RU2843259C2 - Method of capturing cleavage products of crispr endonuclease - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to molecular biology and biotechnology, in particular to a method for capturing DNA ends formed during endonuclease cleavage, such as CRISPR endonuclease cleavage, for subsequent analysis, which includes amplification and sequencing.

EFFECT: invention provides an accurate and efficient method of capturing nucleic acids split by endonucleases, such as restriction endonuclease and CRISPR endonuclease.

16 cl, 7 dwg, 3 tbl, 7 ex

Description

FIELD OF INVENTION

[001] The present invention relates to the field of nucleic acids and endonuclease-associated methods, and more particularly to methods for capturing nucleic acids for subsequent analysis, such as sequencing.

FEDERALLY SPONSORED RESEARCH STATEMENT

[002] None.

LIST OF SEQUENCES

[003] Place to fill.

LEVEL OF TECHNOLOGY

[004] Capture of nucleic acids by cloning vectors or adaptors is based on ligation. More efficient ligation involves compatible sticky ends of the nucleic acids to be ligated, i.e., the nucleic acids have sticky ends that are capable of hybridizing to the sticky ends of the ligation partner. Sticky end ligation is kinetically more favorable than blunt end ligation, where the ligation partners do not form any hybrids prior to ligation. For this reason, a common ligation method involves creating single base sticky ends by dA tailing. Enzymatic or chemical methods are used to add a dA sticky end to one of the ligation partners and a dT sticky end to the other ligation partner. dA tailing or TA cloning is used in cloning and sequencing library preparation.

[005] Although the dA tailing method is preferred due to its simplicity, it has several disadvantages. First, the incorporated A nucleotide is undesirable if the captured sequence is a coding sequence or part of a coding sequence. Second, capture via dA tailing is non-specific. All nucleic acids in the sample that have an accessible 3' end will be modified by the terminal transferase to incorporate the 3' terminal A, and thus both desired and undesired nucleic acids will become substrates for the ligation reaction.

[006] There is a need for an accurate and efficient method for capturing nucleic acids cleaved by endonucleases such as restriction endonucleases and CRISPR endonucleases.

ESSENCE OF THE INVENTION

[007] The present invention relates to a method for capturing the ends of endonuclease-cleaved nucleic acids by forming sticky 5'-ends using controlled cleavage with exonucleases such that an adapter with a sticky 5'-end will be effectively ligated. The method is an alternative to the method for producing a dA tail, which is more efficient and more specific.

[008] In some embodiments, the present invention relates to a method of capturing a nucleic acid from a sample comprising a target nucleic acid and non-target nucleic acids, comprising: contacting a double-stranded target nucleic acid having a 3' end with a catalyst having 3'-5' exonuclease activity and thereby producing a first 5' overhang at the first 5' end and a first 3' recessed end in the target nucleic acid; contacting the nucleic acid with an adapter having a second 5' overhang at a second 5' end, a second 5' overhang capable of hybridizing to the first 5' overhang, and thereby producing a hybrid between the first and second 5' overhangs; and linking at least one of the first 3' ends to the second 5' end and thereby capturing the target nucleic acid by forming an adaptor-equipped nucleic acid. In some embodiments, the adaptor further comprises a second recessed 3' end, and the linking further comprises linking the first 5' end to the second 3' end. In some embodiments, the 3' end of the double-stranded target nucleic acid is part of a blunt end. In some embodiments, the 3' end of the double-stranded target nucleic acid is part of a 3' overhang. In some embodiments, the 3' end of the double-stranded target nucleic acid is obtained by cleaving the nucleic acid with a sequence-specific endonuclease.

[009] In some embodiments, the sequence-specific endonuclease is a restriction endonuclease. In some embodiments, the sequence-specific endonuclease is a CRISPR endonuclease. In some embodiments, the sequence-specific endonuclease is a catalytically inactive CRISPR endonuclease fused to Fok I. In some embodiments, the sequence-specific endonuclease is a transcription activator-like effector nuclease (TALEN) or a TALEN-Fok I fusion protein. In some embodiments, the sequence-specific endonuclease is a zinc finger nuclease (ZFN) or a ZFN-Fok I fusion protein. In some embodiments, the catalyst having 3'-5' exonuclease activity is a DNA polymerase.

[0010] In some embodiments, the DNA polymerase having 3'-5' exonuclease activity is a family A polymerase, a family B polymerase, or a family C polymerase comprising the Exo I, Exo II, and Exo III domains of the Klenow fragment of Pol I.E. coli. In some embodiments, the DNA polymerase comprises aspartate residues corresponding to residues D355, D424, and D501 of the Pol I Klenow fragmentE. coli. In some embodiments, the DNA polymerase is selected from the group consisting of T4 DNA polymerase, RB69 DNA polymerase, Klenow fragment, T7 DNA polymerase, delta Pol IIIE. coli, eukaryotic polymerase epsilon, eukaryotic polymerase delta and mitochondrial polymerase gamma.

[0011] In some embodiments, 3'-5' exonuclease activity is demonstrated in the presence of less than four of dATP, dCTP, dGTP, and dTTP, and the contacting is with less than four of dATP, dCTP, dGTP, and dTTP, and the 3'-5' exonuclease activity is terminated at a nucleotide identical to one of less than four of dATP, dCTP, dGTP, and dTTP. In some embodiments, a single dNTP is used, and the 3'-5' exonuclease activity is terminated at a nucleotide identical to the single dNTP.

[0012] In some embodiments, the 5' sticky end is 2-15 nucleotides in length. In some embodiments, the adapter comprises at least one of a nucleic acid barcode, a sequencing primer binding site, and an amplification primer binding site. In some embodiments, the barcode is selected from a unique molecular ID (UMI) and a sample ID (SID), or both.

[0013] In some embodiments, the adapter is produced by annealing two strands of nucleic acid. In some embodiments, the adapter is produced by exonuclease cleavage of the 3' end to form an oligonucleotide with a sticky 5' end. In some embodiments, the adapter comprises a nucleic acid modification that increases the melting temperature of a nucleic acid duplex comprising the modification. In some embodiments, the adapter comprises a sequence selected from SEQ ID NO: 7-11.

[0014] In some embodiments, the linking is accomplished by ligation and is preceded by phosphorylation of the nucleic acids or adapter, i.e., using polynucleotide kinase.

[0015] In some embodiments, the method further comprises amplifying the captured nucleic acid, such as with an amplification primer that anneals to a primer binding site in the adapter. In some embodiments, the amplification primer comprises a sequence selected from SEQ ID NO: 12-16. In some embodiments, the amplification is a linear amplification. In some embodiments, the amplification is an exponential amplification by PCR, such as comprising 5, 10, 20, 30, or 40 cycles.

[0016] In some embodiments, the adapter is linked to a functional capture moiety, and the method further comprises capturing the functional capture moiety in the adapter-equipped nucleic acid and thereby capturing the nucleic acid. In some embodiments, the functional capture moiety is selected from biotin, an antigen capable of binding to a capture antibody, and a capture sequence capable of hybridizing to the capture nucleic acid.

[0017] In some embodiments, the method further comprises sequencing the captured nucleic acid. In some embodiments, the sequencing primer anneals to a primer binding site in the adapter. In some embodiments, sequencing is performed after amplification. In some embodiments, the sequencing primer anneals to a primer binding site present in the amplification primer. In some embodiments, the captured nucleic acid is 1, 10, or 100 kb in length.

[0018] In some embodiments, the method does not include intermediate purification steps.

[0019] In some embodiments, the present invention relates to a method of sequencing a target nucleic acid in a sample comprising the target nucleic acid and non-target nucleic acids, comprising: contacting a double-stranded target nucleic acid having a 3' end with a catalyst having a 3'-5' exonuclease activity, thereby producing a first 5' overhang at the first 5' end and a first 3' recessed end in the target nucleic acid; contacting the nucleic acid with an adapter having a second 5' overhang at a second 5' end, a second 5' overhang capable of hybridizing to the first 5' overhang, thereby producing a hybrid between the first and second 5' overhangs; linking at least one of the first 3'-end to the second 5'-end and thereby obtaining an adapter-equipped nucleic acid; and sequencing the adapter-equipped nucleic acid.

[0020] In some embodiments, the method further comprises amplifying the captured nucleic acid prior to sequencing. In some embodiments, the sequenced nucleic acid is 1, 10, or 100 kb in length.

[0021] In some embodiments, the sequencing method does not include intermediate purification steps.

[0022] In some embodiments, the present invention relates to a method of enriching a nucleic acid from a sample comprising a target nucleic acid and non-target nucleic acids, comprising: contacting a double-stranded target nucleic acid having a 3' end with a catalyst having a 3'-5' exonuclease activity, thereby producing a first 5' overhang at a first 5' end and a first 3' recessed end in the target nucleic acid; contacting the nucleic acid with an adaptor having a second 5' overhang at a second 5' end, a second 5' overhang capable of hybridizing to the first 5' overhang, thereby producing a hybrid between the first and second 5' overhangs; wherein the adaptor further comprises a functional capture moiety; linking at least one of the first 3'-ends to the second 5'-end and thereby obtaining an adapter-equipped nucleic acid; and capturing a functional capture moiety on the adapter and thereby enriching target nucleic acids from the sample.

[0023] In some embodiments, the enriched nucleic acid is 1, 10, or 100 kb in length.

[0024] In some embodiments, the enrichment method does not include intermediate purification steps.

[0025] In some embodiments, the present invention relates to a kit for enriching a nucleic acid from a sample comprising a target nucleic acid and non-target nucleic acids, comprising: a catalyst having 3'-5' exonuclease activity and capable of forming a 5' sticky end in nucleic acids; an adapter designed to have a 5' sticky end at the 5' end, the 5' sticky end capable of hybridizing to a 5' sticky end expected in the target nucleic acid after treatment with the catalyst; and a DNA ligase.

[0026] In some embodiments, the adapter is double-stranded and further comprises a second recessed 3' end.

[0027] In some embodiments, the catalyst having 3'-5' exonuclease activity is a DNA polymerase. In some embodiments, the DNA polymerase having 3'-5' exonuclease activity is a family A polymerase, a family B polymerase, or a family C polymerase comprising the Exo I, Exo II, and Exo III domains of the Klenow fragment of Pol IE. coli. In some embodiments, the DNA polymerase comprises aspartate residues corresponding to residues D355, D424, and D501 of the Pol I Klenow fragmentE. coli. In some embodiments, the DNA polymerase is selected from the group consisting of T4 DNA polymerase, RB69 DNA polymerase, Klenow fragment, T7 DNA polymerase, delta Pol IIIE. coli, eukaryotic polymerase epsilon, eukaryotic polymerase delta and mitochondrial polymerase gamma.

[0028] In some embodiments, the kit further comprises one or more of dATP, dCTP, dGTP, and dTTP.

[0029] In some embodiments, the 5' sticky end in the adapter is 2-15 nucleotides in length. In some embodiments, the adapter comprises at least one of a nucleic acid barcode, a sequencing primer binding region, and an amplification primer binding region. In some embodiments, the barcode is selected from a unique molecular ID (UMI) and a sample ID (SID), or both. In some embodiments, the adapter is produced by annealing two strands of a nucleic acid. In some embodiments, the adapter is produced by exonuclease cleavage of the 3' end to produce an oligonucleotide with a 5' sticky end. In some embodiments, the adapter comprises a nucleic acid modification that increases the melting temperature of a nucleic acid duplex comprising the modification. In some embodiments, the adapter comprises a sequence selected from SEQ ID NOs: 7-11. In some embodiments, the adapter is linked to a functional capture moiety, i.e., biotin, an antigen capable of binding to the capture antibody, and a capture sequence capable of hybridizing to the capture nucleic acid.

