodiments 620-624, wherein the RGN polypeptide is fused to a base-editing polypeptide.

631. The method of embodiment 630, wherein the base-editing polypeptide comprises a deaminase.

632. A method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) of any one of embodiments 1-126 and a CRISPR RNA (crRNA); and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

633. The method of embodiment 632, wherein a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence.

634. The method of embodiment 632, wherein the RGN is fused to a prime editing polypeptide.

635. The method of embodiment 634, wherein the prime editing polypeptide comprises a DNA polymerase.

636. The method of embodiment 635, wherein the DNA polymerase comprises a reverse transcriptase. 637. The method of any one of embodiments 634-636, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

638. The method of embodiment 632, wherein the RGN polypeptide is fused to a baseediting polypeptide.

639. The method of embodiment 6338, wherein the base-editing polypeptide comprises a deaminase.

640. The method of embodiment 632, wherein the polynucleotide encoding the Type II RGN is an mRNA.

641. A method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) the guide RNA (gRNA) of any one of embodiments 127-386; and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

642. The method of embodiment 641, wherein the assembled RNP complex directs cleavage of the target sequence.

643. The method of embodiment 641, wherein the RGN polypeptide is fused to a baseediting polypeptide.

644. The method of embodiment 643, wherein the base-editing polypeptide comprises a deaminase.

645. The method of embodiment 641, wherein the RGN is fused to a prime editing polypeptide.

646. The method of embodiment 645, wherein the prime editing polypeptide comprises a DNA polymerase.

647. The method of embodiment 646, wherein the DNA polymerase comprises a reverse transcriptase.

648. The method of any one of embodiments 645-647, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

649. A method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) the guide RNA (gRNA) of any one of embodiments 127-386; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN. 650. The method of embodiment 649, wherein a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence.

651. The method of embodiment 649, wherein the RGN polypeptide is fused to a baseediting polypeptide.

652. The method of embodiment 651, wherein the base-editing polypeptide comprises a deaminase.

653. The method of embodiment 649, wherein the RGN is fused to a prime editing polypeptide.

654. The method of embodiment 653, wherein the prime editing polypeptide comprises a DNA polymerase.

655. The method of embodiment 654, wherein the DNA polymerase comprises a reverse transcriptase.

656. The method of any one of embodiments 653-655, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

657. The method of embodiment 649, wherein the polynucleotide encoding the Type II RGN is an mRNA.

658. A method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) of any one of embodiments 397-550 and a tracrRNA; and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

659. The method of embodiment 658, wherein the assembled RNP complex directs cleavage of the target sequence.

660. The method of embodiment 658, wherein the RGN polypeptide is fused to a baseediting polypeptide.

661. The method of embodiment 660, wherein the base-editing polypeptide comprises a deaminase.

662. The method of embodiment 658, wherein the RGN is fused to a prime editing polypeptide.

663. The method of embodiment 662, wherein the prime editing polypeptide comprises a DNA polymerase. 664. The method of embodiment 663, wherein the DNA polymerase comprises a reverse transcriptase.

665. The method of any one of embodiments 662-664, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

666. A method for binding a target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) of any one of embodiments 387-540 and atracrRNA; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

667. The method of embodiment 666, wherein a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence.

668. The method of embodiment 666, wherein the RGN polypeptide is fused to a baseediting polypeptide.

669. The method of embodiment 668, wherein the base-editing polypeptide comprises a deaminase.

670. The method of embodiment 666, wherein the RGN is fused to a prime editing polypeptide.

671. The method of embodiment 670, wherein the prime editing polypeptide comprises a DNA polymerase.

672. The method of embodiment 671, wherein the DNA polymerase comprises a reverse transcriptase.

673. The method of any one of embodiments 670-672, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

674. The method of embodiment 666, wherein the polynucleotide encoding the Type II RGN is an mRNA.

675. The method of any one of embodiments 620-674, wherein the target sequence comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712.

676. The method of any one of embodiments 624-675, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 69, 93, and 252.

677. The method of any one of embodiments 624-676, wherein the RGN comprises the amino acid sequence set forth as any one of SEQ ID NOs: 1, 69, 93, and 252.

678. The method of any one of embodiments 624-677, wherein the assembled RNP complex directs cleavage of the target sequence.

679. A method of increasing efficiency of cleaving and/or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering the RGN system of any one of embodiments 551-606 or the RNP complex of embodiment 607 to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering to the target sequence or to a cell comprising the target sequence a reference RGN system or RNP complex, wherein a tracrRNA, a gRNA, or a crRNA in the reference RGN system or RNP complex does not comprise a bridged nucleic acid (BNA) modification or does not comprise any chemical modification.

680. The method of embodiment 679, wherein all nucleotides of the first stem of the antirepeat of the tracrRNA of the RGN system of any one of embodiments 551-606 or the RNP complex of embodiment 607 comprise BNA modifications.

681. The method of embodiment 680, wherein at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat comprise BNA modifications.

682. The method of embodiment 681, wherein the BNA modifications comprise LNA modifications.

683. The method of embodiment 681, wherein the BNA modifications comprise cEt modifications.

684. The method of any one of embodiments 679-683, wherein the efficiency of cleaving and/or modifying the target sequence is increased by 15-fold to 30-fold.

685. The method of embodiment 684, wherein the efficiency of cleaving and/or modifying the target sequence is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.

686. The method of embodiment 685, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.

687. A method of engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat and the tracrRNA comprises an anti-repeat; and b) adding or substituting one or more nucleotides in the crRNA repeat and one or more nucleotides in the anti-repeat, wherein the one or more nucleotides added or substituted in the repeat and the one or more nucleotides added or substituted in the anti -repeat are capable of hybridizing to each other, wherein the added or substituted one or more nucleotides comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a).

688. The method of embodiment 687, wherein the one or more nucleotides are 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. 689. The method of embodiment 687 or 688, wherein the added or substituted one or more nucleotides are in the 3' region of the crRNA repeat and in the 5' region of the anti-repeat, and wherein the 3' region of the crRNA repeat and the 5' region of the anti -repeat comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

690. The method of any one of embodiments 687-689, wherein the gRNA is a dgRNA.

691. The method of any one of embodiments 687-689, wherein the gRNA is a sgRNA.

692. The method of any one of embodiments 687-691, further comprising: c) modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA modification.

693. The method of embodiment 692, wherein the at least one chemical modification is in the crRNA, the tracrRNA, or both.

694. The method of embodiment 692, wherein the at least one chemical modification is in: the crRNA repeat; the anti-repeat; a tail of the tracrRNA; the crRNA repeat and the anti-repeat; or the crRNA repeat, the anti-repeat, and the tail of the tracrRNA.

695. The method of embodiment 692, wherein the at least one chemical modification is in: a first stem of the crRNA repeat; a first stem of the anti-repeat; a tail of the tracrRNA; the first stem of the crRNA repeat and the first stem of the anti-repeat; or the first stem of the crRNA repeat, the first stem of the anti-repeat, and the tail of the tracrRNA.

696. The method of embodiment 695, wherein the at least one chemical modification is in the first stem of the anti-repeat.

697. The method of embodiment 696, wherein the at least one chemical modification is on 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in the first stem of the anti-repeat.

698. The method of embodiment 696, wherein the at least one chemical modification is on consecutive nucleotides in the first stem of the anti-repeat.

699. The method of embodiment 696, wherein the at least one chemical modification is on alternate nucleotides in the first stem of the anti-repeat.

700. The method of embodiment 696, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat.

701. The method of claim 700, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat and on at least three terminal nucleotides at the 3’ region of the first stem of the crRNA repeat.

702. The method of claim 700, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat, on at least three terminal nucleotides at the 3’ region of the first stem of the crRNA repeat, and on three terminal nucleotides at the 3’ region of the tail of the tracrRNA.

703. The method of embodiment 696, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA.

704. The method of embodiment 696, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat and on at least one nucleotide in the first stem of the crRNA repeat.

705. The method of embodiment 696, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat, on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA, and on at least one nucleotide at the 3 ’ region of the first stem of the crRNA repeat.

706. The method of any one of embodiments 687-6705, wherein the at least one chemical modification comprises a BNA modification.

707. The method of embodiment 706, wherein the BNA modification comprises a 2', 4' BNA modification.

708. The method of embodiment 707, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'- O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

709. The method of embodiment 708, wherein the 2', 4' BNA is a LNA modification.

710. The method of embodiment 708, wherein the 2', 4' BNA is a cEt modification.

711. The method of any one of embodiments 687-710, wherein the editing efficiency of the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5 -fold, 10-fold, 20-fold, 50-fold, 100-fold, or more compared to the gRNA provided in step a).

712. The method of any one of embodiments 687-711, wherein efficiency of cleaving and/or modifying a target sequence by an RGN system comprising the engineered gRNA is increased at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5 -fold, 10- fold, 20-fold, 50-fold, 100-fold, or more compared to the RGN system comprising the gRNA provided in step a).

713. The method of embodiment 712, wherein the efficiency is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.

714. The method of embodiment 713, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.

715. The method of any one of embodiments 687-714, wherein the engineered gRNA further comprises an extension comprising an edit template for prime editing.

716. An engineered gRNA produced by the method of any one of the 687-715.

717. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

718. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1. Gene editing efficiency in primary human T cells with modified dual guide RNAs (dgRNAs)

The efficiency of gene editing for different combinations of modified dual guide RNAs (dgRNAs; FIGs. 3A, 3B) were tested in primary human T cells. The dgRNAs were incubated with APG07433.1 RNA guided nuclease (RGN) at room temperature to form ribonucleoprotein (RNP) prior to delivering into the cells. This method of delivery - using the RGN as its protein form complexed with gRNA - is referred to as RNP delivery hereinafter. A total of 60 pmol of APG07433.1 protein and 60 pmol of dgRNA were used per 1 million cells. For positive controls, the same amount of single guide (sgRNA) and dgRNA were used. Note that in the control conditions, the sgRNA and dgRNA are modified with MS (a combination of 2'-O-methyl and 3' phosphorothioate modifications) at the 3 nt (herein after called 3X MS) of both 5’ and 3’ ends. The target gene in this test is the TRAC (T cell receptor alpha constant) locus; therefore, the editing can be evaluated by flow cytometry after staining the T cells with CD3 antibodies. The results demonstrate that the modifications at stem-loop 3, the stem loop closest to the 3’ end of the gRNA, abolishes editing activity, while modifications at stem loop 1, the stem loop formed by hybridization between the crRNA repeat of the crRNA and the anti-repeat of the tracrRNA, alone preserve editing ability of the dgRNA (FIG. 4).

