HK40021189B - Method for manufacturing dna-edited eukaryotic cell, and kit used in method - Google Patents

Description

Methods for manufacturing DNA-edited eukaryotic cells, and kits used in such methods.

Technical Field

This invention relates to methods for manufacturing DNA-edited eukaryotic cells, animals, and plants, and kits used in such methods.

Background Technology

Bacteria and archaea possess adaptive immune mechanisms that specifically recognize and exclude invading organisms such as bacteriophages. The CRISPR-Cas system first inserts the genomic information of the invading organism into its own genome (adaptation). Then, when the same invading organism attempts to invade again, it utilizes the complementarity between the information inserted into its own genome and the genome sequence to cleave and exclude the invading genome (interference).

Recently, genome editing (DNA editing) technology using the aforementioned CRISPR-Cas system as a "tool for DNA editing" has been developed (Non-Patent Literature 1).

The effectors in CRISPR-Cas systems that function during DNA cutting are broadly classified into "Type 1" systems, which consist of multiple Cas cells, and "Type 2" systems, which consist of a single Cas cell. In particular, Type 1 CRISPR-Cas systems, particularly those involving Cas3 and a cascade complex (referring to the complex cascaded with crRNA, hereinafter the same), are widely known ("Type I"). Similarly, Type 2 CRISPR-Cas systems, involving Cas9, are widely known (hereinafter, regarding CRISPR-Cas systems, "Type I" and "Type II" are sometimes simply referred to as "Type I" and "Type II," respectively). Furthermore, Type 2 CRISPR-Cas systems involving Cas9 (hereinafter sometimes also called "CRISPR-Cas9 systems") are widely used in current DNA editing technologies. For example, Non-Patent Literature 1 reports a Type 2 CRISPR-Cas system that uses Cas9 to cut DNA.

On the other hand, despite significant efforts in using the CRISPR-Cas system (hereinafter sometimes referred to as the "CRISPR-Cas3 system"), which uses Cas3 and a cascade complex to cut DNA, successful examples of genome editing in eukaryotic cells have not yet been reported. For example, Non-Patent Documents 2 and 3 report instances where target DNA was completely degraded in a cell-free system and specific E. coli strains were selectively removed using only the CRISPR-Cas3 system, but these do not indicate successful genome editing, and there has been no evidence of success in eukaryotic cells. Furthermore, Patent Document 1 proposes using the FokI nuclease instead of Cas3 for genome editing in eukaryotic cells, based on the CRISPR-Cas3 system's ability to degrade target DNA in E. coli via the helicase and exonuclease activities of Cas3 (Example 5, Figure 6). In addition, Patent Document 2 proposes to retarget target DNA by decomposing target DNA in E. coli using the CRISPR-Cas3 system (Figure 4) by deleting Cas3 and using inactive Cas3 (Cas3’ and Cas3”) (e.g., Example 15, claim 4(e)).

Existing technical documents

Patent documents

Patent Document 1: Japanese Patent Publication No. 2015-503535

Patent Document 2: Japanese Patent Publication No. 2017-512481

Non-patent literature

Non-patent literature 1: Jinek M et al. (2012) A Programmable Dual-RNA Guided DNA Endonuclease in Adaptive Bacterial Immunity, Science, Vol. 337 (Issue 6096), pp. 816-821

Non-patent literature 2: Mulepati S&Bailey S(2013)In Vitro Reconstitution of anEscherichia coli RNA-guided Immune System Reveals Un idirectional,ATP-dependent Degradation of DNA Target,Journal of Biological Chemistry,Vol.288(No.31),pp.22184-22192

Non-patent literature 3: Ahmed A. Gomaa et al. (2014) Programmable Reomoval of Bacterial Strains by Use of Genome Targeting CRISPR-Cas Systems, mbio.asm.org, Volume 5, Issue 1, e00928-13

Summary of the Invention

The problem that the invention aims to solve

This invention was made in view of the circumstances in which the purpose is to establish a CRISPR-Cas3 system in eukaryotic cells.