[0030] In some embodiments, the kit further comprises a polynucleotide kinase, reagents for amplifying nucleic acids (e.g., an amplification primer capable of annealing to a primer binding site in an adapter), and reagents for sequencing nucleic acids (e.g., a sequencing primer capable of annealing to a primer binding site in an adapter). In some embodiments, the amplification primer comprises a sequence selected from SEQ ID NO: 12-16.

[0031] In some embodiments, the present invention relates to a reaction mixture for enriching a nucleic acid from a sample comprising a target nucleic acid and non-target nucleic acids, comprising: a double-stranded target nucleic acid having a 3' end; a catalyst having 3'-5' exonuclease activity capable of forming a first 5' overhang at a first 5' end and a first 3' recessed end in the target nucleic acid; and an adapter having a second 5' overhang at a second 5' end, a second 5' overhang capable of hybridizing to the first 5' overhang, and, optionally, a DNA ligase.

BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a schematic of the method used for a Cas9 CRISPR endonuclease-cleaved target sequence. The steps of double-stranded DNA cleavage, exonuclease cleavage, and adapter ligation are shown.

[0033] Figure 2 is a schematic of a target DNA locus showing the location of the endonuclease cleavage site, the CRISPR guide RNA recognition sequences (spacer sequences), and the location of the amplification primer binding sites.

[0034] Figure 3 shows the results of a T/A adaptor ligation experiment (prior art) used to target plasmid DNA, and wherein the Cas9 ribonucleoprotein (RNP) complex includes the CrA and CrB guide rRNAs.

[0035] Figure 4 shows the results of an experiment with the method of the invention used to target plasmid DNA, and wherein the Cas9 ribonucleoprotein (RNP) complex includes the guide rRNAs CrA and CrB.

[0036] Figure 5 shows the results of a T/A adaptor ligation experiment (prior art) used to target genomic DNA, and wherein the Cas9 ribonucleoprotein (RNP) complex includes the CrA and CrB guide rRNAs.

[0037] Figure 6 shows the results of an experiment with the method of the invention used to target genomic DNA, and wherein the Cas9 ribonucleoprotein (RNP) complex includes the guide rRNAs CrA and CrB.

[0038] Figure 7 shows the results of an experiment with the method of the invention used for target genomic DNA, wherein the Cas9 ribonucleoprotein (RNP) complex includes the guide rRNAs CrC and CrD.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Definitions

[0040] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, Sambrook et al ., Molecular Cloning, A Laboratory Manual , ^4th Ed. Cold Spring Harbor Lab Press (2012).

[0041] The following definitions are provided to better understand the present invention.

[0042] The term "endonuclease" refers to an enzyme that catalyzes the hydrolysis of a phosphodiester bond between two nucleoside residues in a polynucleotide (DNA or RNA) where neither nucleoside residue is terminal. An endonuclease may introduce a single-strand break or a double-strand break. A single-strand endonuclease is referred to as a "nickase". A double-strand break may be blunt (forming blunt ends) or staggered (forming overhanging or recessed ends).

[0043] The term "exonuclease" refers to an enzyme that catalyzes the hydrolysis of the phosphodiester bond between the terminal nucleoside residue and the penultimate nucleoside residue in a polynucleotide (DNA or RNA). Exonucleases may be processive or capable of stepwise removal of multiple nucleoside residues from the end of a nucleic acid chain.

[0044] The term "CRISPR repeat" or "CRISPR repeat sequence" refers to a minimal CRISPR repeat sequence.

[0045] The term "endoribonuclease" refers to an enzyme that can hydrolyze a phosphodiester bond in RNA. In some embodiments, the endoribonuclease can be a site-specific polypeptide. The endoribonuclease can be a member of the CRISPR system (e.g., type I, type II, type III). The term "endoribonuclease" can refer to the Repeat Associated Mysterious Protein (RAMP) superfamily of proteins (e.g., Cas6, Cas6, Cas5 families). Endoribonucleases can also include RNase A, RNase H, RNase I, RNase III family members (e.g., Drosha, Dicer, RNase N), RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V1, RNase V.

[0046] The term "inhibitory" refers to the ability of a chemical structure to partially or completely inhibit a chemical reaction. Those skilled in the art will appreciate that whether the inhibition is partial or complete depends on the sensitivity of the detection methods. The term "inhibitory cleavage" with respect to a nuclease refers to the ability to detectably reduce the amount of a cleavage product. The term "preventive cleavage" with respect to a nuclease refers to the ability to reduce the amount of a cleavage product below the detection level.

[0047] The term "NATNA" refers to a nucleic acid that targets nucleic acids. The NATNA may be part of a programmable endonuclease system, such as the CRISPR system. The NATNA may comprise two nucleic acid-targeting polynucleotides ("dual guides"), including a CRISPR RNA (cRNA) and a CRISPR transactivating RNA (tracRNA). The NATNA may comprise a single nucleic acid-targeting polynucleotide ("single guide"), comprising a cRNA and a tracRNA connected by a fusion region (linker). The cRNA may comprise a guide region and an activating region. The tracRNA may comprise a region capable of hybridizing to the activating region of the cRNA. The term "guide region" refers to a region capable of hybridizing to a sequence in a target nucleic acid. The term "activating region" refers to a region that interacts with a polypeptide, such as a CRISPR nuclease.

[0048] The term "adapter" refers to a nucleic acid added to nucleic acids in a sample. Typically, the same adapter is added to multiple nucleic acids in a sample. The term "same adapter" includes adapters that have the same sequence, except for different barcodes (defined below). The term "adapter" includes single-stranded adapters, partially single-stranded (interchangeable with partially double-stranded) adapters, and double-stranded adapters. The term "overhang" with respect to a nucleic acid refers to the single-stranded terminal portion of a partially double-stranded nucleic acid. The terms "recess" and "recessed end" with respect to a nucleic acid refer to the end of the double-stranded portion of a partially double-stranded nucleic acid. In a partially double-stranded nucleic acid, the recessed end of one strand marks the beginning of the sticky end on the opposite strand.

[0049] The term "barcode" refers to a nucleic acid sequence inserted into another sequence for identification purposes. Barcodes may typically be 2 or more and up to about 50 nucleotides in length. Barcodes are designed to be at least a minimal amount different from other barcodes in a sample. Barcodes may be unique to each molecule in a sample or unique to a sample and common to multiple molecules in a sample. The term "multiplex identifier," "MID," or "sample identifier," "SID" refers to a barcode that identifies a sample or source of a sample. As such, all or substantially all MID-barcoded polynucleotides from a single source or sample will share a common MID of a single sequence; while all or substantially all (e.g., at least 90% or 99%) of the MID barcoded polynucleotides from different sources or samples will have a different MID barcode sequence. Polynucleotides from different sources having different MIDs can be mixed and sequenced in parallel while maintaining sample information encoded in the MID barcode. The terms "unique molecular identifier," "UMI," or "UID" refer to a barcode that identifies the polynucleotide to which it is attached. In some embodiments, all or substantially all (e.g., at least 90% or 99%) of the UMI barcodes are unique such that each or substantially each of the UMI barcoded polynucleotides in a sample has a unique UMI. In other embodiments, the polynucleotides in a sample are non-uniquely labeled such that 1-50% of the polynucleotides in the sample have UMIs that are common to at least one other polynucleotide, as described, for example, in U.S. Patent No. 9,902,992.

[0050] The term "primer" refers to an oligonucleotide that binds to a specific region of a single-stranded template nucleic acid molecule and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction. Typically, a primer comprises less than about 100 nucleotides, and preferably comprises less than about 30 nucleotides. A target-specific primer specifically hybridizes to a target polynucleotide under hybridization conditions. Such hybridization conditions may include, but are not limited to, hybridization in isothermal amplification buffer (20 mM Tris-HCl, 10 mM ( _NH4 ) _2SO4 ), 50 mM KCl, 2 mM MgSO4, 0.1% TWEEN® ₂₀ , pH 8.8 at 25°C) at a temperature _of from about 40°C to about 70°C. In addition to the target binding region, the primer may comprise additional regions, typically in the 5' portion. The additional region may include a universal primer binding site or a barcode.

[0051] The term "universal primer" refers to a primer that can hybridize to a universal primer binding site present in a plurality of different nucleic acids in a sample. A universal primer, in contrast to a target-specific primer, is capable of priming the amplification of all nucleic acids in a sample without bias in favor of any sequence. Universal primer binding sites may be natural or artificial sequences, typically added to a target sequence in a non-target-specific manner.

[0052] The term "target" or "target nucleic acid" refers to a nucleic acid of interest in a sample. A sample may contain multiple targets, as well as multiple copies of each target.

[0053] Prior art capture methods and nucleic acids include dA tailing and dT-tailed adaptor ligation. The dA tailing and tether ligation method includes adding an A nucleotide to the 3' end of a first nucleic acid (e.g., a target or insert) and a T nucleotide to the 3' end of a second nucleic acid (e.g., an adaptor or vector). The dA tailing method is used in sequencing and cloning. For example, PCR cloning utilizes the natural dA tailing by Taq polymerase and uses a dT-tailed vector to allow sticky end ligation. In the production of sequencing libraries, the dA tailing method is used for randomly fragmented DNA (e.g., sonicated DNA or naturally fragmented cell-free DNA). The dA tailing method is also used for nucleic acids cleaved by endonucleases, including CRISPR endonucleases, see Gilpatrick et al., (2020)Targeted nanopore sequencing with Cas9-guided adapter ligation, Nat. Biotechnol. 38:433. In addition, SITE-Seq®, a method for genomic sequencing of CRISPR-mediated genome modifications, also uses the dA tailing method, see Cameron, P.,et al., (2017).Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nature Methods, 14(6), 600-606.

[0054] Alternative methods for producing sticky ends for ligation include exonuclease digestion. For example, Gibson assembly involves the assembly of large DNA fragments by limited nuclease digestion, creating compatible or semi-compatible DNA ends, see Gibson, D.G.,et al., (2009)Enzymatic assembly of DNA molecules up to several hundred kilobases,Nat. Methods 6, 343-345, and Gibson, D. G., et al., (2010)Creation of Bacterial Cell Controlled by a Chemically Synthesized Genome,Science 329, 52-56. Previously, labeled PCR primers and controlled exonuclease digestion were used to generate compatible ends for ligation-independent cloning of PCR products in bacterial cells, see Aslanidis, C. and de Jong, P., (1990)Ligation-independent cloning of PCR products (LIC-PCR), Nucleic Acids Res. 18:6069.

[0055] The present invention relates to an alternative to producing a dA tail and an improved method for capturing a DNA end formed by a sequence-specific endonuclease (e.g., a restriction endonuclease or a CRISPR endonuclease). The method uses controlled exonuclease activity on a DNA end to be captured to excise a specific segment from the 3' end so as to produce a sticky 5' end. An adapter is designed with a sticky 5' end capable of hybridizing to a sticky 5' end in the DNA to be captured. The adapter (or other nucleic acid partner) forms a specific and stable hybrid to the DNA to be captured. The adapter can then be covalently linked using a ligase. Direct comparison of the invention with the commonly used 3'-A-sticky end adaptor ligation ("dA-tailing method") demonstrates improvement in ligation specificity and efficiency.