Example 2. LNA-modified dgRNA provides similar editing levels as sgRNA

A dose-dependent analysis of modified dgRNA vs. control conditions was performed. In this experiment, 1 million primary T cells were nucleofected with different RNP amount (ranging from 30 pmole to 90 pmole) and/or with different ratio of protein to gRNA (1 :2 vs 1:6). The target gene and the editing evaluation were as described in FIG. 4. The results shown in FIG. 5 indicate that while all combinations of modified dgRNAs tested can edit the TRAC gene, the LNA-modified tracrRNAs provide higher editing than the MS modified conditions. LNA-modification increases editing efficiency compared to standard/original modification scheme of 3X MS at the 5’ and 3’ ends (labeled 3x MS @ 5' + 3' dgRNA in FIG. 5). In addition, the highest editing level detected with LNA- modified dgRNA achieves the same level as the sgRNA condition.

Example 3. LNA-modified dgRNA facilitates high rate of gene disruption with mRNA delivery method

RGN-encoding mRNA was nucleofected into the cell along with the gRNA tested. This method of delivery is referred to as mRNA delivery hereinafter. The target gene and the editing evaluation were as described in FIG. 4. In the mRNA delivery, APG07433.1 edits TRAC loci well with sgRNA but not with dgRNA using the standard modification scheme - 3X MS at the 5’ and 3’ ends (labeled as “3x MS @ 5' + 3' dgRNA” in FIG. 6) or even with the use of additional MS modifications at the first stem of stem loop 1 (labeled as “MS/PS mod” in FIG. 6). The use of LNA modifications as described herein (labeled as “LNA mod.” in FIG. 6) when the RGN-encoding mRNA is delivered with dgRNA enables similar levels of editing to use of sgRNA. FIG. 6 provides data using two different spacer sequences, Spacer Option 1 and Spacer Option 2, to demonstrate the consistency of editing effect on the TRAC loci using APG07433. 1 with different modification schemes.

Example 4. The potency of LNA-modified dgRNA in gene editing/disru ption

To compare the potency of sgRNA vs. LNA-modified dgRNA, different amounts of gRNA were tested as indicated in FIG. 7. RGN-encoding mRNA was delivered at a constant amount of 1 pg. The results demonstrate that the LNA-modified dgRNA provides a stable level of editing even after 10-fold of dilution (from 50 pmole to 5 pmole), whereas the editing from sgRNA dropped more than 50% using the same condition. Similar results were seen using two different spacer sequences, Spacer Option 1 and Spacer Option 2. Example 5. Demonstration of broad applicability of LNA modification technology for diverse gRNAs and RGNs.

LNA modification of the first stem of the anti-repeat of stem loop 1 enhances the editing efficiency of sgRNAs for two different RGN systems (FIGs. 8A and 8B) with either RNP delivery or mRNA delivery. Editing evaluation was as described in Examples 1-4. The APG07433.1 RGN system recognizes a NNNNCC consensus PAM sequence (FIG. 8A), while the APG01604 RGN system recognizes a NNGRR consensus PAM sequence (FIG. 8B). Similar results were seen using two different spacer sequences for the APG07433.1 RGN system targeting TRAC (sg_1880 and sg_l 881) and two different spacer sequences for the APG01604 RGN system targeting TRAC (sg_2275) and beta 2 microglobulin (B2M; sg_1989). Editing of the B2M target gene was evaluated by flow cytometry after staining the T cells with B2M antibodies.

Example 6. MS modification of stem loop 1 does not enhance gene editing and chemical modification of stem loop 3 abolishes gene editing.

MS modification of stem loop 1 does not enhance editing efficiency (FIGs. 9A and 9B). FIG. 9A depicts eight scenarios of 2'-0-Me and/or MS modifications in stem loop 1 and/or stem loop 3 for APG07433.1 sgRNA. None of the tested sgRNAs with 2'-0-Me and/or MS modifications enhanced sgRNA editing as compared to a control sgRNA (FIG. 9B). Chemical modification at stem loop 3 abolished sgRNA activity. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). Editing evaluation was as described in Examples 1-4.

Example 7. The amount of LNA modification of stem loop 1 correlates with gene editing efficiency in multiple RGN systems.

The amount of LNA modifications correlates with guide RNA editing efficiency for three different RGN systems (APG07433.1, FIG. 10A; APG01604, FIG. 10B; APG05586, FIG. IOC). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining of B2M (for editing of B2M target sequences) using flow cytometry as described in Examples 1-5. Different amounts of nucleotides within a region of the anti-repeat forming the first stem of stem loop 1 were LNA-modified: 1, 3, 6, or 11 LNA-modified nucleotides for APG07433.1 dgRNA; 3 or 7 LNA-modified nucleotides for APG01604 dgRNA; and 4 or 9 LNA-modified nucleotides for APG05586 dgRNA. The highest editing for each RGN system was achieved when all nucleotides within the region of the anti-repeat forming the first stem of stem loop 1 were LNA-modified. Two different spacers were tested for each RGN system: two TRAC spacers for APG07433.1; and TRAC and B2M spacers for APG01604 and APG05586. Each RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). Gene editing was more robust with RNP delivery compared to mRNA delivery for the APG01604 and APG05586 systems. These experiments also demonstrate that LNA modification of the first stem of stem loop 1 improves gene editing efficiency for a third RGN system (APG05586) recognizing aNNRYA consensus PAM sequence.

Example 8. LNA modification maintains or increases gene editing efficiency of shortened guide RNAs.

The effect of LNA modification on gene editing efficiency of shortened sgRNAs was assessed using the APG07433. 1 system (FIGs. 11A-11C). The full-length APG07433. 1 sgRNA was shortened by a combination of truncations in various regions of the sgRNA: 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1 and 6 nt deleted from the tail (-10 first stem SL1, -6 tail); 5 nt pairs (10 nt) deleted from the first stem of stem loop 1, 4 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, -4 tail, -2 first stem SL3); and 5 nucleotide (nt) pairs (10 nt) deleted from the first stem of stem loop 1, 6 nt deleted from the tail, and 1 nt pair (2 nt) deleted from the first stem of stem loop 3 (-10 first stem SL1, -6 tail, -2 first stem SL3) (FIG. 11A; top). These shortened APG07433. 1 sgRNAs have MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the sgRNA and serve as controls to assess additional chemical modifications introduced into the first stem of stem loop 1. Shortened APG07433.1 sgRNAs that additionally included LNA and MS modifications in the first stem of stem loop 1 and an extra 2’- O-Me modification on the fourth nucleotide from the 3’ end are shown at bottom of FIG. 11A. LNA and MS modifications in the first stem of stem loop 1 (labeled as “modified”) maintains or improves editing efficiency for shortened APG07433. 1 sgRNAs (FIG. 1 IB). With serial dilutions of the sgRNA, it was observed that LNA and MS modifications in the first stem of stem loop 1 (labeled as “modified shortened”) increased editing potency for shortened APG07433.1 sgRNAs having only MS modifications at the three terminal nucleotides at both the 5' region and 3' region (labeled as “3 MS shortened”) to a level comparable to full-length APG07433.1 sgRNAs having only MS modifications at the three terminal nucleotides at both the 5' region and 3' region (labeled as “3 MS full length”)(FIG. 11C). These results suggest that LNA and MS modification of the first stem of stem loop 1 may allow a reduction in gRNA length and/or concentration for efficient gene editing.

The effect of LNA modification on gene editing efficiency of shortened dgRNAs was assessed using the APG07433.1 system (FIGs. 12A and 12B). Chemical modification and shortening schemes for crRNA and tracrRNA are shown in FIG. 12A. The crRNA was shortened by 5 terminal nt at the 3' region and chemically modified with either: MS modifications at the three terminal nucleotides at the 5' and 3' regions (left; O and Q represent two exemplary TRAC spacers used), or MS modifications at the three terminal nucleotides at the 5' and 3' regions plus 2'-O-Me modifications within the crRNA repeat forming the first stem of stem loop 1 (right; P and R represent two exemplary TRAC spacers used). The tracrRNA was shortened and chemically modified in the following ways: ‘tracr(L)’, the anti -repeat forming the first stem of stem loop 1 was shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprised LNA modifications, the tail was shortened by 6 nt, and the three terminal nucleotides at the 3’ region comprised MS modifications; ‘tracr(M)’, the anti-repeat forming the first stem of stem loop 1 was shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprised LNA modifications, the tail was shortened by 4 nt, 1 nt pair (2 nt) was deleted from the first stem of stem loop 3, and the three terminal nucleotides at the 3’ region comprised MS modifications; ‘tracr(N)’, the anti-repeat forming the first stem of stem loop 1 was shortened by 5 terminal nt at the 5' region, all nucleotides of the anti-repeat forming the first stem of stem loop 1 comprised LNA modifications, the tail was shortened by 6 nt, 1 nt pair (2 nt) was deleted from the first stem of stem loop 3, and the three terminal nucleotides at the 3’ region comprised MS modifications. FIGs. 12A and 12B show that LNA modification maintains or increases gene editing efficiency for shortened APG07433.1 dgRNAs with RNP delivery. The ‘M’ shortening and chemical modification scheme performs the best for sgRNA and dgRNA.

Editing evaluation was as described in Examples 1-4. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery). RNP delivery results in more efficient editing than mRNA delivery for dgRNA. The sgRNAs and dgRNAs used two exemplary TRAC spacers.

Example 9. LNA modification of the first stem of the anti-repeat of stem loop 1 enhances the editing efficiency of guide RNAs for other RGN systems.