Methods for solving problems

To achieve the aforementioned objectives, the inventors conducted repeated and in-depth research, ultimately successfully establishing the CRISPR-Cas3 system in eukaryotic cells. The most widely used CRISPR-Cas9 system has successfully performed genome editing in various eukaryotic cells, but in this system, mature crRNA is typically used as the crRNA. Surprisingly, however, genome editing in eukaryotic cells is difficult in the CRISPR-Cas3 system when using mature crRNA; efficient genome editing is only possible by using pre-crRNA, which is not typically used as a component of the system. That is, it was determined that for the CRISPR-Cas3 system to function in eukaryotic cells, the cleavage of crRNA by proteins forming a cascade is crucial. The CRISPR-Cas3 system using this pre-crRNA can be applied not only to type I-E systems but also extensively to type I-F and I-G systems. Furthermore, by adding nuclear localization signals, particularly bipartite nuclear localization signals, to Cas3, the genome editing efficiency of the CRISPR-Cas3 system in eukaryotic cells can be further improved. Furthermore, the inventors discovered that, unlike the CRISPR-Cas9 system, the CRISPR-Cas3 system may be able to cause large deletions in the upstream region due to the inclusion of PAM sequences, thus completing this invention.

That is, the present invention relates to a CRISPR-Cas3 system in eukaryotic cells, and provides the following invention in more detail.

[1] A method for manufacturing DNA-edited eukaryotic cells, the method comprising the step of introducing eukaryotic cells into a CRISPR-Cas3 system, the CRISPR-Cas3 system comprising the following (A) to (C),

(A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide.

(B) cascade proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and

(C)crRNA, the polynucleotide encoding the crRNA, or the expression vector containing the polynucleotide.

[2] A method for producing DNA-edited animals (but not humans) or plants, including the step of introducing an animal (but not humans) or plant into a CRISPR-Cas3 system, the CRISPR-Cas3 system comprising the following (A) to (C),

(A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide.

(B) cascade proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and

(C)crRNA, the polynucleotide encoding the crRNA, or the expression vector containing the polynucleotide.

[3] The method according to [1] or [2] includes the step of cutting crRNA by proteins that constitute a cascade protein after introducing the CRISPR-Cas3 system into eukaryotic cells.

[4] According to the method described in [1] or [2], the crRNA is pre-crRNA.

[5] Nuclear localization signals were added to the Cas3 protein and/or cascade proteins according to any one of [1] to [4].

[6] According to the method described in [5], the nuclear localization signal is a bipartite nuclear localization signal.

[7] A kit for use in any of the methods described in [1] to [6], comprising the following (A) and (B),

(A) Cas3 protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide, and

(B) Cascade protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide.

[8] The kit according to [7] further comprises crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[9] According to the kit described in [8], the crRNA is pre-crRNA.

[10] The kit described in any of [7] to [9] adds nuclear localization signals to the Cas3 protein and/or cascade proteins.

[11] According to the kit described in [10], the nuclear localization signal is a bipartite nuclear localization signal.

Furthermore, in this specification, the term "polynucleotide" refers to a polymer of nucleotides and is used with the same meaning as the terms "gene," "nucleic acid," or "nucleic acid molecule." Polynucleotides can exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). Additionally, the term "protein" is used with the same meaning as "peptide" or "polypeptide."

The effects of the invention

DNA can be edited in eukaryotic cells by using the CRISPR-Cas3 system of this invention.

Attached Figure Description

Figure 1 shows the results of SSA assay for determining the cleavage activity of exogenous DNA.

Figure 2 is a schematic diagram showing the location of the target sequence in the CCR5 gene.

Figure 3A shows the CCR5 gene (clone 1) with a portion of its base sequence deleted using the CRISPR-Cas3 system.

Figure 3B shows the CCR5 gene (clone 2) with a portion of its base sequence deleted using the CRISPR-Cas3 system.

Figure 3C shows the CCR5 gene (clone 3) with a portion of its base sequence deleted using the CRISPR-Cas3 system.

Figure 3D shows the CCR5 gene (clone 4) with a portion of its base sequence deleted using the CRISPR-Cas3 system.

Figure 4: (a) is a schematic diagram showing the structure of a cascaded plasmid. (b) is a schematic diagram showing the structure of a Cas3 plasmid. (c) is a schematic diagram showing the structure of a pre-crRNA plasmid. (d) is a schematic diagram showing the structure of a reporter vector (containing the target sequence).

Figure 5 is a schematic diagram showing the location of the target sequence in the EMX1 gene.

Figure 6A shows the EMX1 gene (clone 1) with a portion of its base sequence deleted using the CRISPR-Cas3 system.

Figure 6B shows the EMX1 gene (clone 2) with a portion of its base sequence deleted using the CRISPR-Cas3 system.

Figure 7 is a schematic diagram showing the structure of the Cas3/cascade plasmid with added bpNLS.

Figure 8 is a schematic diagram showing the structure of the cascaded (2A) plasmid.

Figure 9 shows the results of SSA assay for determining the cleavage activity of exogenous DNA.