[0056] The diagram in Figure 1 shows one embodiment of the method. In the example shown in Figure 1, a blunt-ended double-strand break is generated in the target DNA by a Cas9CRISPR endonuclease. The end is processed using controlled 3'-5' exonuclease activity. In some embodiments, the controlled exonuclease activity is exhibited by the DNA polymerase in the absence of dNTPs or in the presence of less than all 4 dNTPs, such as in the presence of only one dNTP that defines the position at which exonuclease cleavage is terminated. In the example in Figure 1, the exonuclease is T4 DNA polymerase in the presence of only dGTP. In this example, exonuclease cleavage is terminated when T4 DNA polymerase encounters the first G in the strand being cleaved. In some embodiments, the sequence at the 3' end to be ligated comprises a 2-15 nucleotide fragment with only 3 of the 4 bases present. Thus, a 4-base dNTP can be added to the exonuclease reaction to terminate the exonuclease activity at the end of the 2-15 nucleotide fragment. The adapters are designed to anneal at the sticky 5' end formed by the controlled exonuclease activity. The annealed adapters are ligated using ligase.

[0057] In some embodiments, the method of the invention is compared to a method for producing a dA tail known in the art. In some embodiments, the selected target is a human T cell receptor locus (T Cell Receptor Alpha Constant or TRAC locus). In some embodiments, the sequence-specific endonuclease is Cas9 CRISPR endonuclease, and the reaction mixture further comprises a rRNA and a trac rRNA. The rRNAs targeting the TRAC locus are added to a Cas9 sequence-specific endonuclease guide for the TRAC locus. In some embodiments, multiple rRNAs are designed for the TRAC locus. A map of the locus, the position of the rRNAs (Cr-A, B, C, and D), and the cleavage site (arrow) are shown in Figure 2. In some embodiments, each reaction includes one of the rRNAs (e.g., Cr-A, B, C, or D, Figure 2).

[0058] Following exonuclease digestion, the end of the DNA below the double-strand break is targeted for ligation. For ligation according to the invention, the reaction mixture is incubated with a DNA polymerase having 3'-5' exonuclease activity (e.g., T4 DNA polymerase) and less than 4 dNTPs (e.g., one dNTP, dGTP for the target sequence shown in Figure 1).

[0059] The preparation of the dA tail and the T/A ligation are carried out in a parallel reaction according to the state of the art.

[0060] After ligation of the adapter, PCR is optionally performed to amplify the adapter-equipped DNA. In some embodiments, the PCR primers comprise a primer that anneals to the adapter. In some embodiments, the adapter comprises a universal primer binding site. In some embodiments, the universal primer pairs with a downstream primer capable of hybridizing to a target sequence downstream of an endonuclease cleavage site. In some embodiments, one of the reverse primers shown in Figure 2 (rv-1, 2, 3, or 4) is used for the TRAC locus.

[0061] In some embodiments, the amplified adaptor-tipped DNA is visualized, for example, by gel electrophoresis as shown herein. The results in the examples provided herein, as assessed by gel electrophoresis (Figures 3-7), indicate that both methods resulted in PCR products of the expected size. Notably, in the dA-tailing method known in the art, multiple amplicons are observed (Figure 3, lanes 3, 4, 7, and 8), indicating that multiple adapters are likely ligated to the target end of the DNA. In the case of the method of the invention, multimers are not observed (Figures 2, 6, and 7), indicating increased specificity of the method due to the longer sticky 5' ends of the adapters. Furthermore, comparison of Figure 5 (genomic DNA, dA tailing according to the prior art) with Figures 6 and 7 (genomic DNA, present invention) demonstrates a higher amount of specific product and less unwanted molecules, indicating the higher specificity and efficiency achieved by the present invention.

[0062] The present invention includes a method for processing nucleic acids from a sample. In some embodiments, the sample is obtained from an individual or a patient. In some embodiments, the sample may comprise a solid tissue fragment or a solid tumor obtained from the individual or patient, such as by biopsy. The sample may also comprise bodily fluids that may contain nucleic acids (e.g., urine, sputum, serum, blood or blood fractions, i.e., plasma, lymph, saliva, sputum, sex, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cyst contents, bile, gastric fluid, intestinal fluid, or stool samples). In other embodiments, the sample is a culture sample, such as a tissue culture containing cells and fluids from which nucleic acids can be isolated. In some embodiments, the sample includes formalin-fixed, paraffin-embedded (FFPET) tissues and nucleic acids isolated therefrom. The sample may also include acellular material, such as a cell-free fraction of blood, containing cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). In some embodiments, the nucleic acids of interest in the sample are derived from infectious agents, such as viruses, bacteria, protozoa, or fungi, infecting the patient serving as the source of the sample. The sample may be collected from a human, a non-human individual, or the environment. In some embodiments, the sample is a reaction mixture containing nucleic acids that have undergone one or more in vitro biochemical reactions.

[0063] The present invention includes manipulation of nucleic acids isolated or extracted from a sample. Methods for isolating nucleic acid are well known in the art. See J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, 2 ^nd Ed., Cold Spring Harbor Laboratory Press: New York, NY Various reagents and kits are commercially available for isolating nucleic acids (DNA or RNA) from biological samples, including products from BD Biosciences (San Jose, Cal.), Clontech (TaKaRa Bio.); Epicentre Technologies (Madison, Wisc.); Gentra Systems, (Minneapolis, Minn.); Qiagen (Valencia, Cal.); Ambion (Austin, Tex.); BioRad Laboratories (Hercules, Cal.); KAPA Biosystems (Roche Sequencing Solutions, Pleasanton, Cal.), etc.

[0064] In some embodiments, the present invention includes intermediate purification or separation steps, such as to remove unused primers and adapters or unligated sample DNA. Purification or separation can be accomplished by a size exclusion method selected from gel electrophoresis, affinity chromatography, and size exclusion chromatography. In some embodiments, size selection can be accomplished using the reversible solid phase immobilization (SPRI) technology from Beckman Coulter (Brea, Cal.).

[0065] In some embodiments, the use of compatible buffer systems allows for the omission of purification or separation steps between the 3' deep end formation step, the adaptor ligation step, and the step of further processing the captured nucleic acid, such as by amplification and/or sequencing.

[0066] In some embodiments, the present invention utilizes a catalyst (enzyme) having 3'-5' exonuclease activity. Such activity is present in nucleic acid polymerases and is sometimes referred to as "proofreading activity," such as in DNA polymerases active at a DNA replication fork. This activity occurs in the presence of magnesium ions and in the absence of dNTPs. Compared to exonucleases such as, for example, exonuclease III, the rate and extent of hydrolysis is slow and programmable. If less than all four dNTPs are present, 3'-5' exonuclease activity occurs until the first nucleotide identical to the dNTP in the reaction mixture is encountered in the strand being cleaved. In the example shown in Figure 1, it is desirable to remove the sequence 3'- TCCACC -5' from the longer sequence 3'- TCCACC GTG...-5'. The reaction mixture contains an enzyme with 3'-5' proofreading exonuclease activity and only dGTP. The exonuclease removes six TCCACC nucleotides until a G (corresponding to the dGTP in the reaction mixture) is encountered. The example shows that, knowing the sequence to be hydrolyzed at the 3' end, one skilled in the art can prepare a method and a reaction mixture by omitting the correct dNTP or dNTPs such that hydrolysis by the 3'-5' exonuclease occurs to the desired position and the desired overhanging 5' end is formed.

[0067] 3'-5' proofreading exonuclease activity is present in many DNA polymerases of viral, bacterial, and eukaryotic origin. In some embodiments, the catalyst is a DNA polymerase. DNA polymerases with 3'-5' proofreading activity comprise the Exo I, Exo II, and Exo III domains of the Klenow fragment of E. coli Pol I. In some embodiments, the DNA polymerase is selected from a family A polymerase, a family B polymerase, and a family C polymerase, polymerases comprising the Exo I, Exo II, and Exo III domains of the Klenow fragment of E. coli Pol I.

[0068] In some embodiments, the DNA polymerase is selected from a family A polymerase, a family B polymerase, and a family C polymerase, a polymerase comprising a DNA polymerase containing aspartate residues corresponding to residues D355, D424, and D501 of the Klenow fragment of E. coli Pol I.

[0069] In some embodiments, the DNA polymerase is selected from T4 DNA polymerase, RB69 DNA polymerase, Klenow fragment, T7 DNA polymerase, E. coli Pol III δ (delta) fragment, eukaryotic polymerase ε (epsilon), eukaryotic polymerase δ (delta), and mitochondrial polymerase γ (gamma).

[0070] In some embodiments, the DNA polymerase is a wild-type DNA polymerase. In some embodiments, the DNA polymerase is a mutant DNA polymerase having increased 3'-5' exonuclease activity. In some embodiments, the mutant DNA polymerase is a T4 DNA polymerase having one or more mutations selected from R335C, L412I, I417V, P424L, Q730S, A737V, and A777V.

[0071] In some embodiments, the DNA polymerase is a mutant DNA polymerase having reduced 3'-5' exonuclease activity. In some embodiments, the mutant DNA polymerase is a T4 DNA polymerase having one or more mutations selected from D219A, D112A, and E114A.

[0072] In some embodiments, the end of the double-stranded nucleic acid is generated by a sequence-specific endonuclease. In some embodiments, the endonuclease is encoded by a CRISPR locus. The genomic CRISPR (clustered regularly interspaced short palindromic repeats) locus is found in many prokaryotic genomes and confers resistance to invasion by foreign nucleic acids. A review of the structure, nomenclature, and classification of CRISPR loci is provided in Makarova et al. , Evolution and classification of the CRISPR-Cas systems . Nature Reviews Microbiology. 2011 Jun; 9(6): 467-477.

[0073] Briefly, a typical CRISPR locus includes a series of short repeats regularly interspersed with spacers. A CRISPR locus also includes coding sequences for CRISPR-associated (Cas) genes. A unit of spacer-repeat sequence encodes a CRISPR RNA (crRNA). In vivo, mature crRNAs are processed from a polycistronic transcript referred to as a pre-crRNA or pre-crRNA array. Repeats in the pre-crRNA array are recognized by Cas proteins, which bind to and cleave the repeats, releasing mature crRNAs. CRISPR systems cleave the target nucleic acid, where the Cas proteins and crRNAs form CRISPR ribonucleoproteins (crRNPs). The crRNA molecule directs the crRNP to a target nucleic acid (e.g., a foreign nucleic acid invading a bacterial cell), and Cas nuclease proteins cleave the target nucleic acid.

[0074] Type I CRISPR systems include means for processing a pre-rRNA array that includes a multiprotein complex called Cascade (CRISPR-associated complex for antiviral defense), consisting of CasA, B, C, D, and E subunits. The Cascade-rRNA complex recognizes a target nucleic acid by hybridizing the target nucleic acid to the rRNA. The associated nucleoprotein complex recruits the Cas3 helicase/nuclease to facilitate cleavage of the target nucleic acid.

[0075] Type II CRISPR systems include a transactivating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a repeat of the sRNA in the pre-sRNA array and recruits endogenous RNase III to cleave the pre-sRNA array. The tracrRNA/sRNA complex can associate with a nuclease, such as Cas9. The sRNA-tracrRNA-Cas9 complex recognizes a target nucleic acid by hybridizing the target nucleic acid to the sRNA. Hybridization of the sRNA to the target nucleic acid activates the Cas9 nuclease to cleave the target nucleic acid.

[0076] Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave pre-sRNA arrays using one or more CRISPR polymerase-like proteins.