The effect of LNA modification of the first stem of the anti -repeat of stem loop 1 was assessed for the APG05586 system. The APG05586 system recognizes an NNRYA consensus PAM sequence. Editing evaluation was conducted as described in Examples 1-5. Two different spacer sequences (TRAC and B2M) were tested. Experiments show that LNA modification increased the potency of the sgRNA editing for APG05586 (FIG. 25).

The effect of LNA modification of the first stem of the anti -repeat of stem loop 1 are assessed in other RGN systems including APG07991. The APG07991 system recognizes a nGG consensus PAM sequence. Editing evaluation is conducted as described in Examples 1-5. At least two different spacer sequences for each RGN system are tested. Both RNP delivery and mRNA delivery are tested. Experiments show that gene editing efficiency of LNA-modified guide RNAs is enhanced for other RGN systems including APG07991.

Example 10. LNA modification maintains or increases gene editing efficiency of shortened guide RNAs for different RGN systems. The effect of LNA modification on gene editing efficiency of shortened guide RNAs for other RGN systems including APG01604, APG05586, and APG07991 are assessed. Shortening and chemical modifications of guide RNAs are conducted as described in Example 8. Editing evaluation is conducted as described in Examples 1-5. At least two different spacer sequences for each RGN system are tested. Both RNP delivery and mRNA delivery are tested. Experiments show that gene editing efficiency of LNA-modified shortened guide RNAs is maintained or enhanced for other RGN systems including APG01604, APG05586, and APG07991.

Example 11. LNA modification maintains or increases gene editing efficiency in reverse transcriptase (RT) mediated gene editing.

The utility of LNA modified dgRNAs for use in reverse transcriptase (RT) mediated gene editing (Anzalone, AV et al. Nature 2019, Nelson JW et al. Nat Biotechnol 2021, Chen PJ, et al. Cell 2021) is evaluated. Guide RNAs for RT mediated gene editing tend to be very long in length. Using an LNA-modified dgRNA has the advantage of reducing the maximum length to be synthesized. RT mediated gene editing uses a reverse transcriptase fused to an RNA guided nickase that selectively nicks the non-target strand. A primer binding site (PBS) and an RT template are fused to the gRNA. The PBS hybridizes with the nicked genomic DNA, allowing the genomic DNA to serve as a primer for reverse transcription of the RT template, which templates the desired changes. After reverse transcription of the RT template proceeds, a “flap” of DNA containing the edits is produced. Upon repair it is stably incorporated into the genome.

The 3’ end of the tracrRNA is extended to include an RT template region that provides a template (with any desired edits) for the fused RT and a PBS. Alternatively, the RT template and PBS can be fused to the 5’ end of the crRNA. The RT template is located on the 5’ end of the PBS in both cases.

Both of these configurations are evaluated in cell-based assays, with RT templates directing changes in one or several genomic base pairs. The RT mediated gene editing composition is tested by sequencing the targeted genomic site using targeted amplicon sequencing, by reversion of a stop codon that is introduced into a green fluorescent protein gene (or other fluorescent protein), by knocking out an endogenous gene, or by other means.

Example 12. LNA and LNA+PS modification at the three terminal nucleotides at the 5' region and 3' region of a guide RNA are as effective as MS modification.

The effect on gene editing efficiency by replacing MS modifications with either LNA modifications or LNA + PS modifications at the three terminal nucleotides at the 5' region and 3' region of a guide RNA was assessed using the APG07433.1 system. FIG. 13 shows the designs for testing gene editing efficiency of a shortened (‘M’ backbone, see Example 8, FIG. 11A), chemically modified APG07433.1 gRNA in a sgRNA format. Gene editing efficiencies of shortened ‘M’ APG07433.1 sgRNAs were equally effective with MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (FIGs. 14, 15).

FIG. 16A shows a design for testing gene editing efficiency of a wild-type (WT, full-length), chemically modified APG07433.1 gRNA in a dgRNA format. Gene editing efficiencies of WT APG07433.1 dgRNAs were equally effective with MS, LNA, or LNA+PS modifications at the three terminal nucleotides at the 5' region and 3' region of the sgRNA (FIG. 16B). All tested dgRNAs had LNA modifications at all nucleotides of the anti-repeat forming the first stem of stem loop 1.

Therefore, these experiments show that LNA or LNA+PS at the three terminal nucleotides at the 5' region and 3' region of a gRNA, in both sgRNA and dgRNA formats, can replace MS modifications for efficient gene editing.

Editing evaluation was as described in Examples 1-4. The RGN was delivered as a protein complexed with guide RNA (RNP delivery) or as mRNA encoding the RGN (mRNA delivery).

Example 13. Gene editing efficiency is improved by lengthening the first stem of stem loop 1 for RGN systems having dgRNAs with less than 11 nucleotide pairs in the first stem of stem loop 1 and modifying all nucleotides in the lengthened first stem of the anti-repeat with LNA.

Gene editing efficiency of a dgRNA using mRNA delivery of RGN is enhanced in RGN systems such as the APG07433.1 system (see Example 7, FIG. 10A) by LNA modification of nucleotides in the first stem of the anti-repeat. By contrast, this LNA modification did not improve gene editing using mRNA delivery for RGN systems having dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1, including APG01604, APG05586, APG08167, and APG07991 systems (see FIGs. 10B, IOC, 27, and 28).

Thus, strategies to rescue gene editing for these RGN systems were explored. It was noted that the LNA-modified APG07433.1 dgRNA, which was effective for gene editing, contained 11 LNA-modified nucleotides in the first stem of the anti-repeat. Without being bound by any one theory, more LNA modifications and/or a longer first stem of stem loop 1 may contribute to the effectiveness of the APG07433.1 dgRNA.

FIGs. 17A and 17B show strategies for rescuing gene editing of RGN systems having dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1. FIG. 17A shows a strategy that includes lengthening the first stem at the end most distal to the first bubble of stem loop 1 of APG05586 dgRNA (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) by 2 nucleotide pairs using native sequence of APG05586 pre-crRNA and LNA modification of all nucleotides in the lengthened first stem of the anti -repeat. FIG. 17B shows strategies to lengthen the first stem at the end most distal to the first bubble of stem loop 1 of APG05586 dgRNA or APG08167 dgRNA (i.e. lengthening at the 3’ end of the crRNA and the 5’ end of the tracrRNA) by 2 nucleotide pairs using native sequence of the respective pre-crRNAs. Gene editing could be rescued for four RGN systems having WT (original) dgRNAs with < 11 nucleotide pairs in the first stem of stem loop 1 by lengthening the first stem at the end most distal to the first bubble of stem loop 1 (i.e. lengthening at the 3’ terminal nucleotide of the crRNA and the 5 ’ terminal nucleotide of the tracrRNA) and modifying all nucleotides of the first stem of the antirepeat with LNA (FIGs. 18, 19, and 27). Lengthening the first stem of stem loop 1 to at least 9 nucleotide pairs (with all 9 nucleotides of the first stem of the anti-repeat LNA-modified) or to at least 11 nucleotide pairs (with all 11 nucleotides of the first stem of the anti-repeat LNA-modified) could rescue gene editing (FIGs. 18 and 19). The added nucleotides could either be from native sequence of a respective pre-crRNA or the APG07433.1 gRNA sequence (FIGs. 18, 19, and 27). For the APG07991 dgRNA, lengthening the first stem of stem loop 1 to at least 11 nucleotide pairs (with all 11 nucleotides of the first stem of the anti -repeat LNA-modified) could rescue gene editing (FIG. 27). The rescue of gene editing by lengthened and LNA-modified APG07991 dgRNA was clearly demonstrated in FIG. 28, comparing gene editing efficiency of lengthened and LNA-modified APG07991 dgRNAs against WT (original) APG07991 dgRNA having 6 nucleotide pairs at the first stem of stem loop 1.

The APG07991 RGN can use SpyCas9 guide RNA for gene editing. However, SpyCas9 dgRNA has < 11 nucleotide pairs in the first stem of stem loop 1 and presents the same challenge for efficient gene editing as dgRNAs for other RGN systems discussed in this Example. Similar to what had been demonstrated for other dgRNAs, gene editing using SpyCas9 dgRNA (and APG07991 RGN delivered as mRNA) could be rescued by lengthening the first stem of stem loop 1 to at least 11 nucleotide pairs and modifying all nucleotides of the first stem of the anti-repeat with LNA (FIG. 29).

It was noted that APG07433. 1 nucleotide pairs most distal to the first bubble in the first stem of stem loop 1 (i.e. the nucleotides most proximal to the 3’ terminal nucleotide of the crRNA and the 5’ terminal nucleotide of the tracrRNA) were G:C rich (FIG. 17B), but gene editing could still be rescued by lengthening the first stem of stem loop 1 with non-G:C rich nucleotide sequences in the lengthened and modified dgRNAs tested (FIGs. 18, 19, and 27-29).

Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining of B2M (for editing of B2M target sequences) using flow cytometry as described in Examples 1-5. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

Thus, the strategies discussed in this Example allow gene editing with an RGN delivered as mRNA and using dgRNAs that had previously failed in gene editing in this context (e.g., the RGN systems discussed in this Example, Cas9, and other commercially available RGN systems). As discussed in Example 11, using an LNA-modified dgRNA has the advantage of reducing the maximum length of gRNA to be synthesized, and therefore, the strategies of this Example further enables the use of dgRNAs in RT-mediated gene editing (i.e. prime editing), where very long gRNAs in an sgRNA format would be difficult to produce. Importantly, this Example demonstrates a method to rescue gene editing of dgRNA with diverse gRNA secondary structures, as APG07433. 1, APG01604, APG05586, and APG08167 gRNAs have total three stem loops (e.g., see FIGs. 10A-10C, and 18), while APG07991 and SpyCas9 gRNAs have total four stem loops (e.g., see FIGs. 27-29).

Example 14. Nucleotide substitutions at the 3' end of the crRNA and 5' end of the tracrRNA improve gene editing efficiency for a shortened dgRNA.