[0077] Type V CRISPR systems contain another set of Cas-like genes, including Csf1, Csf2, Csf3, and Csf4, which are distant homologs of the Cas genes in Type I-III CRISPR systems.

[0078] CRISPR endonucleases require a nucleic acid targeting nucleic acid (NATNA), also known as a guide RNA. The endonuclease may form a ribonucleoprotein complex (RNP) with one or more guide RNAs. In some embodiments, the endonuclease is a type II CRISPR endonuclease and the NATNA comprises tracRNA and crRNA.

[0079] In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, the NATNA may comprise, in order from 5′ to 3′, a spacer extension, a spacer, a minimal CRISPR repeat, a single guide connector, a minimal tracrRNA, a 3′ tracrRNA sequence, and a tracrRNA extension. In some cases, the nucleic acid targeted to the nucleic acids may comprise a tracrRNA extension, a 3′ tracrRNA sequence, a minimal tracrRNA, a single guide connector, a minimal CRISPR repeat, a spacer, and a spacer extension in any order.

[0080] In some embodiments, a guide nucleic acid targeting nucleic acids may comprise a single NATNA guide. The NATNA comprises a spacer sequence that can be designed to hybridize to a target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence. Optionally, the NATNA may have a spacer extension segment and a tracrRNA extension segment. These elements may include elements that can contribute to the stability of the NATNA. The CRISPR repeat and the tracrRNA sequence may interact to form a paired, double-stranded structure. The structure may facilitate binding of an endonuclease to the NATNA.

[0081] In some embodiments, a single guide NATNA comprises a spacer sequence located 5' of a first duplex comprising a region of hybridization between a minimal CRISPR repeat and a minimal tracrRNA sequence. The first duplex may be interrupted by a loop. The loop facilitates recruitment of an endonuclease to NATNA. The loop may be followed by a first stem comprising a linker connecting the minimal CRISPR repeat and the minimal tracrRNA sequence. The last paired nucleotide at the 3' end of the first duplex may be connected to a second linker connecting the first duplex to the mid-tracrRNA. The mid-tracrRNA may comprise one or more additional hairpins.

[0082] In some embodiments, a NATNA may comprise a dual guide nucleic acid structure. The dual guide NATNA comprises a spacer extension segment, a spacer, a minimal CRISPR repeat, a minimal tracrRNA sequence, a 3′ tracrRNA sequence, and a tracrRNA extension segment. The dual guide NATNA does not include a single guide connector. Instead, the minimal CRISPR repeat sequence comprises a 3′ CRISPR repeat sequence, and the minimal tracrRNA sequence comprises a 5′ tracrRNA sequence, and the dual guide NATNAs can hybridize through the minimal CRISPR repeat and the minimal tracrRNA sequence.

[0083] In some embodiments, the NATNA is an engineered guide RNA comprising one or more DNA residues (CRISPR hybrid RDNA or chRDNA). In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. Briefly, some chRDNA for use with a Type II CRISPR system may be comprised of two strands forming a secondary structure comprising an activating region comprising an upper duplex region, a lower duplex region, a loop, a targeting region, a ligament, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may comprise varying proportions of DNA and RNA. Another chRDNA may be a single guide D(R)NA for use with a type II CRISPR system that comprises a targeting region and an activating region consisting of a lower duplex region, an upper duplex region, a fusion region, a loop, a linkage, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain varying proportions of DNA and RNA. For example, the targeting region may contain DNA or a mixture of DNA and RNA, and the activating region may contain RNA or a mixture of DNA and RNA.

[0084] In some embodiments, the endonuclease that forms the 3' end of the double-stranded target nucleic acid is a restriction endonuclease, such as a type II restriction endonuclease. In some embodiments, the restriction endonuclease results in the formation of a blunt end. For each endonuclease, the adaptor of the invention comprises a 5' sticky end capable of hybridizing to the 5' sticky end formed in the method provided herein in the excised nucleic acid to be captured.

[0085] In embodiments, the endonuclease that forms the 3' end of the double-stranded target nucleic acid is a catalytically inactive CRISPR endonuclease (e.g., a catalytically inactive Cas9 or Cas12a) conjugated to the cleavage domain of the restriction endonuclease Fok I (see, for example, Guilinger, J. P.,et al.,(2014).Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification, Nature biotechnology,32(6), 577-582).

[0086] In embodiments wherein the endonuclease that forms the 3' end of the double-stranded target nucleic acid is a zinc finger nuclease (ZFN) or a ZFN-Fok I fusion protein, the target sequence is approximately 22-52 bases in length and comprises a pair of ZFN recognition sequences, each 9-18 nucleotides in length, separated by a spacer of 4-18 nucleotides in length (see, e.g., Kim Y. G., et al., (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl. Acad. Sci USA. 93(3): 1156-1160).

[0087] In embodiments wherein the endonuclease that forms the 3' end of the double-stranded target nucleic acid is a transcription activator-like effector (TALEN) nuclease or a TALEN-Fok I fusion protein, the target sequence is approximately 48-85 nucleotides in length and comprises a pair of TALEN recognition sequences, each 18-30 bases in length, separated by a spacer of 12-25 bases in length (see, e.g., Christian M. et al., (2010) Targeting DNA double-strand breaks with TAL effector nucleases, Genetics. 186 (2): 757-61).

[0088] As shown in the figure 1, the methods and compositions provided herein utilize a sequence associated with a recognition site and a cleavage site of each endonuclease. In some embodiments, the recognition sequence and the cleavage site are the same. For example, in the case of type II restriction endonucleases, the recognition site is a palindromic sequence and cleavage occurs at the palindromic sequence. In some embodiments, the recognition site is different from the cleavage site. In some embodiments, the endonuclease is a CRSIPR endonuclease and the recognition site is a sequence containing a protospacer adjacent motif (PAM), while the cleavage site is in a region that hybridizes to the targeting region of the CRISPR guide RNA (spacer region). The PAM is adjacent to a known fragment of the target sequence.

[0089] CRISPR nucleases do not cleave a fixed sequence, but are instead guided by a guide nucleic acid, as described above. However, CRISPR nucleases recognize an additional sequence called a protospacer adjacent motif (PAM). The present invention utilizes a PAM sequence as well as a target nucleic acid sequence known to affect the capture of an excised target nucleic acid. As shown in Figure 1, the PAM and a short fragment of the adjacent target sequence are used to design an adapter oligonucleotide. In some embodiments, the PAM and a short fragment between the PAM and the cleavage site are used to design a 5' overhang capable of hybridizing to a 5' overhang generated by the method described herein in an excised nucleic acid to be captured.

[0090] In some embodiments, the endonuclease that forms the 3' end of the double-stranded target nucleic acid is a class I CRISPR endonuclease (CASCADE), and the protospacer adjacent motif (PAM) consists of a sequence selected from 5'-AAG-3', 5'-AGG-3', 5'-ATG-3', 5'-GAG-3', 5'-CAG-3', 5'-GTG-3', 5'-TAA-3', 5'-TGG-3', 5'-AAA-3', 5'-AAC-3', 5'-AAT-3', 5'-ATA-3', 5'-TAG-3', and 5'-TTG-3'.

[0091] In some embodiments, the endonuclease that forms the 3' end of the double-stranded target nucleic acid is a class II CRISPR endonuclease, and the protospacer adjacent motif (PAM) consists of a sequence selected from 5'-NGG-3', 5'-NGGNG-3', 5'-NNAAAAW-3', 5'-NNNNGATT-3', 5'-GNNNCNNA-3', and 5'-NNNACA-3',5'-TTN-3', 5'-TTTN-3', and 5'-TTTV-3'.

[0092] In some embodiments, the present invention utilizes an adapter added to at least one end of a nucleic acid or a nucleic acid strand. The adapter may be a single-stranded, double-stranded, or partially double-stranded adapter. The adapter comprises a double-stranded portion, wherein the double-stranded portion is ligated to the double-stranded nucleic acid in a manner described herein. Adapters of various shapes and functions are known in the art, see, for example, U.S. Patent Nos. 8,822,150 (Y-shaped adapter); 8,455,193 (stem-loop/hairpin adapter); and 11,085,084 (various shapes of partially double-stranded adapters). In some embodiments, the function of the adapter is to incorporate certain useful elements into the nucleic acid. Examples of adapter-derived elements include nucleic acid barcodes, amplification primer binding sites, sequencing primer binding sites, enzyme recognition sites, and ligation sites.

[0093] In some embodiments, the adapter molecules are in vitro synthesized artificial sequences. In other embodiments, the adapter molecules are in vitro synthesized natural sequences. In other embodiments, the adapter molecules are isolated natural molecules or isolated non-natural molecules.

[0094] In some embodiments, the adapter is single-stranded and the 5' sequence of the adapter hybridizes to the sticky 5' end of the target nucleic acid and the 5' end of the adapter is ligated to the recessed 3' end of the target nucleic acid.

[0095] In some embodiments, the adapter is at least partially double-stranded and comprises a 5' sticky end. The 5' sticky end of the adapter hybridizes to the 5' sticky end of the target nucleic acid, and the 5' end of the adapter is ligated to the recessed 3' end of the target nucleic acid, while the recessed 3' end of the adapter is ligated to the 5' end of the target nucleic acid.

[0096] In the case of a double-stranded adaptor (or an adaptor having a double-stranded portion), the 5' sticky end at the 5' end of the adaptor may hybridize or hybridizes to the 5' sticky end of the target nucleic acid and may ligate to the 3' end of the target nucleic acid.

[0097] In some embodiments, the present invention includes designing an adapter. The term "designing" refers to identifying different sequences in a target nucleic acid that can be captured by an adapter as provided herein, and further designing a 5' sticky end of an adapter oligonucleotide to allow hybridization to such sequences. As described in detail herein, the method includes providing a 5' sticky end near a cleavage site in a target nucleic acid. The sequence near the cleavage site in the target nucleic acid includes known and unknown sequences. The known sequences include at least a portion of a recognition sequence for an endonuclease near the cleavage site. The known sequence is used to design a method for exonuclease cleavage. The known sequence is further used to design the adapter oligonucleotide.

[0098] For example, Figure 1 shows a target nucleic acid cleaved by a CRISPR endonuclease, wherein known sequences adjacent to the end of the nucleic acid include a target sequence, a protospacer adjacent motif (PAM), and a portion of the spacer sequence prior to the cleavage site. In this example, the known sequence 5'-AGGTGG(N) _n -3' (including the adjacent target sequence) is used to design an exonuclease reaction comprising T4 DNA polymerase and dGTP to produce a 5'-AGGTGG overhang on the target nucleic acid. In this example, the adaptor is designed to have a 5' overhang comprising the sequence 5'-CCACCT-3'.

[0099] In the example shown in Figure 1, the cleavage occurs between the 3rd and 4th nucleotide of the 5' PAM, which is equivalent to a position between the 17th and 18th nucleotides of the spacer. In other embodiments, other CRISPR endonucleases cleave at different positions in the spacer sequence. In some embodiments, the PAM is downstream (3' side) of the spacer sequence containing the cleavage site. In some embodiments, the PAM is upstream (5' side) of the spacer sequence containing the cleavage site.