A shortened ‘M’ APG07433.1 gRNA backbone provides low gene editing efficiency in a dgRNA format, where the RGN is delivered as mRNA, even with LNA modifications of nucleotides of the first stem of the anti-repeat (Example 8, FIG. 12B). Therefore, strategies were explored to improve gene editing efficiency for shortened LNA-modified dgRNAs.

FIG. 20 shows a strategy to rescue gene editing by a shortened dgRNA. Gene editing efficiency was improved for a shortened ‘M’ APG07433.1 dgRNA by substituting the 3’ 3 terminal nucleotides of the crRNA and the 5’ 2 terminal nucleotides of the tracrRNA with G nucleotides (FIGs. 20, 21). Editing evaluation was as described in Examples 1-4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

Thus, substituting nucleotide sequences of the first stem at the end distal to the first bubble of stem loop 1 with new nucleotide sequences can improve gene editing efficiency for shortened dgRNAs. Without being bound by any one theory, new nucleotide sequences engineered at the first stem at the end distal to the first bubble of stem loop 1 can include nucleotides that render stem loop 1 more stable, such as G:C rich sequences.

Example 15. LNA modification of the first stem of the repeat of a crRNA permits editing for gRNAs as long as the first stem of the anti-repeat of the tracrRNA is also LNA modified.

It has been observed that a gRNA having LNA modifications in the first stem of the antirepeat performed well in gene editing (see, e.g., Examples 1-5, 7, and 9). Strategies were explored to see if LNA modification to other parts of a gRNA improved gene editing efficiency.

FIG. 22A shows a modified APG07433.1 dgRNA that performed well in gene editing, having all 11 nucleotides LNA modified in the first stem of the anti -repeat (Tracr(J); see FIG. 10A). APG07433.1 tracrRNAs having LNA modifications at: all nucleotides in the first stem and in the second stem of the anti-repeat (Tracr(Jb)); all nucleotides in the second stem of the anti-repeat (Tracr(Jc)); or all nucleotides in the anti-repeat, including nucleotides of the first stem, the bubble, and the second stem (Tracr(Jd)), were tested for efficiency in gene editing (FIG. 22B). LNA modification of all nucleotides of the first stem of the anti-repeat in the tracrRNA of a gRNA is most effective for gene editing as compared to other LNA modification strategies for the anti-repeat (FIG. 23), consistent with what has been observed in previous Examples.

APG07433.1 dgRNAs having LNA modifications at all nucleotides of the first stem or the second stem of the crRNA repeat were tested for efficiency in gene editing (FIG. 24). It was found that LNA modification of all nucleotides of the first stem of the crRNA repeat of a gRNA was effective for gene editing as long as all nucleotides of the first stem of the anti-repeat of the gRNA were also LNA-modified (FIG. 24, top graph, right side). Having LNA modification of all nucleotides of the second stem of the crRNA repeat of a gRNA worsened gene editing for a gRNA having LNA modification of all nucleotides of the first stem of the anti -repeat (FIG. 24, bottom graph, right side). Two exemplary spacers (1880 and 1881) were used. Editing evaluation was as described in Examples 1-4. The RGN was delivered as mRNA encoding the RGN (mRNA delivery).

Example 16. The amount of LNA modification at the first stem of the anti-repeat correlates with editing efficiency and stability of a guide RNA.

It had been previously observed that the amount of LNA modification at the first stem of the anti -repeat correlated with guide RNA editing efficiency (see Example 7, FIG. 10A). The melting temperature of a DNA/tracrRNA heteroduplex, representing a hybridized crRNA repeat/anti-repeat, was determined for APG07433. 1. The melting temperature can be a measure of the stability of a hybridized double -stranded nucleic acid molecule, such as a crRNA repeat/anti-repeat hybridized molecule. The amount of LNA modification at the first stem of stem loop 1 correlates with guide RNA editing efficiency and melting temperature (Tm) of a DNA/tracrRNA anti-repeat heteroduplex. The highest editing and highest Tm were achieved when all nucleotides within the region of the antirepeat forming the first stem of stem loop 1 were LNA-modified (FIG. 26).

Determination of tracrRNA melting temperature with different amounts of LNA modification at the first stem of the anti -repeat was performed as described in Cromwell et al. (2018) Nature Communications 9: 1448. In brief, an equimolar amount of single stranded DNA containing the sequence of APG07433.1 crRNA repeat was mixed with APG07433. 1 tracrRNA in annealing buffer (30mM potassium chloride, 3mM HEPES, pH 7.0) to a final concentration of 2 pM. SYBR Green I was added to a final concentration of 0.1X. The reaction was performed in QuantStudio 3 (Thermo Fisher) with a program that first incubated the solution for 5 min at 95°C, then ramp cooled (0. EC s’¹) to 25°C for annealing of the two oligos, then heated to 95°C at O.LC s’¹. The corresponding SYBR Green signal was used to determine melting temperature.

The ability of chemical modifications at the first stem of stem loop 1 of a gRNA to stabilize the gRNA was further demonstrated by staggered delivery of mRNA encoding an RGN and a gRNA to primary T cells (FIG. 30). The deliveries included: mRNA delivery 8 hr prior to gRNA delivery; co-delivery of mRNA and gRNA; gRNA delivery 8 hr prior to mRNA delivery; and gRNA delivery 24 hr prior to mRNA delivery. As assessed by gene editing efficiency, LNA modifications of all nucleotides of the anti-repeat forming the first stem of stem loop 1 confer more stability to an sgRNA as compared to an sgRNA without the LNA modifications (FIG. 30). MS/LNA modified dgRNA enables effective gene editing with co-delivery of the mRNA and gRNA components, but is not as stable as the chemically modified sgRNAs, as demonstrated in the case of staggered delivery. Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker using flow cytometry as described in Examples 1-4.

Example 17. Chemical modification increases efficiency of guide RNAs in base editing.

The effect of chemical modifications (MS and/or LNA) on efficiency of gRNAs in base editing was assessed using fusions of an adenine deaminase and the APG07433.1 RGN (FIGs. 31 and 32). A fusion of an adenine deaminase and an RGN polypeptide is called an adenine base editor (ABE). Two types of deaminase-RGN fusions were tested: one fusion was an end-to-end fusion of a variant deaminase (SEQ ID NO: 542) with a DI 1A nickase version of APG07433.1 (SEQ ID NO: 543). The variant adenine deaminase was created through the directed evolution and optimization of the parental LPG50148 adenine deaminase (SEQ ID NO: 544). The variant adenine deaminase has improved base editing activity as compared to parental LPG50148 and has the following mutations with respect to the parental enzyme: L35N, V81S, N156R, L162W. (The parental LPG50148 and the evolved variant are disclosed in WO 2022/056254 and U.S. provisional application no. 63/382,344 (filed November 4, 2022), respectively, each of which is incorporated in its entirety by reference herein.)

Another deaminase-RGN fusion tested was an “inlaid” ABE (SEQ ID NO: 550). An inlaid ABE has an adenine deaminase inserted within the RGN. (Inlaid ABEs are disclosed in U.S. provisional application no. 63/485,642 (filed February 17, 2023), which is incorporated in its entirety by reference herein.)

Assays to measure base editing activity of ABEs were performed as described in WO 2019/236566, except that each ABE was delivered as an mRNA, and the gRNA was provided as synthetic gRNA with different chemical modifications. The mRNA and gRNA were transfected into human primary cells using standard techniques, for example with electroporation. The cells were cultured for 3 days after nucleofection. Genomic DNA was isolated using standard techniques. The editing efficiency was determined by performing next generation sequencing on the purified genomic DNA, as described in WO 2019/236566. Fifty pmole (about 2 pg) gRNA and 1 pg mRNA encoding the ABE was used in experiments for FIG. 31, while 100 pmole (about 4 pg) gRNA and 2 pg mRNA encoding the ABE was used in experiments for FIG. 32. Two exemplary TRAC spacers were used for the experiments of FIG. 32.

The following gRNAs were tested in base editing with the two types of ABEs (FIG. 31): (A) End modified sgRNA has MS modifications of the three terminal nucleotides at both the 5 ’ and 3 ’ regions of the sgRNA; (B) LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5 ’ and 3 ’ regions and LNA modifications at all nucleotides of the first stem of the anti-repeat; (C) MS/LNA modified sgRNA has MS modifications of the three terminal nucleotides at both the 5 ’ and 3 ’ regions, MS modifications of the three terminal nucleotides at the 3 ’ region of the crRNA repeat, and LNA modifications at all nucleotides of the first stem of the anti-repeat; and (D) MS/LNA modified dgRNA has MS modifications of the three terminal nucleotides at both the 5 ’ and 3’ regions of the crRNA, LNA modifications at all nucleotides of the first stem of the anti -repeat, and MS modifications of the three terminal nucleotides at the 3’ region of the tracrRNA.

It was demonstrated that LNA (or LNA and MS) modifications of the first stem of stem loop 1 maintained or increased base editing efficiency for both APG07433.1 sgRNA and dgRNA as compared to the end modified APG07433. 1 sgRNA (FIGs. 31 and 32). Moreover, LNA (or LNA and MS) modifications in the first stem of stem loop 1 maintained or increased base editing efficiency for a shortened APG07433. 1 sgRNA (FIG. 32, ‘shrt’ sgRNA in graphs). These results demonstrated that LNA (or LNA and MS) modifications of the first stem of stem loop 1 maintains or increases base editing efficiency for sgRNA, dgRNA, and shortened sgRNA using two types of ABEs.

Example 18. Guide RNAs modified with another BNA are equally effective at gene editing as compared to LNA modification.

The gene editing efficiency for two dgRNAs chemically modified with either LNA or S- constrained ethyl (cEt) at all nucleotides of the anti-repeat forming the first stem of stem loop 1 were compared: APG07433.1 dgRNA (described in Example 7 and FIG. 10A); and APG05586 dgRNA lengthened at the first stem distal to the first bubble of stem loop 1 to 11 nucleotide pairs (described in Example 13 and FIGs. 18 and 19). Gene editing efficiency was measured in primary human T cells by assessing knockout of the CD3 surface marker (for editing of TRAC target sequences) or immunostaining of B2M (for editing of B2M target sequences) using flow cytometry as described in Examples 1-5.