[00100] Many restriction endonucleases produce sticky ends that allow easy ligation to a compatible end. However, many restriction endonucleases produce blunt ends, for example, Afe I, AluI, BsaAI, BsaBI, BstuI, DpnI, DraI, Eco53kI, EcoRV, FspI, HaeIII, HincII, HpaI, HpyCH4V, Hpy166II, MscI, NaeII, NlaVI, NruI, PmlI, PvuII, RsaI, ScaI, SfoI, SnaBI, SspI, StuI, SwaI and ZraI are some of the blunt end forming enzymes that can be used in the method of the present invention. In the case of blunt-ending restriction endonuclease, the known sequences include a target sequence and a palindromic recognition sequence, which in the case of blunt-ending enzymes is cleaved via a double-strand break in the middle of the palindrome. The known sequence is used to design an exonuclease cleavage method. The known sequence is further used to design an adapter oligonucleotide. For example, in the case of EcoRV, the recognition sequence is 5'-GATATC-3', which can occur in the target sequence 5'-GATATC-3'. Cleavage with EcoRV endonuclease leaves a nucleic acid fragment with 5'-ATC(N) _n -3'. The 5'-ATC-3' sequence and any known adjacent sequence are used to design an exonuclease reaction containing T4 DNA polymerase and a nucleotide adjacent to C to form a 5'-ATC sticky end on the target nucleic acid. In this example, the adapter is designed to have a sticky 5' end containing at least the sequence 5'-GAT-3'.

[00101] One skilled in the art will appreciate that hybridization between nucleic acid strands does not require 100% complementarity or 100% Watson-Crick base pairing. In some embodiments, a stable hybrid between the 5' sticky end of the adapter and the 5' sticky end of the target nucleic acid includes the formation of canonical (Watson-Crick) base pairs as well as non-canonical base pairs and mismatches. In some embodiments, the adapter is designed to contain chemically modified nucleic acids, wherein the modification facilitates the formation of a stable hybrid. For example, the 5' sticky end of the adapter may contain a chemical modification that increases the melting temperature of the double-stranded region. In addition, one skilled in the art will appreciate that the formation of a stable hybrid between nucleic acid strands depends on reaction conditions, including the ionic strength of the buffer and the temperature. Thus, the adaptor and reaction conditions are prepared and formulated such that the sticky 5' end of the adaptor forms a stable hybrid with the sticky 5' end of the target nucleic acid under the selected reaction conditions.

[00102] Custom synthetic oligonucleotides are readily available from many commercial suppliers using phosphorasmidite chemistry, including ThermoFisher Scientific, Applied Biosystems (Waltham, Mass.), Biolytic (Newark, Cal.), BioAutomation (Plano, Tex.), and Integrated DNA Technologies (Coralville, Ia.). Other synthetic methods are used by Twist Bioscience (South San Francisco, Cal.) and Custom Array/GenScript (Redmond, Wash.). Optionally, the custom synthetic oligonucleotides used to prepare the adapter described herein can be further purified by any standard method, such as high performance liquid chromatography (HPLC). Optionally, protecting groups such as DMT (dimethoxytrityl) can be used in the purification. For example, a crude oligonucleotide preparation can be purified using an RP 18 HPLC column (Hypersil, 8 x 240 mm) using a gradient of 0.1 M triethylammonium acetate, pH 7.1, acetonitrile. The DMT at the peak can be collected, desalted by dialysis, evaporated, and dissolved in 10 mM Tris, pH 8.0. The OD measured at 260 nm can be determined to estimate the yield.

[00103] A double-stranded adapter oligonucleotide can be prepared by combining two nucleic acid strands (a strand with an overhang and a strand without an overhang) in a reaction mixture containing a suitable buffer (e.g., 1x TE or 1.5x TE). To ensure proper annealing without any unwanted secondary structure, the reaction mixture can be heated, for example, to 90°C or more, and slowly cooled. In some embodiments, a partially double-stranded adapter comprises a single strand that adopts a secondary structure, such as a dumbbell, a stem-loop, or a hairpin. Such adapters can also be prepared by subjecting a single strand to heating followed by slow cooling in a suitable buffer (e.g., 1x TE or 1.5x TE) to ensure formation of the desired secondary structure.

[00104] In some embodiments, the adapter includes one or more chemical modifications. In some embodiments, the modification causes increased stability of the double-stranded region of the adapter. In some embodiments, the modification confers resistance to exonuclease cleavage or inhibition of exonucleases.

[00105] In some embodiments, the modification is a backbone modification. One type of backbone modification is a modified internucleoside linkage. For example, the modification includes phosphorothioate linkages and heteroatom internucleoside linkages.

[00106] Another type of backbone modification is a modification of the sugar moiety. In some embodiments, the modification comprises the insertion of a 6-membered morpholine ring in place of the ribose or deoxyribose ring. Another backbone modification comprises the insertion of a cyclohexenyl ring in place of the ribose or deoxyribose (ceNA). Another backbone modification includes the insertion of closed nucleic acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′-carbon of the ribose, thereby forming a bicyclic structure having a 2′-C,4′-C-oxymethylene bond. LNAs are characterized by duplex stability and resistance to cleavage by 3′-5′ exonucleases.

[00107] In some embodiments, the modification is a nitrogenous base modification. For example, the double-stranded nucleic acid substrate may include one or more of 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and -cytosine, 5-propynyl (-C=C-CH3)-uracil and -cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, -cytosine and -thymine, 5-uracil (pseudouracil), 4-thiouracil-, 8-halo-, 8-amino-, 8-thio-, 8-thioalkyl-, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo-, 5-trifluoromethyl- and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-fixing bases such as substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one), 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases can be used to enhance the binding affinity of a polynucleotide compound. They can include 5-substituted pyrimidines, 6-azapyrimidines and N-2-, N-6- and 0-6-substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions can increase nucleic acid duplex stability by 0.6-1.2°C and can be useful base substitutions (e.g. when combined with 2′-O-methoxyethyl sugar modifications).

[00108] In some embodiments, the double-stranded portion of the adapter includes a modified nucleotide that increases the melting temperature of the double-stranded portion, such as 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutynyl-2'-deoxyuridine, 8-aza-7-deazaguanosine, a ribonucleotide, 2'O-methyl-ribonucleotide, or a locked nucleic acid. In another aspect, the adapter is modified to inhibit nuclease cleavage, such as by including a phosphorothioate nucleotide.

[00109] In some embodiments, the present invention includes the use of a barcode. Analysis of individual nucleic acid molecules by massively parallel sequencing typically requires a separate level of barcoding to identify samples and correct for errors. The use of molecular barcodes is described, for example, in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368. A unique molecular identifier barcode (UMI or UID) is added to each molecule to be sequenced to label the molecule and its progeny (e.g., the original molecule and its PCR amplicons). In some embodiments, the UMI is in the 5' portion of the amplification primer. In some embodiments, the UMI is in an adapter ligated to the nucleic acid.

[00110] UMI has multiple uses, including enumeration of the number of parent target molecules in a sample and error correction (Newman, A., et al ., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage , Nature Medicine doi:10.1038/nm.3519). Briefly, all progeny of a single target molecule are labeled with the same UMI barcode and form a barcoded family. Sequence differences that are not common to all (or most) members of a barcoded family are discarded as an artifact. UMI barcodes can also be used for positional deduplication and target quantification, since the entire family represents a single molecule in the original sample (Newman, A., et al ., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA , Nature Biotechnology 34:547). An adapter containing the UMI barcode can be conjugated to the nucleic acid to be sequenced.

[00111] A sample identification barcode is used for multiplex sequencing. A multiplex sample ID barcode (MID or SID) is used to identify a nucleic acid source where two or more nucleic acid samples are mixed prior to being added to a flow cell for sequencing.

[00112] In some embodiments, a nucleic acid molecule to be sequenced includes a UMI and a MID. In some embodiments, a single barcode is used as both a UMI and a MID. In some embodiments, a barcode consists of multiple parts. For example, the unique identification information consists of a barcode sequence and a nucleic acid end sequence. In some embodiments, a barcode consists of multiple subcodes, as described in U.S. Patent Application Publication No. 20200109397, " Modular Nucleic Acid Adaptors ."

[00113] In some embodiments, each barcode comprises a predetermined sequence. In other embodiments, the barcode comprises a random sequence. The barcodes are approximately 4-20 bases long, such that between 96 and 384 different adapters, each containing a different pair of identical barcodes, can be added to a human genomic sample. In some embodiments, the number of UMIs in a reaction can be in excess of the number of molecules to be labeled. One skilled in the art will appreciate that the number of barcodes depends on the complexity of the sample (i.e., the expected number of unique target molecules), and can design an appropriate number of barcodes of an appropriate length for each experiment.

[00114] In some embodiments, the present invention includes a ligation step. According to methods well known in the art (Sambrook et al ., Molecular Cloning, A Laboratory Manual , ^4th Ed. Cold Spring Harbor Lab Press (2012)), a ligase enzyme catalyzes the addition of a 5'-phosphate residue (5'-end) to a 3'-hydroxyl residue (3'-end). The addition forms a covalent bond, referred to as a phosphodiester bond, between the ends of the nucleic acids. The ligation is catalyzed by a single-stranded or double-stranded DNA ligase, such as a single-stranded DNA ligase such as CircLigase™ ssDNA ligase (Epicentre Biotechnologies, Madison, Wisc. or Lucigen, Middleton, Wisc.), or a double-stranded DNA ligase selected from T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, or any other enzyme capable of efficiently catalyzing the formation of phosphodiester bonds between two nucleic acids.

[00115] In some embodiments, the 5'-phosphate residue is naturally present in the products of endonuclease cleavage of a target nucleic acid. In some embodiments, the 5'-phosphate residue is added to the exonuclease-cleaved target nucleic acid or adapter oligonucleotide. The addition of the 5'-phosphate can be performed on the fragment enzymatically, for example, using a polynucleotide kinase, such as T4 polynucleotide kinase.

[00116] In some embodiments, the method includes forming a library comprising two or more nucleic acids from a sample. For example, the library may consist of a plurality of different nucleic acids conjugated to adapters comprising elements necessary for manipulation of the library, including universal primer binding sites for amplification, universal primer binding sites for sequencing, unique barcodes, or barcodes for identifying samples. The nucleic acid library is ready for sequencing or another type of detection method, such as PCR. The library may be stored and aliquots of the library may be used for multiple rounds of analysis.

[00117] In some embodiments, the nucleic acids in the sample comprise all of the nucleic acids originally found in the sample obtained from the organism, individual (e.g., patient), or environment. In some embodiments, the nucleic acids in the sample are enriched. In some embodiments, enrichment is accomplished by removing, such as capturing and removing, repetitive sequences or abundant sequences or transcripts from the organism's (or individual's) genome (e.g., human Alu sequences, mammalian globin sequences, or rRNA sequences present in all cellular organisms). In some embodiments, enrichment is accomplished by capturing and retaining sequences of interest. In some embodiments, both enrichment methods are used, such as first removing repetitive or redundant sequences to facilitate or improve subsequent capture of sequences of interest from the sample. For enrichment, the capture probes may be free in solution or fixed to a solid support.

[00118] In some embodiments, enrichment by capture uses adapters ligated to target nucleic acids as described herein. As part of the invention, an adapter is ligated to a target nucleic acid that has been specifically excised using a sequence-specific nuclease (e.g., a restriction endonuclease or a CRISPR endonuclease). The adapter serves as a capture probe and separates the target nucleic acids from the sample, thereby enriching the target nucleic acid.

[00119] In some embodiments, the length of the captured nucleic acid is 1, 10, 50 kb, or more.