It was demonstrated that, for two different dgRNAs, modification (e.g., cEt) of all nucleotides at the first stem of the anti-repeat of stem loop 1 of a dgRNA with another bridged nucleic acid (BNA), cEt, was equally effective at enhancing gene editing efficiency as compared to a control dgRNA having MS modifications at the three terminal nucleotides at both the 5' region and 3' region of the crRNA and tracrRNA (3MS) but without chemical modifications elsewhere in the dgRNA (FIGs. 33 and 34).

Example 19. LNA modification improves editing efficiency of dgRNA comprising an extended tracrRNA.

Experiments were conducted to investigate whether the presence of an extension added on to a guide RNA, making the guide RNA greater in length, would interfere with guide RNA folding and/or function. The function of a dual guide RNA + 32 nt extension at the tail of the tracrRNA was assessed by measuring gene editing efficiency in a system comprising the dual guide RNA + 32 nt extension and an RGN. The original dual guide RNA comprises a 46 nt crRNA and a 79 nt tracrRNA. The extended dual guide RNA comprises a 46 nt crRNA and a 111 nt tracrRNA. A single guide RNA corresponding to the original dual guide RNA without extension was used as a control. FIG. 35 (top) shows three chemically modified dgRNAs with 32 nt extensions at the tail of the tracrRNA that were tested: (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5 ' region and at the three terminal nucleotides at the 3' region of the tracrRNA + 32 nt extension (3MS ends); (b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of stem loop 1 (i.e. the stem loop formed by hybridization of the crRNA repeat of the crRNA and the anti-repeat of the tracrRNA) on tracrRNA + 32 nt extension, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + 32 nt extension; (c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of stem loop 1 on tracrRNA + 32 nt extension, LNA modifications at 4 nucleotides of the first stem of stem loop 3 (i.e., the stem loop most proximal to the tail) of the tracrRNA + 32 nt extension, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + 32 nt extension.

As seen previously (see, e.g., FIG. 10A) dgRNA that is not LNA-modified does not have high gene editing efficiency (with delivery of the RGN as mRNA). Consistent with this, dgRNA + 32 nt extension that is not LNA-modified has low editing efficiency (FIG. 35, (a) bar) as compared to the control single guide RNA without extension. However, LNA-modified dgRNAs + 32nt extensions (FIG. 35, (b) and (c) bars) have gene editing efficiencies comparable to the control single guide RNA without extension.

Therefore, these experiments show that LNA modification can improve editing efficiency of dgRNA with a 32 nt extension, which is promising for using these long guide RNAs in gene editing.

Editing evaluation was as described in Examples 1-4. The RGN was delivered as a mRNA encoding the RGN (mRNA delivery).

Example 20. LNA modification and nucleotide engineering at 3' end of crRNA and 5' end of tracrRNA improve editing efficiency of dgRNA that comprises a shortened first stem and an extended tail.

As shown in Example 14 and FIGs. 20 and 21, engineering nucleotide substitutions (e.g., G-C rich substitutions) at the 3' end of the crRNA and 5' end of the tracrRNA improves gene editing efficiencies for shortened guide RNAs having LNA modifications at the anti-repeat forming the first stem of stem loop 1. Shorter guide backbones would be beneficial in gene editing to minimize challenges facing synthesis of very long guide RNAs, such as obtaining sufficient purity and yield of the guide RNAs. Therefore, LNA-modified, longer guide RNAs (guide RNA + 32 nt extension) having engineered, shortened backbones were tested for gene editing efficiency.

FIGs. 36A and 36B show schematics of guide RNAs + extensions to illustrate that engineering nucleotide substitutions at the 3' end of the crRNA and 5' end of the tracrRNA allows use of a shortened guide RNA backbone in a guide RNA having an extension. FIG. 36A shows a dgRNA + extension that has a 46 nt crRNA and a 111 nt tracrRNA, while FIG. 36B shows that a shortened dgRNA + extension, wherein the first stem of stem loop 1 was reduced by 5 bp and the first stem of stem loop 3 was reduced by 1 bp, has a 41 nt crRNA and a 104 nt tracrRNA. FIG. 36C shows details of the nucleotide substitutions at the 3' end of the crRNA and 5' end of the tracrRNA for the shortened dgRNA + extension. Stars indicate 3 nucleotide substitutions at the 3 ’ end of crRNA and 2 nucleotide substitutions at the 5’ end of tracrRNA. For both dgRNAs + extensions shown in FIGs. 36A and 36B, the first stem of the anti-repeat is LNA modified. A single guide RNA corresponding to the original dgRNA without 32 nt extension or shortened backbone or nucleotide engineering was used as a control.

Similar to what has been seen previously (see, e.g., FIG. 10A and FIG. 35), a dgRNA + extension having a shortened backbone engineered as described in FIG. 36C but that is not LNA- modified has low editing efficiency as compared to the control single guide RNA without extension (FIG. 37, ‘a’ in graph). However, an LNA-modified, dgRNA + extension having a shortened backbone engineered as described in FIG. 36C has gene editing efficiency comparable to the control single guide RNA without extension (FIG. 37, ‘b’ in graph).

Therefore, these experiments show that LNA modification improved editing efficiency of gRNAs with an engineered shortened backbone and extended tail.

Example 21. LNA modification of the first stem of the anti-repeat of tracrRNA is the key for editing efficiency of guide RNAs with extension.

FIG. 38 show schematics of dual guide RNAs + extensions having various chemical modifications: (a) MS modifications at the three terminal nucleotides at both the 5' region and 3' region (3MS ends) of the crRNA, and MS modifications at the three terminal nucleotides at the 5' region and at the three terminal nucleotides at the 3' region of the tracrRNA + extension (3MS ends);

(b) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension;

(c) 3MS ends for the crRNA, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of the stem loop most proximal to the tail (stem loop 3 in this system), and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (d) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, and 3MS ends for the tracrRNA + extension; (e) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the anti-repeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (f) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the anti-repeat, LNA modifications at 4 nucleotides of the first stem of stem loop 3, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension; (g) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, and 3MS ends for the tracrRNA + extension; (h) MS modifications at the three terminal nucleotides at the 5' region of the crRNA, LNA modifications at all nucleotides of the first stem of the crRNA repeat, LNA modifications at all nucleotides of the first stem of the antirepeat, and MS modifications at the three terminal nucleotides at the 3' region of the tracrRNA + extension, (g) and (h) dgRNAs + extensions have the shortened backbone engineered as described in FIG. 36C. A single guide RNA corresponding to the original dual guide RNA without extension or shortened backbone or nucleotide engineering was used as a control. dgRNAs + extensions having no LNA modifications or having LNA modifications only at the first stem of the crRNA repeat have low gene editing efficiencies (FIG. 38, (a), (d), and (g) bars in graph). However, dgRNAs + extensions having LNA modifications at the first stem of the anti -repeat have gene editing efficiencies comparable to the control single guide RNA, whether the crRNA repeat has only 3 MS modifications at the 3 ’ end or LNA modification on the entire first stem, engineered shortened or not (FIG. 38, (b), (e), and (h) bars in graph). Adding LNA modifications at the first stem of stem loop 3 did not further improve editing efficiencies once the first stem of the anti-repeat was fully LNA-modified (FIG. 38, compare (b) with (c), (e) with (f) bars in graph). Therefore, these experiments show that LNA modification of all nucleotides of the first stem of the anti-repeat of a guide RNA + extension was the key for gene editing efficiency, whether the crRNA repeat has only 3 MS modifications at the 3’ end or LNA modifications throughout the first stem. This was true for both non-engineered dgRNA + extension and engineered shortened dgRNA + extension. In summary, LNA modification can improve editing efficiency of longer guide RNAs (e.g., guide RNAs comprising more than 80, more than 100, or more than 120 nt of tracrRNA, or guide RNAs with a total length of more than 110, more than 120, more than 130, more than 140, more than 150, or more than 160 nt).

Editing evaluation was as described in Examples 1-4. The RGN was delivered as a mRNA encoding the RGN (mRNA delivery). Table 2: Sequences of the application

N: (A, C, G, U)

2'-0-Me residues: (A, C, G, U) s: phosphorothioate backbone modification

LNA (locked nucleic acid) modification: Ab, Cb, Gb, Ub

S-constrained ethyl (cEt) modification: (cetA), (cetC), (cetG), (cetU)

343

Claims

THAT WHICH IS CLAIMED:

(a) an anti-repeat;

(b) a tail; and

2. The nucleic acid molecule of claim 1, wherein the at least one BNA modification is within the anti-repeat.

3. The nucleic acid molecule of claim 1 or 2, wherein the at least one BNA modification is within the first stem of the anti-repeat.

4. The nucleic acid molecule of claim 3, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive mucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti -repeat.

5. The nucleic acid molecule of claim 3 or 4, wherein all nucleotides within the first stem of the anti -repeat comprise BNA modifications.

6. The nucleic acid molecule of claim 1 or 2, wherein the at least one BNA modification is not within the second stem of the anti -repeat.

7. The nucleic acid molecule of any one of claims 1-6, wherein the at least one BNA modification is not within a bulge of the tracrRNA.

8. The nucleic acid molecule of any one of claims 1-7, wherein three terminal nucleotides of the tail of the tracrRNA comprise BNA modifications.

9. The nucleic acid molecule of any one of claims 1-7, wherein three terminal nucleotides of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

10. The nucleic acid molecule of any one of claims 1-9, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

11. The nucleic acid molecule of claim 10, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4’-C- ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

12. The nucleic acid molecule of claim 10 or 11, wherein the 2', 4' BNA is a LNA modification.

13. The nucleic acid molecule of claim 10 or 11, wherein the 2', 4' BNA is a cEt modification.

14. The nucleic acid molecule of any one of claims 1-13, wherein the tracrRNA further comprises at least one other chemical modification.