[00120] In some embodiments, the present invention relates to a method of enriching a nucleic acid from a sample comprising a target nucleic acid and non-target nucleic acids, comprising: contacting a double-stranded target nucleic acid having a 3' end with a catalyst having a 3'-5' exonuclease activity, thereby producing a first 5' overhang at the first 5' end and a first 3' recessed end; contacting the nucleic acid with an adaptor having a second 5' overhang at a second 5' end, a second 5' overhang capable of hybridizing to the first 5' overhang, thereby producing a hybrid between the first and second 5' overhangs; wherein the adaptor further comprises a functional capture moiety; linking at least one of the first 3'-end to the second 5'-end and thereby obtaining an adapter-equipped nucleic acid; and capturing a functional capture moiety on the adapter and thereby enriching target nucleic acids from the sample.

[00121] If enriched, the capture probe may comprise a functional capture moiety. The functional capture moiety may be any functional moiety capable of specifically interacting with another capture molecule. Functional capture moieties-capture molecule pairs include avidin (streptavidin)-biotin, antigen-antibody, magnetic (paramagnetic) particle-magnet, or oligonucleotide-complementary oligonucleotide. The capture molecule may be linked to a solid support such that any nucleic acid on which the functional capture moiety is located is captured on the solid support and separated from the rest of the sample or reaction mixture. In some embodiments, the capture molecule comprises a functional capture moiety for a secondary capture molecule. For example, the functional capture moiety may be an oligonucleotide complementary to the capture oligonucleotide (capture molecule). The capture oligonucleotide may be biotinylated and captured on a streptavidin bead. In some embodiments, the adapter-ligated nucleic acid is enriched by capturing the functional capture moiety and separating the adapter-ligated target nucleic acids from unligated nucleic acids in the sample.

[00122] In some embodiments, the method comprises sequencing the nucleic acid provided with the adapter using the methods described herein. Any of a number of sequencing technologies or sequencing assays can be used. As used herein, the term "next generation sequencing (NGS)" refers to sequencing methods that enable massively parallel sequencing of single molecules or clonally amplified single molecules.

[00123] Non-limiting examples of sequence assays suitable for use in the methods provided herein include nanopore sequencing (U.S. Patent Publication Nos. 2013/0244340, 2013/0264207, 2014/0134616, 2015/0119259, and 2015/0337366), Sanger sequencing, capillary sequencing, thermal cycling sequencing (Sears et al., Biotechniques , 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol ., 3:39-42 (1992)), sequencing with mass spectrometry, such as time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/MS; Fu et al., Nature Biotech ., 16:381-384 (1998)), hybridization-mediated sequencing (Drmanac et al ., Nature Biotech ., 16:54-58 (1998)) and NGS methods including, but not limited to, sequencing-by-synthesis (e.g., HiSeq™, MiSeq™ or Genome Analyzer, each available from Illumina), ligation-mediated sequencing (e.g., SOLiD™, Life Technologies), ion semiconductor sequencing (e.g., Ion Torrent™, Life Technologies) and SMRT® sequencing (e.g., Pacific Biosciences).

[00124] Commercially available sequencing technologies include hybridization-based sequencing platforms from Affymetrix Inc. (Sunnyvale, Calif.), synthesis-based sequencing platforms from Illumina/Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), and a ligation-based sequencing platform from Applied Biosystems (Foster City, Calif.). Other sequencing technologies include, but are not limited to, Ion Torrent technology (ThermoFisher Scientific, Waltham, Mass.), single-molecule real-time (SMRT®) sequencing (Pacific Biosciences, Menlo Park, Calif.), and nanopore sequencing (Genia/Stratos Technology from Roche Sequencing Solutions, Santa Clara, Calif.) and Oxford Nanopore Technologies (Oxford, UK).

[00125] In some embodiments, the sequencing step includes aligning the sequence and determining a consensus. In some embodiments, the consensus sequence is determined from a plurality of sequences that all have an identical UMI. Sequences that have an identical UMI are considered to be derived from a single parent molecule as a result of amplification. In other embodiments, the UMI is used to eliminate artifacts, i.e., variations that exist in the progeny of a single molecule (distinguished by a particular UMI). Such artifacts, which are the result of PCR errors or sequencing errors, can be eliminated using the UMI.

[00126] In some embodiments, the amount or representation of each sequence in a sample can be quantitatively analyzed by counting the relative amounts of sequences at each UMI among a population having the same multiplex sample ID (MID). One of skill in the art can determine the number of sequence reads per UMI ("sequence depth") required to determine a consensus sequence. In some embodiments, the desired depth is 5-50 reads per UMI.

[00127] The utility of adapters and amplification primers for introducing additional elements into a nucleic acid library to be sequenced is described, for example, in U.S. Patent Nos. 9,476,095; 9,260,753; 8,822,150; 8,563,478; 7,741,463; 8,182,989; and 8,053,192.

[00128] In some embodiments, the present invention relates to a method of sequencing a target nucleic acid in a sample comprising the target nucleic acid and non-target nucleic acids, comprising: contacting a double-stranded target nucleic acid having a 3' end with a catalyst having 3'-5' exonuclease activity, thereby producing a first 5' overhang at the first 5' end and a first 3' recessed end; contacting the nucleic acid with an adapter having a second 5' overhang at a second 5' end, the second 5' overhang capable of hybridizing to the first 5' overhang and thereby forming a hybrid between the first and second 5' overhangs; linking at least one of the first 3'-end to the second 5'-end and thereby obtaining an adapter-equipped nucleic acid; and sequencing the adapter-equipped nucleic acid.

[00129] In some embodiments, the length of the captured and sequenced nucleic acid is 1 kb, 10 kb, 50 kb, or more. Recent advances in nanopore sequencing allow reads of 5-20 kb targets when the targets are excised with Cas9 CRISPR endonuclease and captured by dA tailing and adapter ligation, see Cas9 Sequencing Kit Brochure, Cat. No. SQK-CS9109 from Oxford Nanopore (Oxford, UK). The sequencing technology allows reads of sequences greater than 50 kb; However, such sequences are not captured by the dA tailing and ligation method, but are instead captured by transposase-directed insertion of adaptors (tagmentation), see the Ultra Long DNA Sequencing Kit brochure, Cat. No. SQK-ULK0001 from Oxford Nanopore. The present invention has been shown to be more efficient and specific than the dA tailing method, and thus can be used to sequence short sequencing templates as well as templates longer than 20 kb.

[00130] In some embodiments, the present invention includes an amplification step. The adapter-equipped nucleic acids produced by the method described herein can be amplified, for example, prior to sequencing or any other subsequent analysis step. The amplification step can include linear or exponential amplification, such as PCR. The amplification can be isothermal or include thermal cycling. In some embodiments, the amplification is exponential and includes PCR or any of its variants, including real-time PCR, digital droplet PCR (ddPCR), emulsion PCR, and the like.

[00131] In some embodiments, a universal primer is used, i.e., a primer that hybridizes to a universal primer binding site present on all nucleic acids in a sample. In some embodiments, the universal primer binding site is in an adapter produced according to the present invention. In some embodiments, the universal primer binding site is added by extending a primer that hybridizes to the adapter or the target nucleic acid and contains the universal primer binding site in the non-hybridizing 5' region of the primer. All nucleic acids having a universal primer binding site can be amplified using the same universal primer. The number of amplification cycles in which universal primers are used can be low, but can also be 10, 20, or even 30 or more cycles, depending on the amount of amplification product needed for subsequent steps. Since Amplification using universal primers has reduced sequence bias, the number of amplification cycles may not be limited due to the problem of amplification bias.

[00132] In some embodiments, a reverse primer is paired with a first universal primer. The reverse primer is designed to hybridize to a target nucleic acid on a strand opposite to the strand to which the first universal primer hybridizes. In some embodiments, more than one reverse primer is designed to optimally amplify the adapter-equipped nucleic acid (see Fig. 2 for an example with four reverse primers rv-1, rv-2, rv-3, and rv-4). In some embodiments, the reverse primer comprises a binding site for a second universal primer. The second universal primer may be the same or different from the first universal primer. In some embodiments, after one round or a limited number of rounds of amplification with the reverse primer, only the second universal primer paired with the first universal primer may be used for amplification.

[00133] In some embodiments, the present invention relates to a kit for enriching a nucleic acid from a sample using the method of the present invention. The kit comprises at least a catalyst having 3'-5' exonuclease activity and capable of forming a 5' sticky end in nucleic acids; an adapter designed to have a 5' sticky end at the 5' end, a 5' sticky end capable of hybridizing to a 5' sticky end expected in a target nucleic acid after treatment with the catalyst; and a DNA ligase. The kit may further comprise a polynucleotide kinase.

[00134] The adapter in the kit may be single-stranded (i.e., have one 5' end) or double-stranded and have a recessed 3' end at the end with a sticky 5' end.

[00135] As provided herein, in some embodiments, the catalyst having 3'-5' exonuclease activity is a DNA polymerase, such as a family A polymerase, a family B polymerase, or a family C polymerase comprising the Exo I, Exo II, and Exo III domains of the Klenow fragment of Pol I.E. coli; or a DNA polymerase containing aspartate residues corresponding to residues D355, D424, and D501 of the Klenow fragment of Pol IE. coli, or the DNA polymerase is selected from the group consisting of T4 DNA polymerase, RB69 DNA polymerase, Klenow fragment, T7 DNA polymerase, fragment delta Pol IIIE. coli, eukaryotic polymerase epsilon, eukaryotic polymerase delta and mitochondrial polymerase gamma.

[00136] In some embodiments, the kit further comprises one or more of dATP, dCTP, dGTP, and dTTP.

[00137] In some embodiments, the 5' sticky end in the adapter is 2-15 nucleotides in length. In some embodiments, the adapter comprises at least one of a nucleic acid barcode, a sequencing primer binding region, and an amplification primer binding region. The barcode can be a unique molecular ID (UMI) and a sample ID (SID). The adapter can comprise a nucleic acid modification that increases the melting temperature of a nucleic acid duplex comprising the modification. In some embodiments, the adapter is linked to a functional capture moiety selected from biotin, an antigen capable of binding to a capture antibody, and a capture sequence capable of hybridizing to a capture nucleic acid.

[00138] In some embodiments, the kit includes reagents for amplifying nucleic acids, such as a primer capable of annealing to a primer binding site in an adapter (universal primer).

[00139] In some embodiments, the kit includes reagents for sequencing nucleic acids, such as a sequencing primer capable of annealing to a primer binding site in an adapter.

[00140] In some embodiments, the kit further comprises instructions for performing a method of capturing excised nucleic acid using the method described herein.

[00141] In some embodiments, the present invention relates to a reaction mixture for enriching a nucleic acid from a sample. The reaction mixture comprises a double-stranded target nucleic acid having a 3' end; a catalyst having 3'-5' exonuclease activity, capable of forming a first 5' sticky end at a first 5' end and a first recessed 3' end in the target nucleic acid; and an adapter having a second 5' sticky end at a second 5' end, the second 5' sticky end capable of hybridizing with the first 5' sticky end. The reaction mixture may further comprise a DNA ligase.

[00142] In some embodiments, the double-stranded target nucleic acid is 1, 10, or 100 kb in length.

[00143] The 3' end of the double-stranded target nucleic acid in the reaction mixture may be part of a blunt end. The 3' end of the double-stranded target nucleic acid may be part of a 3' sticky end. The 3' end of the double-stranded target nucleic acid can be obtained by cleaving the nucleic acid with a sequence-specific endonuclease, such as a restriction endonuclease, a CRISPR endonuclease, a catalytically inactive CRISPR endonuclease fused to Fok I, a transcription activator-like effector nuclease (TALEN), or a TALEN-Fok I fusion protein, or a zinc finger nuclease (ZFN), or a ZFN-Fok I fusion protein.

[00144] The catalyst having 3'-5' exonuclease activity in the reaction mixture may be a DNA polymerase. The DNA polymerase may be a family A polymerase, a family B polymerase, or a family C polymerase containing the Exo I, Exo II, and Exo III domains of the Klenow fragment of Pol IE. coli. DNA polymerase may also be a DNA polymerase with aspartate residues corresponding to residues D355, D424, and D501 of the Pol I Klenow fragmentE. coli. DNA polymerase can be T4 DNA polymerase, RB69 DNA polymerase, Klenow fragment, T7 DNA polymerase, fragment delta Pol IIIE. coli, eukaryotic polymerase epsilon, eukaryotic polymerase delta and mitochondrial polymerase gamma.

[00145] In some embodiments, the reaction mixture also includes less than four of dATP, dCTP, dGTP, and dTTP, such as three, two, or only one dNTP.

[00146] In some embodiments, the adapter is double-stranded and further comprises a second recessed 3' end. In some embodiments, the overhanging 5' end is 2-15 nucleotides in length. The adapter in the reaction mixture may comprise a nucleic acid barcode (e.g., a unique molecular ID (UMI) or a sample ID (SID)), a sequencing primer binding site, and an amplification primer binding site. The adapter may comprise a nucleic acid modification that increases the melting temperature of a nucleic acid duplex comprising the modification. In some embodiments, the adapter is linked to a functional capture moiety selected from biotin, an antigen capable of binding to a capture antibody, and a capture sequence capable of hybridizing to a capture nucleic acid.

EXAMPLES

[00147] Example 1. Obtaining reagents

[00148] Nucleic acids: plasmid and genomic DNA.

[00149] Plasmids 1 and 2 were prepared using the Macherey-Nagel NucleoSpin Plasmide Mini Kit (available from the Analytics Shop, Stockbridge, Ga.). Human genomic DNA was prepared using the Gentra Puregene kit (Qiagen, Germantown, Md.) from human peripheral blood mononuclear cells. Genomic DNA was dephosphorylated using Quick CIP from New England Biolabs (NEB, Ipswich, Mass.) (cat. #0525): 5 μg DNA was incubated with 3 μl QuickCIP at 37°C for 10 min, then 80°C for 2 min in 1× NEB CutSmart® buffer. The target DNA map is shown in Figure 2.

[00150] The sequence of plasmids P1 and P2 differs from the genomic region in that 1) they do not contain sequences recognized by the Cr-C and Cr-D guides or the rv-3 and rv-4 primer binding sites; and 2) they contain an insert of 574 or 2147 bp, respectively, between the Cr guide sites and the rv primer sites that determines the length of the resulting amplicons.

[00151] Endonuclease

[00152] Preparation of Cas9 ribonucleoproteins (RNPs). cRNA and tracRNA (IDT Cat# 1072532) were dissolved in nuclease-free water at 100 μM. 1 μL of cRNA (any of Cr-A, B, C , or D ), 1 μL of tracRNA, and 8 μL of IDT duplex buffer were combined, then heated to 95°C for 2 min, then allowed to cool to room temperature at a rate of 0.1°C per second to yield a 10 μM cr:tracr guide stock. A 500 nM Cas9:1000 nM guide RNP stock was prepared by combining 10 µl NEB CutSmart® buffer, 79.2 µl nuclease-free water, 10 µl 10 µM guide RNPs, and 0.8 µl 10 mg/ml (62 µM) Cas9 protein, then incubated at 37°C for 15 min. Aliquots were stored at -80°C.

[00153] Adapters

[00154] Adapter stocks consisted of 5 μM of each oligonucleotide in water. Aliquots of 100 μL were heated to 95 °C for 2 min, then allowed to cool to room temperature at a rate of 0.1 °C per second, yielding a stock of 5 μM adapter. The dA-tail adapter was prepared from the dA-top and dA-bottom oligonucleotides. CRISPR-LE adapters were prepared from the ABCD-top oligonucleotide paired with A-bot , B-bot , C-bot , or D-bot to generate specific adapters for reactions using the Cr-A, Cr-B, Cr-C, or Cr-D guides, respectively.

[00155] The adapter and end sequences for each experiment are provided in Table 1 and Table 2. Table 2 additionally provides the dNTPs present in the exonuclease digestion reaction mixture that stop further exonuclease digestion.

[00156] Table 1. Experiment with obtaining a dA tail

sRNA Adapter SEQ ID NO: Cr-A, B, C or D ^5' CAGACACTCACACTAATACTCGT ^3'
_3' GTCGTGAGTGTGTTATGAGCp ₅ SEQ ID NO: 5/
SEQ ID NO:6

[00157] Table 1 (continued). Experiment with obtaining a dA tail

sRNA The resulting end of DNA Cr-A _5' pTTGGGGAG… ^3'
_3' AAACCCCTC … _5' Cr-B ^5' pCACTGGCA… ^3'
_3' A GTGACCGT… _5' Cr-C ^5' pCGCAGGTG… ^3'
_3' A GCGTCCAC… _5' Cr-D ^5' pGTAAGGAG… ^3'
_3' A CATTCCTC… _5'

[00158] Table 2. Nuclease digestion experiment

sRNA Adapter SEQ ID NO of adapter dNTP in the reaction mixture Cr-A ^5' CAGACACTCACACTAATACTCG ^3'
_3' GTCTGTGAGTGTGATTATGAGCAACCCCTCTp ₅ SEQ ID NO:7/SEQ ID NO:8 dGTP Cr-B ^5' CAGACACTCACACTAATACTCG ^3'
_3' GTCTGTGAGTGTGATTATGAGCGTGAp _5' SEQ ID NO:7/SEQ ID NO:9 dCTP Cr-C ^5' CAGACACTCACACTAATACTCG ^3'
_3' GTCTGTGAGTGTGATTATGAGCGCGTCCp ₅ SEQ ID NO:7/SEQ ID NO:10 dATP Cr-D ^5' CAGACACTCACACTAATACTCG ^3'
_3' GTCTGTGAGTGTGATTATGAGCCATTCCTCp ₅ SEQ ID NO:7/SEQ ID NO:11 dGTP

[00159] Table 2 (continued). Nuclease digestion experiment

The resulting end of DNA SEQ ID NO dNTP in the reaction mixture Cr-A ^5' pTTGGGGAGACCACT…… ^3'
_3' GGTGA…… _5' SEQ ID NO:17 dGTP Cr-B ^5' pCACTGGCATCTGG …… ^3'
_3' CCGTAGACC …… ₅ SEQ ID NO:18 dCTP Cr-C ^5' pCGCAGGTGTT …… ^3'
_3' ACAA …… _5' SEQ ID NO:19 dATP Cr-D ^5' pGTAAGGAGCTGCT …… ^3'
_3' GACGA …… _5' SEQ ID NO:20 dGTP

[00160] Example 2. RNP cleavage and dA tailing (prior art method) used with plasmid and genomic DNA samples.

[00161] 200 ng of plasmid DNA was combined with 5 µl of RNP (final concentration 100 nM), 1 µl of 10 µM dATP, 1 µl of NEB Taq (Cat# M0273) in 1x CutSmart® buffer in a total of 25 µl. Reaction mixtures were incubated at 37°C for 15 minutes, then 72°C for 5 minutes (to remove Cas9 from the target DNA and add a dA tail with Taq). Samples were purified with SPRI before proceeding to the ligation step. The method was identical for genomic DNA samples except that 1 µg of phosphorylated genomic DNA was replaced with 200 ng of plasmid DNA. The results for plasmid DNA are shown in Figure 3, and the results for genomic DNA are shown in Figure 5.

[00162] Example 3. RNP cleavage and the CRISPR-LE method (according to the invention) used with respect to plasmid and genomic DNA samples.

[00163] 200 ng of plasmid DNA was combined with 5 µl of RNP (final concentration 100 nM) in 1x CutSmart® buffer in a total of 25 µl. Reaction mixtures were incubated at 37°C for 15 min, then 72°C for 5 min (to remove Cas9 from the target DNA). DNA was purified with 0.8 volume of SPRI and eluted with 15 µl of water. All 15 μl of DNA were then treated with 1 μl of 0.067 mg/ml T4 DNA polymerase, 2.5 mM appropriate dNTP, 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM _MgCl2 , 1 mM DTT in a volume of 20 μl and incubated at 22°C for 30 min, then 75°C for 20 min. DNA was purified with 0.8 volume of SPRI and eluted with 15 μl of water before proceeding to the ligation step. The method was identical for genomic DNA samples except that 1 μg of dephosphorylated genomic DNA was replaced by 200 ng of plasmid DNA.

[00164] The results for plasmid DNA are shown in Figure 4, and the results for genomic DNA are shown in Figure 6 (CrA and CrB rRNA) and Figure 7 (CrC and CrD rRNA).

[00165] Example 4. Intermediate stages of purification

[00166] SPRI purification. SPRI purification was performed using the Beckman Coulter SPRIselect reagent kit (Cat. #B23317). Reaction volumes were first adjusted to 100 µL with water, then 0.8 volume of SPRI beads were added. DNA-SPRI mixtures were incubated on a magnetic stand for 4 min, then the supernatant was removed. The beads were washed 2 times with 85% ethanol, then eluted in 15 µL of water.

[00167] Example 5. Ligation of adapters

[00168] SPRI purified DNA samples (15 µl) were combined with 1 µl NEB Quick Ligase (Cat# M2200) and 250 nM adapter in 1x NEB Quick Ligase Buffer (Cat# B6058S) in a 20 µl reaction volume and incubated for 20 min at room temperature. Ligation products were purified with 0.8 volume SPRI and eluted with 20 µl water before proceeding to the PCR step.

[00169] Example 6: Amplification

[00170] 20 µl PCR mixtures consisted of 0.2 µl NEB Q5 Hot Start High Fidelity DNA Polymerase (Cat. #M0493), 2 µl adapter ligated DNA, 500 nM forward primer ( F ), 500 nM appropriate reverse primer ( rv-1, 2, 3, or 4 ), 200 nM each NTP in 1x Q5 reaction buffer. Thermal cycling conditions were 98°C for 30 sec, (98°C for 10 sec, 58°C for 30 sec, 72°C for 45 sec) 32 times, 72°C for 2 minutes.

[00171] Example 7. Gel electrophoresis .

[00172] 10 μl of each PCR product were run on 1% agarose-TAE gels at 90 volts for 60 min and visualized using the Bio-Rad Gel Doc EZ Imaging System. Expected amplicon sizes (in bp) for each configuration are shown in Table 3.

[00173] Table 3. Expected amplicon sizes

sRNA Reverse primer Expected amplicon sizes, bp Genomic DNA Plasmid P1 Plasmid P2 Cr-A rv-1 620 1194 2767 Cr-A rv-2 535 1109 2682 Cr-A rv-3 1078 1652 3225 Cr-A rv-4 1197 1771 3344 Cr-B rv-1 270 844 2417 Cr-B rv-2 185 759 2332 Cr-B rv-3 728 1302 2875 Cr-B rv-4 847 1421 2994 Cr-C rv-1 748 1322 2895 Cr-C rv-2 663 1237 2810 Cr-C rv-3 1206 1780 3353 Cr-C rv-4 1325 1899 3472 Cr-D rv-1 812 1386 2959 Cr-D rv-2 727 1301 2874 Cr-D rv-3 1270 1844 3417 Cr-D rv-4 1389 1963 3536

[00174] Although the present invention has been described in detail with reference to specific examples, it will be obvious to those skilled in the art that various modifications can be made within the scope of the present invention. Accordingly, the scope of the invention is not limited by the examples presented in the present description, but by the claims below.