15. The nucleic acid molecule of claim 14, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

16. The nucleic acid molecule of claim 14 or 15, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

17. The nucleic acid molecule of claim 14, wherein the at least one other chemical modification is within the tail of the tracrRNA.

18. The nucleic acid molecule of any one of claims 14-17, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O- methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di- Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.

19. The nucleic acid molecule of claim 18, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications.

20. The nucleic acid molecule of claim 18, wherein three terminal nucleotides of the tail of the tracrRNA comprise MS modifications and all nucleotides of the first stem of the anti -repeat comprise BNA modifications.

21. The nucleic acid molecule of claim 20, wherein the BNA modifications comprise LNA modifications.

22. The nucleic acid molecule of claim 20, wherein the BNA modifications comprise cEt modifications.

23. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

24. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

25. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of about 11 nucleotides.

26. The nucleic acid molecule of any one of claims 1-22, wherein the first stem of the antirepeat comprises atotal length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

27. The nucleic acid molecule of any one of claims 1-26, wherein the first stem of the antirepeat comprises at the 5' region a nucleotide sequence from a native precursor CRISPR RNA (pre-crRNA) or a GC-rich nucleotide sequence.

28. The nucleic acid molecule of claim 27, wherein the first stem of the anti -repeat comprises at the 5' region a GC-rich nucleotide sequence, wherein the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

29. The nucleic acid molecule of any one of claims 1-28, wherein the tracrRNA comprises a total length of 60-80 nt, 80-100 nt, 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, or more than 180 nt.

30. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, and 383, 709, and 713.

31. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

32. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 102, 103, and 370-373, 710, and 711.

33. The nucleic acid molecule of any one of claims 1-29, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

34. The nucleic acid molecule of any one of claims 1-33, wherein the tracrRNA is part of a gRNA that is capable of binding to an RGN.

35. The nucleic acid molecule of claim 34, wherein the RGN is a Type II RGN.

36. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

37. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

38. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93.

39. The nucleic acid molecule of claim 34 or 35, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

40. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises: i) a spacer; and ii) a crRNA repeat comprising a first stem and a second stem, wherein the tracrRNA comprises: i) a tail; and ii) an anti-repeat comprising a first stem and a second stem, and wherein at least one of the crRNA and the tracrRNA comprises at least one bridged nucleic acid (BNA) modification.

41. The gRNA of claim 40, wherein the gRNA is a single guide RNA (sgRNA).

42. The gRNA of claim 41, wherein the sgRNA comprises a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

43. The gRNA of claim 40, wherein the gRNA is a dual guide RNA (dgRNA).

44. The gRNA of any one of claims 40-43, wherein the at least one BNA modification is within the crRNA repeat.

45. The gRNA of any one of claims 40-43, wherein the at least one BNA modification is within the first stem of the crRNA repeat.

46. The gRNA of any one of claims 40-43, wherein the at least one BNA modification comprises at least two consecutive BNA modifications in the first stem of the crRNA repeat.

47. The gRNA of claim 45 or 46, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications.

48. The gRNA of claim 45 or 46, wherein three terminal nucleotides at the 3' region of the first stem of the crRNA repeat comprise BNA modifications and phosphorothioate (PS) modifications.

49. The gRNA of any one of claims 40-48, wherein the at least one BNA modification is not within the second stem of the crRNA repeat.

50. The gRNA of any one of claims 40-44, wherein the at least one BNA modification is within the anti-repeat.

51. The gRNA of claim 50, wherein the at least one BNA modification is within the first stem of the anti-repeat.

52. The gRNA of claim 51, wherein the at least one BNA modification comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen BNA modifications on consecutive nucleotides, or at least two, three, four, five, six, or seven BNA modifications on alternate nucleotides, within the first stem of the anti-repeat.

53. The gRNA of claim 51 or 52, wherein all nucleotides within the first stem of the anti-repeat comprises BNA modifications.

54. The gRNA of claim 50, wherein the at least one BNA modification is not within the second stem of the anti-repeat.

55. The gRNA of any one of claims 40-54, wherein the at least one BNA modification is not within a bulge of the gRNA.

56. The gRNA of any one of claims 40-55, wherein the at least one BNA modification is within the tail of the tracrRNA.

57. The gRNA of claim 56, wherein the three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA comprise BNA modifications.

58. The gRNA of claim 56, wherein the three terminal nucleotides at the 3’ region of the tail of the tracrRNA comprise both BNA modifications and phosphorothioate (PS) modifications.

59. The gRNA of any one of claims 40-58, wherein at least three terminal nucleotides in the 3’ region of the first stem of the crRNA repeat and all nucleotides in the first stem of the anti-repeat comprise BNA modifications.

60. The gRNA of any one of claims 40-59, wherein all nucleotides in the first stem of the crRNA repeat lack chemical modifications and all nucleotides in the first stem of the anti-repeat comprise BNA modifications.

61. The gRNA of any one of claims 40-60, wherein the at least one BNA modification is within the spacer.

62. The gRNA of claim 61, wherein three terminal nucleotides at the 5' region of the spacer comprise BNA modifications.

63. The gRNA of any one of claims 40-62, wherein the spacer is 18-30 nucleotides in length.

64. The gRNA of any one of claims 40-63, wherein the at least one BNA modification comprises a 2', 4' BNA modification.

65. The gRNA of claim 64, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA^NC[N-Me] modification, 2'-O,4'-C-ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

66. The gRNA of claim 64 or 65, wherein the 2', 4' BNA is a LNA modification.

67. The gRNA of claim 64 or 65, wherein the 2', 4' BNA is a cEt modification.

68. The gRNA of any one of claims 40-67, wherein the gRNA further comprises at least one other modification.

69. The gRNA of claim 68, wherein the at least one other modification is within the crRNA.

70. The gRNA of claim 68 or 69, wherein the at least one other modification is within the 5' region or the 3' region of the crRNA.

71. The gRNA of claim 68 or 69, wherein the at least one other modification is within the 5' region and the 3' region of the crRNA.

72. The gRNA of any one of claims 68-71, wherein the at least one other chemical modification is within the crRNA repeat of the crRNA.

73. The gRNA of any one of claims 68-72, wherein the at least one other chemical modification is within the first stem of the crRNA repeat.

74. The gRNA of any one of claims 68-73, wherein the at least one other chemical modification is within the spacer of the crRNA.

75. The gRNA of claim 68, wherein the at least one other chemical modification is within the tracrRNA.

76. The gRNA of claim 75, wherein the at least one other chemical modification is within the anti-repeat of the tracrRNA.

77. The gRNA of claim 75 or 76, wherein the at least one other chemical modification is within the first stem of the anti-repeat of the tracrRNA.

78. The gRNA of claim 75, wherein the at least one other chemical modification is within the tail of the tracrRNA.

79. The gRNA of any one of claims 68-78, wherein the at least one other chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'- O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification.

80. The gRNA of claim 79, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications.

81. The gRNA of claim 79 or 80, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, and the remaining nucleotides of the first stem of the crRNA repeat comprise 2'-0-Me modifications.

82. The gRNA of any one of claims 40-81, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

83. The gRNA of any one of claims 40-81 , wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

84. The gRNA of any one of claims 40-81, wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of about 11 nucleotides.

85. The gRNA of any one of claims 40-81 , wherein the first stem of the crRNA repeat or the first stem of the anti-repeat comprises a total length of 6-15 nucleotides, 8-13 nucleotides, or 10-12 nucleotides.

86. The gRNA of any one of claims 40-81, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises a nucleotide sequence from a native precursor CRISPR RNA (pre -crRNA) or a GC-rich nucleotide sequence.

87. The gRNA of claim 86, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti-repeat at the 5 ’ region comprises a GC-rich nucleotide sequence, wherein the first stem of the crRNA repeat at the 3' region or the first stem of the anti -repeat at the 5’ region comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

88. The gRNA of claim 79, wherein three terminal nucleotides at both the 5' region and the 3' region of the crRNA comprise MS modifications, BNA modifications, or BNA+PS modifications.

89. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as:

(a) SEQ ID NO: 39 or that differs from SEQ ID NO: 39 by 1 or 2 nucleotides;

(b) SEQ ID NO: 384 or that differs from SEQ ID NO: 384 by 1 or 2 nucleotides;

(c) SEQ ID NO: 385 or that differs from SEQ ID NO: 385 by 1 or 2 nucleotides; (d) SEQ ID NO: 386 or that differs from SEQ ID NO: 386 by 1 or 2 nucleotides;

(f) SEQ ID NO: 397 or that differs from SEQ ID NO: 397 by 1 or 2 nucleotides.

90. The gRNA of claim 89, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 4-9, 42-44, 292, 293, 380-382, 399-401, and 708.

91. The gRNA of claim 89 or 90, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 10, 12, 51-53, 294, 295, 383, 709, and 713.

92. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as

(a) SEQ ID NO: 300 or that differs from SEQ ID NO: 300 by 1 or 2 nucleotides;

(b) SEQ ID NO: 304 or that differs from SEQ ID NO: 304 by 1 or 2 nucleotides;

(c) SEQ ID NO: 308 or that differs from SEQ ID NO: 308 by 1 or 2 nucleotides;

(d) SEQ ID NO: 312 or that differs from SEQ ID NO: 312 by 1 or 2 nucleotides;

(e) SEQ ID NO: 320 or that differs from SEQ ID NO: 320 by 1 or 2 nucleotides;

(f) SEQ ID NO: 344 or that differs from SEQ ID NO: 344 by 1 or 2 nucleotides;

(g) SEQ ID NO: 348 or that differs from SEQ ID NO: 348 by 1 or 2 nucleotides;

(h) SEQ ID NO: 352 or that differs from SEQ ID NO: 352 by 1 or 2 nucleotides;

(i) SEQ ID NO: 356 or that differs from SEQ ID NO: 356 by 1 or 2 nucleotides;

(j) SEQ ID NO: 360 orthat differs from SEQ ID NO: 360 by 1 or 2 nucleotides;

(k) SEQ ID NO: 388 or that differs from SEQ ID NO: 388 by 1 or 2 nucleotides;

(l) SEQ ID NO: 389 orthat differs from SEQ ID NO: 389 by 1 or 2 nucleotides; or

(m) SEQ ID NO: 390 orthat differs from SEQ ID NO: 390 by 1 or 2 nucleotides.

93. The gRNA of claim 92, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 73-75, 301-303, 305-307, 309-311, 313-315, 321-323, 345-347, 349-351, 353- 355, 357-359, and 361-363.

94. The gRNA of claim 92 or 93, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 80, 81, 364-367, 369, and 375-379.

95. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 324 orthat differs from SEQ ID NO: 324 by 1 or 2 nucleotides;

(b) SEQ ID NO: 328 or that differs from SEQ ID NO: 328 by 1 or 2 nucleotides;

(c) SEQ ID NO: 332 orthat differs from SEQ ID NO: 332 by 1 or 2 nucleotides;

(d) SEQ ID NO: 336 orthat differs from SEQ ID NO: 336 by 1 or 2 nucleotides; (e) SEQ ID NO: 391 or that differs from SEQ ID NO: 391 by 1 or 2 nucleotides;

(g) SEQ ID NO: 393 or that differs from SEQ ID NO: 393 by 1 or 2 nucleotides.

96. The gRNA of claim 95, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 97-99, 325-327, 329-331, 333-335, and 337-339.

97. The gRNA of claim 95 or 96, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 102, 103, 370-373, 710, and 711.

98. The gRNA of any one of claims 40-88, wherein the crRNA repeat has a nucleotide sequence set forth as any one of:

(a) SEQ ID NO: 465 or that differs from SEQ ID NO: 465 by 1 or 2 nucleotides;

(b) SEQ ID NO: 469 or that differs from SEQ ID NO: 469 by 1 or 2 nucleotides;

(c) SEQ ID NO: 473 or that differs from SEQ ID NO: 473 by 1 or 2 nucleotides;

(d) SEQ ID NO: 477 or that differs from SEQ ID NO: 477 by 1 or 2 nucleotides;

(e) SEQ ID NO: 481 or that differs from SEQ ID NO: 481 by 1 or 2 nucleotides;

(f) SEQ ID NO: 508 or that differs from SEQ ID NO: 508 by 1 or 2 nucleotides;

(h) SEQ ID NO: 516 or that differs from SEQ ID NO: 516 by 1 or 2 nucleotides.

99. The gRNA of claim 98, wherein the crRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 466-468, 470-472, 474-476, 478-480, 482-484, 509-511, 513-515, and 517-519.

100. The gRNA of claim 98 or 99, wherein the tracrRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of any one of SEQ ID NOs: 499-501, 504, 505, 534, 535, and 537.

101. The gRNA of any one of claims 40-100, wherein the crRNA and the tracrRNA are linked by a linker between 3 ’ terminal nucleotide of the crRNA repeat and 5 ’ terminal nucleotide of the anti -repeat.

102. The gRNA of claim 101, wherein the linker comprises an azide functional group or an alkyne functional group.

103. The gRNA of claim 101, wherein the linker is a polynucleotide.

104. The gRNA of claim 103, wherein the linker has a nucleotide sequence set forth as AAAG, GAAA, ACUU, or CAAAGG.

105. The gRNA of claim 103 or 104, wherein the linker has a nucleotide sequence set forth as AAAG.

106. The gRNA of any one of claims 103-105, wherein the gRNA is a sgRNA comprising the crRNA and the tracrRNA, wherein the sgRNA comprises a backbone and the spacer, and wherein the backbone of the sgRNA comprises the crRNA repeat, the linker, and the tracrRNA.

107. The gRNA of claim 106, wherein the backbone of the sgRNA has a nucleotide sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 35-37, 296, and 297.

108. The gRNA of claim 106, wherein the sgRNA has the nucleotide sequence set forth as any one of SEQ ID NOs: 25-30, 60-68, 86-88, 108-110, 298, 299, and 405-407.

109. The gRNA of any one of claims 40-108, wherein the gRNA is capable of binding to an RGN.

110. The gRNA of claim 109, wherein the RGN is a Type II RGN.

111. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 1.

112. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 69.

113. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 93.

114. The gRNA of claim 109 or 110, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to SEQ ID NO: 252.

115. The gRNA of any one of claims 40-114, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

116. A nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising:

(a) a spacer; and

(b) a crRNA repeat, wherein the crRNA repeat is capable of hybridizing to an anti-repeat of a tracrRNA to form a guide RNA (gRNA) comprising a stem loop comprising a first stem and a second stem formed by hybridization of the crRNA repeat and the anti-repeat, and wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-O- Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O- m ethyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA.

117. A nucleic acid molecule comprising a CRISPR RNA (crRNA) comprising:

(a) a spacer; and

(b) a crRNA repeat comprising a first stem and a second stem, wherein the crRNA comprises at least one chemical modification, wherein the at least one chemical modification is selected from the group consisting of: 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy- ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and a BNA modification; and wherein the at least one chemical modification is within three terminal nucleotides at the 5’ region or 3’ region of the crRNA.

118. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the transactivating crRNA (tracrRNA) of any one of claims 1-39; b) a crRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

119. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the CRISPR RNA (crRNA) of claim 116 or 117; b) a tracrRNA; and c) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

120. The RGN system of claim 118 or 119, wherein the crRNA and the tracrRNA form a guide RNA.

121. An RNA-guided nuclease (RGN) system, wherein the RGN system comprises: a) the gRNA of any one of claims 40-115; and b) a Type II RGN polypeptide, or a polynucleotide comprising a nucleotide sequence encoding the Type II RGN polypeptide.

122. The RGN system of any one of claims 118-121, wherein the RGN polypeptide recognizes a consensus protospacer adjacent motif (PAM) having a nucleotide sequence set forth as NNNNCC, NNGRR, NNRYA, orNGG.

123. The RGN system of any one of claims 118-122, wherein the gRNA is a sgRNA comprising a total length of 100-120 nt, 120-140 nt, 140-160 nt, 160-180 nt, 180-200 nt, or more than 200 nt.

124. The RGN system of any one of claims 118-123, wherein the RGN polypeptide comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

125. The RGN system of any one of claims 118-124, wherein the RGN polypeptide and the gRNA are not found complexed to one another in nature.

126. The RGN system of any one of claims 118-125, wherein the RGN system binds a target sequence in a target nucleic acid molecule.

127. The RGN system of calim 126, wherein the target sequence is a eukaryotic target sequence.

128. The RGN system of claim 126 or 127, wherein the target sequence has the nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

129. The RGN system of any one of claims 126-128, wherein the target sequence is within a cell.

130. The RGN system of any one of claims 126-129, wherein a complex of the gRNA and the RGN polypeptide directs cleavage of the target sequence.

131. The RGN system of claim 130, wherein the cleavage generates a double-stranded break.

132. The RGN system of claim 130, wherein the cleavage generates a single-stranded break.

133. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is nuclease inactive.

134. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is a nickase.

135. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is fused to a base-editing polypeptide.

136. The RGN system of claim 135, wherein the base-editing polypeptide comprises a deaminase.

137. The RGN system of any one of claims 118-129, wherein the RGN polypeptide is fused to a prime editing polypeptide.

138. The RGN system of claim 137, wherein the prime editing polypeptide comprises a DNA polymerase.

139. The RGN system of claim 138, wherein the DNA polymerase comprises a reverse transcriptase.

140. The RGN system of any one of claims 137-139, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

141. The RGN system of any one of claims 118-140, wherein the RGN polypeptide is fused to a detectable label.

142. The RGN system of any one of claims 118-132, wherein the RGN system further comprises a donor polynucleotide.

143. The RGN system of any one of claims 118-142, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN is an mRNA.

144. The RGN system of any one of claims 118-142, wherein the nucleotide sequence encoding the RGN polypeptide is operably linked to a heterologous promoter.

145. The RGN system of any one of claims 118-142, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is within a vector.

146. A ribonucleoprotein (RNP) complex comprising the RGN system of any one of claims 118- 145.

147. A cell comprising the nucleic acid molecule comprising a tracrRNA of any one of claims 1- 39, the gRNA of any one of claims 40-115, the crRNA of claim 116 or 117, the RGN system of any one of claims 118-145, or the RNP complex of claim 146.

148. The cell of claim 147, wherein the cell comprises a target sequence capable of being bound by a formed crRNA/tracrRNA/RGN polypeptide or gRNA/RGN polypeptide complex of the RGN system of any one of claims 118-145, or by the RNP complex of claim 146.

149. The cell of claim 147 or 148, wherein the target sequence comprises a nucleotide sequence set forth as any of SEQ ID NOs: 273-278, and 712.

150. The cell of any one of claims 147-149, wherein the cell is a prokaryotic cell.

151. The cell of any one of claims 147-149, wherein the cell is a eukaryotic cell.

152. The cell of claim 151, wherein the eukaryotic cell is a primary cell.

153. The cell of claim 152, wherein the primary cell is a T cell.

154. The cell of claim 151, wherein the eukaryotic cell is a plant cell.

155. A plant comprising the cell of claim 154.

156. A seed comprising the cell of claim 154.

157. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the tracrRNA of any one of claims 1-39, the gRNA of any one of claims 40-115, the crRNA of claim 116 or

117, the RGN system of any one of claims 118-145, the RNP complex of claim 146, or the cell of any one of claims 147-153.

158. A method for binding a target sequence in a target nucleic acid molecule comprising delivering the RGN system of any one of claims 118-145, or the RNP complex of claim 146 to the target sequence or to a cell comprising the target sequence.

159. The method of claim 158, wherein the RGN polypeptide or the gRNA further comprises a detectable label, thereby allowing for detection of the target sequence.

160. The method of claim 158 or 159, wherein the RGN polypeptide or the gRNA further comprises an expression modulator, thereby modulating expression of a target gene comprising the target sequence.