[00175] LIST OF SEQUENCES IN ARBITRARY FORM

Oligonucleotide name SEQ ID NO Sequence in 5'-3' direction Cr-A SEQ ID NO:1 CTAATGCCCAGCCTAAGTTG Cr-B SEQ ID NO:2 GCCCCGCCCTTGTCCATCAC Cr-C SEQ ID NO:3 TAGTGCTGGGGCTTAGACGC Cr-D SEQ ID NO:4 GGTGAAATTCCTGAGATGTA dA-top SEQ ID NO:5 CAGACACTCACACTAATACTCGT dA-bottom SEQ ID NO:6 pCGAGTATTAGTGTGAGTGTCTG ABCD top SEQ ID NO:7 CAGACACTCACACTAATACTCG A-bottom SEQ ID NO:8 pTCTCCCCAACGAGTATTAGTGTGAGTGTCTG B-bottom SEQ ID NO:9 pAGTGCGAGTATTAGTGTGAGTGTCTG C-bottom SEQ ID NO:10 pCCTGCGCGAGTATTAGTGTGAGTGTCTG D-bottom SEQ ID NO:11 pCTCCTTACCGAGTATTAGTGTGAGTGTCTG rv-1 SEQ ID NO:12 TGAAGTCCATAGACCTCATGTC rv-2 SEQ ID NO:13 ATCAAAATCGGTGAATAGGCAG rv-3 SEQ ID NO:14 GGAGAAATAAGGAGAGGCAAC rv-4 SEQ ID NO:15 TAATTCCTCCACTTCAACACC F SEQ ID NO:16 CAGACACTCACACTAATACTCG

Legend:

Oligonucleotide name Description Cr-A Targeting region of sRNA A Cr-B Targeting region of sRNA B Cr-C Targeting region of rRNA C Cr-D Targeting region of sRNA D dA-top Top strand, adapter used in the dA-tailing experiment dA-bottom Bottom strand, adapter used in the dA-tailing experiment ABCD top Upper strand common to adapters A-D (endonuclease experiment) A-bot Bottom strand, adapter A (endonuclease experiment) B-bot Bottom strand, adapter B (endonuclease experiment) C-bot Bottom strand, adapter C (endonuclease experiment) D-bot Bottom strand, adapter D (endonuclease experiment) rv-1 Reverse primer for PCR 1 rv-2 Reverse primer for PCR 2 rv-3 Reverse primer for PCR 3 rv-4 Reverse primer for PCR 4 F Forward primer for PCR

[00176] SEQ ID NO: Genomic sequence of 21TRAC (1600 bp) shown in Figure 2:

GGCTCCAACTAACATTTGTTTGGTACTTTACAGTTTATTAAATAGATGTTTATATGGAGAAGCTCTCATTTCTTTCTCAGAAGAGCCTGGCTAGGAAGGTGGATGAGGCACCATATTCATTTTGCAGGTGAAATTCCCTGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAAACGGTAGTGCTGGG GCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTG CCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCAC GAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGC TGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAAC AACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGC CTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTCTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGT CTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACT

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<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q5">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: Cr-C</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q6">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tagtgctggggcttagacgc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q7">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: Cr-D</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q8">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggtgaaattcctgagatgta</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>23</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q9">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: dA-top</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q10">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cagacactcacactaatactcgt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="6">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q11">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic:

dA-bottom</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q12">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q13">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cgagtattagtgtgagtgtctg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="7">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q14">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: ABCD-top</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q15">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cagacactcacactaatactcg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="8">

<INSDSeq>

<INSDSeq_length>31</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..31</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q16">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: A-bottom</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q17">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..31</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q18">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tctccccaacgagtattagtgtgagtgtctg</INSDSeq_sequence

>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>26</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q19">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: B-bottom</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q20">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q21">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>agtgcgagtattagtgtgagtgtctg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>28</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..28</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q22">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: C-bottom</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q23">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..28</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q24">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cctgcgcgagtattagtgtgagtgtctg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="11">

<INSDSeq>

<INSDSeq_length>30</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..30</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q25">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: D-bottom</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q26">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..30</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q27">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctccttaccgagtattagtgtgagtgtctg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="12">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q28">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: rv-1</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q29">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tgaagtccatagacctcatgtc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="13">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q30">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: rv-2</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q31">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atcaaaatcggtgaataggcag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>21</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q32">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: rv-3</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q33">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggagaaataaggagaggcaac</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="15">

<INSDSeq>

<INSDSeq_length>21</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q34">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: rv-4</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q35">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>taattcctccacttcaacacc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="16">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q36">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: F</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q37">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cagacactcacactaatactcg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="17">

<INSDSeq>

<INSDSeq_length>14</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..14</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q38">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: Cr-A</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q39">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..14</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q40">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ttggggagaaccact</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="18">

<INSDSeq>

<INSDSeq_length>13</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..13</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q41">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: Cr-B</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q42">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..13</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q43">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cactggcatctgg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="19">

<INSDSeq>

<INSDSeq_length>10</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..10</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q44">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: Cr-C</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q45">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..10</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q46">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cgcaggtgtt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="20">

<INSDSeq>

<INSDSeq_length>13</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..13</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q47">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: Cr-D</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>modified_base</INSDFeature_key>

<INSDFeature_location>1</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mod_base</INSDQualifier_name>

<INSDQualifier_value>OTHER</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q48">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>5&apos;-phosphate</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..13</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q49">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gtaaggagctgct</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="21">

<INSDSeq>

<INSDSeq_length>1600</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..1600</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q50">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Synthetic: TRAC genomic

sequence</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1600</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q51">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggctccaactaacatttgtttggtactttacagtttattaaatagatgt

ttatatggagaagctctcatttctttctcagaagagcctggctaggaaggtggatgaggcaccatattca

ttttgcaggtgaaattcctgagatgtaaggagctgctgtgacttgctcaaggccttatatcgagtaaacg

gtagtgctggggcttagacgcaggtgttctgatttatagttcaaaacctctatcaatgagagagcaatct

cctggtaatgtgatagatttcccaacttaatgccaacataccataaacctcccattctgctaatgcccag

cctaagttggggagaccactccagattccaagatgtacagtttgctttgctgggcctttttcccatgcct

gcctttactctgccagagttatattgctggggttttgaagaagatcctattaaataaaagaataagcagt

attattaagtagccctgcatttcaggtttccttgagtggcaggccaggcctggccgtgaacgttcactga

aatcatggcctcttggccaagattgatagcttgtgcctgtccctgagtcccagtccatcacgagcagctg

gtttctaagatgctatttcccgtataaagcatgagaccgtgacttgccagccccacagagccccgccctt

gtccatcactggcatctggactccagcctgggttggggcaaagagggaaatgagatcatgtcctaaccct

gatcctcttgtcccacagatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccag

tgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgat

gtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcct

ggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttctt

ccccagcccaggtaagggcagctttggtgccttcgcaggctgtttccttgcttcaggaatggccaggttc

tgcccagagctctggtcaatgatgtctaaaactcctctgattggtggtctcggccttatccattgccacc

aaaaccctctttttactaagaaacagtgagccttgttctggcagtccagagaatgacacgggaaaaaagc

agatgaagagaaggtggcaggagagggcacgtggcccagcctcagtctctccaactgagttcctgcctgc

ctgcctttgctcagactgtttgccccttactgctcttctaggcctcattctaagccccttctccaagttg

cctctccttatttctccctgtctgccaaaaaatctttcccagctcactaagtcagtctcacgcagtcact

cattaacccaccaatcactgattgtgccggcacatgaatgcaccaggtgttgaagtggaggaattaaaaa

gtcagatgaggggtgtgcccagaggaagcaccattctagttgggggagcccatctgtcagctgggaaaag

tccaaataact</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (19)

1. A method for capturing nucleic acid from a sample containing a target nucleic acid and non-target nucleic acids, comprising: a. bringing a double-stranded target nucleic acid having a first 3'-end obtained by cleaving the nucleic acid with a CRISPR endonuclease into contact with a DNA polymerase having a 3'-5'-exonuclease activity, and thereby obtaining a first 5'-sticky end at the first 5'-end and a first 3'-deep end in the target nucleic acid; b. contacting the nucleic acid with an adaptor having a second 5' sticky end at the second 5' end, wherein the second 5' sticky end is capable of hybridizing to the first 5' sticky end to thereby form a hybrid between the first and second 5' sticky ends, wherein the adaptor is linked to a functional capture moiety; and c. ligating the first 3' end to the second 5' end and thereby capturing the target nucleic acid by capturing the functional fragment in the adapter-equipped nucleic acid. 2. The method of claim 1, wherein the adapter further comprises a second recessed 3' end, and the ligation in step c further comprises ligating the first 5' end to the second 3' end. 3. The method of claim 1, wherein the 3' end of the double-stranded target nucleic acid is part of the sticky 3' end. 4. The method of claim 1, wherein the CRISPR endonuclease is a catalytically inactive CRISPR endonuclease fused to Fok I. 5. The method according to claim 1, wherein the DNA polymerase having 3'-5' exonuclease activity is selected from the group consisting of a family A polymerase, a family B polymerase or a family C polymerase containing the Exo I, Exo II and Exo III domains of the Klenow fragment of Pol I of E. coli , a DNA polymerase containing aspartate residues corresponding to residues D355, D424 and D501 of the Klenow fragment of Pol I of E. coli, T4 DNA polymerase, RB69 DNA polymerase, the Klenow fragment, T7 DNA polymerase, the delta fragment of Pol III of E. coli , eukaryotic polymerase epsilon, eukaryotic polymerase delta and mitochondrial polymerase gamma. 6. The method of claim 1, wherein the 3'-5' exonuclease activity is exhibited in the presence of less than four of dATP, dCTP, dGTP and dTTP, and the 3'-5' exonuclease activity stops at a nucleotide identical to one of less than four of dATP, dCTP, dGTP and dTTP. 7. The method according to claim 1, wherein the sticky 5'-end has a length of 2-15 nucleotides. 8. The method according to claim 1, wherein the adapter comprises at least one of a nucleic acid barcode selected from a unique molecular ID (UMI) and a sample ID (SID), a sequencing primer binding region and an amplification primer binding region. 9. The method according to claim 1, wherein the adapter comprises a sequence selected from SEQ ID NO: 7-11. 10. The method of claim 1, further comprising amplifying the captured nucleic acid. 11. The method of claim 10, wherein the amplification primer anneals to a primer binding site in the adapter, and the amplification primer comprises a sequence selected from SEQ ID NO: 12-16. 12. The method according to claim 1, wherein the functional capture fragment is selected from biotin, an antigen capable of binding to a capture antibody, and a capture sequence capable of hybridizing to a capture nucleic acid. 13. The method of claim 1, further comprising sequencing the captured nucleic acid. 14. The method of claim 13, wherein the sequencing primer anneals to a primer binding site present in the amplification primer. 15. The method according to claim 1, wherein the captured nucleic acid has a length of 1, 10 or 100 kb. 16. The method according to claim 1, wherein no purification steps are carried out between steps a. and b. or a. and c.