161. A method for cleaving and/or modifying a target nucleic acid molecule that comprises a target sequence comprising delivering the RGN system of any one of claims 118-145, or the RNP complex of claim 146 to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

162. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) of any one of claims 1-39 and a CRISPR RNA (crRNA); and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

163. The method of claim 162, wherein the assembled RNP complex directs cleavage of the target sequence.

164. The method of any one of claims 158-162, wherein the RGN is fused to a prime editing polypeptide.

165. The method of claim 164, wherein the prime editing polypeptide comprises a DNA polymerase.

166. The method of claim 165, wherein the DNA polymerase comprises a reverse transcriptase.

167. The method of any one of claims 164-166, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

168. The method of any one of claims 158-162, wherein the RGN polypeptide is fused to a baseediting polypeptide.

169. The method of claim 168, wherein the base-editing polypeptide comprises a deaminase.

170. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising the transactivating crRNA (tracrRNA) of any one of claims 1-39 and a CRISPR RNA (crRNA); and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

171. The method of claim 170, wherein a formed complex of the gRNA and the Type II RGN directs cleavage of the target sequence.

172. The method of claim 170, wherein the RGN is fused to a prime editing polypeptide.

173. The method of claim 172, wherein the prime editing polypeptide comprises a DNA polymerase.

174. The method of claim 173, wherein the DNA polymerase comprises a reverse transcriptase.

175. The method of any one of claims 172-174, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

176. The method of claim 170, wherein the RGN polypeptide is fused to a base-editing polypeptide.

177. The method of claim 176, wherein the base-editing polypeptide comprises a deaminase.

178. The method of claim 170, wherein the polynucleotide encoding the Type II RGN is an mRNA.

179. A method for binding a target sequence in a target nucleic acid molecule with RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) the guide RNA (gRNA) of any one of claims 40-115; and ii) a Type II RNA-guided nuclease (RGN), thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

180. The method of claim 179, wherein the assembled RNP complex directs cleavage of the target sequence.

181. The method of claim 179, wherein the RGN polypeptide is fused to a base-editing polypeptide.

182. The method of claim 181, wherein the base-editing polypeptide comprises a deaminase.

183. The method of claim 179, wherein the RGN is fused to a prime editing polypeptide.

184. The method of claim 183, wherein the prime editing polypeptide comprises a DNA polymerase.

185. The method of claim 184, wherein the DNA polymerase comprises a reverse transcriptase.

186. The method of any one of claims 183-185, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

187. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) the guide RNA (gRNA) of any one of claims 40-115; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

188. The method of claim 187, wherein a formed complex ofthe gRNA and the Type II RGN directs cleavage of the target sequence.

189. The method of claim 187, wherein the RGN polypeptide is fused to a base-editing polypeptide.

190. The method of claim 189, wherein the base-editing polypeptide comprises a deaminase.

191. The method of claim 187, wherein the RGN is fused to a prime editing polypeptide.

192. The method of claim 191, wherein the prime editing polypeptide comprises a DNA polymerase.

193. The method of claim 192, wherein the DNA polymerase comprises a reverse transcriptase.

194. The method of any one of claims 191-193, wherein the gRNA further comprises an extension comprising an edit template for prime editing.

195. The method of claim 187, wherein the polynucleotide encoding the Type II RGN is an mRNA.

196. A method for binding a target sequence in a target nucleic acid molecule with RNA-guided nuclease (RGN), the method comprising: a) combining under conditions suitable for formation of a ribonucleoprotein (RNP) complex: i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) of claim 116 or 117 and a tracrRNA; and ii) a Type II RGN, thereby assembling an RNP complex; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RNP complex, thereby binding the target sequence with the RGN.

197. A method for binding a target sequence in a target nucleic acid molecule with an RNA- guided nuclease (RGN), the method comprising contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with i) a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) of claim 116 or 117 and a tracrRNA; and ii) a Type II RGN, or a polynucleotide encoding a Type II RGN, thereby binding the target sequence with the RGN.

198. The method of claim 197, wherein the polynucleotide encoding the Type II RGN is an mRNA.

199. The method of any one of claims 158-198, wherein the target sequence comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 273-278, and 712.

200. The method of any one of claims 162-199, wherein the RGN comprises an amino acid sequence having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity to any one of SEQ ID NOs: 1, 69, 93, or 252.

201. A method of increasing efficiency of cleaving and/or modifying a nucleic acid molecule comprising a target sequence, the method comprising delivering the RGN system of any one of claims 118- 145 or the RNP complex of claim 146 to the target sequence or to a cell comprising the target sequence, wherein cleavage or modification of the nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the nucleic acid molecule by a method comprising delivering to the target sequence or to a cell comprising the target sequence a reference RGN system or RNP complex, wherein a tracrRNA, a gRNA, or a crRNA in the reference RGN system or RNP complex does not comprise a bridged nucleic acid (BNA) modification or does not comprise any chemical modification.

202. The method of claim 201, wherein all nucleotides of the first stem of the anti-repeat of the tracrRNA of the RGN system of any one of claims 118-145 or the RNP complex of claim 146 comprise BNA modifications.

203. The method of claim 202, wherein at least three terminal nucleotides at the 3’ region of the first stem of the crRNA repeat of the crRNA comprise BNA modifications.

204. The method of claim 201, wherein the BNA modifications comprise LNA modifications.

205. The method of claim 201, wherein the BNA modifications comprise cEt modifications.

206. The method of any one of claims 201-205, wherein the efficiency of cleaving and/or modifying the target sequence is increased by 15-fold to 30-fold.

207. The method of claim 206, wherein the efficiency of cleaving and/or modifying the target sequence is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.

208. The method of claim 207, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immuno staining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.

209. A method of engineering a gRNA, the method comprising: a) providing a gRNA comprising a crRNA and a tracrRNA, wherein the crRNA comprises a crRNA repeat and the tracrRNA comprises an anti-repeat; and b) adding or substituting one or more nucleotides in the crRNA repeat and one or more nucleotides in the anti-repeat, wherein the one or more nucleotides added or substituted in the repeat and the one or more nucleotides added or substituted in the anti -repeat are capable of hybridizing to each other, wherein the added or substituted one or more nucleotides comprises at least 2, at least 3, at least 4, or at least 5 Gs or Cs, and wherein the engineered gRNA has an increased editing efficiency as compared to the gRNA provided in step a).

210. The method of claim 209, wherein the one or more nucleotides are 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides.

211. The method of claim 209 or 210, wherein the added or substituted one or more nucleotides are in the 3' region of the crRNA repeat and in the 5' region of the anti-repeat, and wherein the 3' region of the crRNA repeat and the 5' region of the anti -repeat comprise at least 2, at least 3, at least 4, or at least 5 Gs or Cs.

212. The method of any one of claims 209-211, wherein the gRNA is a dgRNA.

213. The method of any one of claims 209-211, wherein the gRNA is a sgRNA.

214. The method of any one of claims 209-213, further comprising: c) modifying at least one nucleotide in the engineered gRNA with at least one chemical modification selected from the group consisting of: 2’-O-methyl (2’-0-Me) modification; 2’-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'- O-methyl 3'phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; and BNA modification.

215. The method of claim 214, wherein the at least one chemical modification is in the crRNA, the tracrRNA, or both.

216. The method of claim 215, wherein the at least one chemical modification is in: the crRNA repeat; the anti-repeat; a tail of the tracrRNA; the crRNA repeat and the anti -repeat; or the crRNA repeat, the anti-repeat, and the tail of the tracrRNA.

217. The method of claim 215, wherein the at least one chemical modification is in: a first stem of the crRNA repeat; a first stem of the anti -repeat; a tail of the tracrRNA; the first stem of the crRNA repeat and the first stem of the anti-repeat; or the first stem of the crRNA repeat, the first stem of the anti -repeat, and the tail of the tracrRNA.

218. The method of claim 217, wherein the at least one chemical modification is in the first stem of the anti-repeat.

219. The method of claim 218, wherein the at least one chemical modification is on 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in the first stem of the anti-repeat.

220. The method of claim 218, wherein the at least one chemical modification is on consecutive nucleotides in the first stem of the anti-repeat.

221. The method of claim 218, wherein the at least one chemical modification is on alternate nucleotides in the first stem of the anti-repeat.

222. The method of claim 218, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat.

223. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA.

224. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti-repeat and on at least one nucleotide in the first stem of the crRNA repeat.

225. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat and on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat.

226. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat, on at least three terminal nucleotides at the 3 ’ region of the first stem of the crRNA repeat, and on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA.

227. The method of claim 222, wherein the at least one chemical modification is on all nucleotides in the first stem of the anti -repeat, on three terminal nucleotides at the 3 ’ region of the tail of the tracrRNA, and on at least one nucleotide at the 3’ region of the first stem of the crRNA repeat.

228. The method of any one of claims 214-227, wherein the at least one chemical modification comprises a BNA modification.

229. The method of claim 228, wherein the BNA modification comprises a 2', 4' BNA modification.

230. The method of claim 229, wherein the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C -ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.

231. The method of claim 230, wherein the 2', 4' BNA is a LNA modification.

232. The method of claim 230, wherein the 2', 4' BNA is a cEt modification.

233. The method of any one of claims 209-232, wherein efficiency of cleaving and/or modifying a target sequence by an RGN system comprising the engineered gRNA is increased at least 10%, at least

30%, at least 50%, at least 70%, at least 90%, at least 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100- fold, or more compared to the RGN system comprising the gRNA provided in step a).

234. The method of claim 233, wherein the efficiency is determined by measuring the percentage of the target sequence or cells comprising the target sequence that has altered expression of the target sequence or of a polypeptide encoded by the target sequence.

235. The method of claim 234, wherein the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immuno staining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.

236. The method of any one of claims 209-235, wherein the engineered gRNA further comprises an extension comprising an edit template for prime editing.

237. An engineered gRNA produced by the method of any one of the claims 209-236.

238. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises a total length of about 11 base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.

239. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), wherein the crRNA comprises a crRNA repeat, wherein the tracrRNA comprises an anti-repeat, wherein the gRNA comprises a stem loop comprising a first stem and a second stem, wherein the first stem comprises at least 3, 4, 5, 6, or 7 GC base pairs, and wherein the first stem comprises at least one bridged nucleic acid (BNA) modification.