Figure 10A is a diagram showing the structure of the pre-crRNA (LRSR and RSR) and mature crRNA used in this embodiment. The underline in the figure indicates the 5’ handle (Cas5 handle), and the double underline indicates the 3’ handle (Cas6 handle).

Figure 10B shows the results of SSA assays using pre-crRNA (LRSR and RSR) and mature crRNA.

Figure 11 shows the results of SSA assays performed using one or two NLS (bpNLS) in plasmids for the expression of the Cas3/cascade gene.

Figure 12 shows the effect of the PAM sequence on the DNA cleavage activity of the CRISPR-Cas3 system.

Figure 13 is a graph showing the effect of a single mispairing of the spacer on the DNA cleavage activity of the CRISPR-Cas3 system.

Figure 14 shows the effect of mutations in Cas3 in the HD nuclease domain (H74A), SF2 helicase domain motif 1 (K320A), and motif 3 (S483/T485A).

Figure 15 is a graph comparing the DNA cleavage activities of CRISPR-Cas3 systems of types I-E, I-F, and I-G.

Figure 16 is a graph showing the size of the deletion generated by the CRISPR-Cas3 system as detected by sequencing of TA clone samples of PCR products.

Figure 17 is a diagram showing the locations of deletions generated by the CRISPR-Cas3 system, detected by mass sequencing of TA clones (n=49).

Figure 18A is a graph showing the number of deletions detected by the CRISPR-Cas3 system for each deletion size obtained by microarray-based capture sequencing of more than 1000 kb around the target EMX1 locus.

Figure 18B is a graph showing the number of detections for each deletion size generated by the CRISPR-Cas3 system, obtained by microarray-based capture sequencing of more than 1000 kb around the target CCR5 locus.

Detailed implementation method:

[1] Methods for manufacturing eukaryotic cells, animals, and plants with edited DNA

The method of the present invention includes the step of introducing a CRISPR-Cas3 system into eukaryotic cells, the CRISPR-Cas3 system comprising the following methods (A) to (C).

(A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide.

(B) cascade proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and

(C) crRNA, the polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

The CRISPR-Cas system classifies CRISPR-Cas into types I and III. Type I is further classified into six subtypes—I-A, I-B, I-C, I-D, I-E, and I-F—based on the types of proteins that make up the cascade (hereinafter referred to simply as "cascade" or "cascade proteins"), and subtypes I-G as subtypes of type I-B (see, for example, [van der Oost Je et al. (2014) Unravelling the structural and mechanistic basis of CRISP)). R-Cas systems,NatureReviews Microbiologym,Vol.12(No.7),pp.479-492],[Jackson RN et al.(2014)Fitting CRISP R-associated Cas3 into the Helicase Family Tree,Current Opinionin Structural Biology,Vol.24,pp.106-114]).

The type I CRISPR-Cas system enables DNA cleavage through Cas3 (a protein with nuclease and helicase activity), cascade, and crRNA synergy. Because Cas3 is used as the nuclease, it is referred to as the "CRISPR-Cas3 system" in this invention.

By using the CRISPR-Cas3 system of the present invention, the following advantages can be obtained, for example.

First, the crRNA used in the CRISPR-Cas3 system generally recognizes target sequences of 32–37 bases (Ming Li et al., Nucleic Acids Res. 2017 May 5; 45(8):4642-4654). In contrast, the crRNA used in the CRISPR-Cas9 system generally recognizes target sequences of 18–24 bases. Therefore, it is believed that the CRISPR-Cas3 system can recognize target sequences more accurately than the CRISPR-Cas9 system.

Furthermore, the PAM sequence of the CRISPR-Cas9 system, a type II system of class 2, is "NGG" (where N is any base) adjacent to the 3' side of the target sequence. Similarly, the PAM sequence of the CRISPR-Cpf1 system, a type V system of class 2, is "AA" adjacent to the 5' side of the target sequence. In contrast, the PAM sequence of the CRISPR-Cas3 system of the present invention is "AAG" or a similar base sequence (e.g., "AGG", "GAG", "TAC", "ATG", "TAG", etc.) adjacent to the 5' side of the target sequence (Figure 12). Therefore, it is believed that if the CRISPR-Cas3 system of the present invention is used, regions that cannot be recognized by existing methods can be targeted for DNA editing.

Furthermore, unlike the two types of CRISPR-Cas systems mentioned above, the CRISPR-Cas3 system generates DNA cuts at multiple sites. Therefore, if the CRISPR-Cas3 system of this invention is used, it is possible to generate large-scale deletion mutations ranging from hundreds to thousands of bases, and in some cases, more than thousands of bases (Figures 3, 6, 16-18). It is believed that this function can be utilized for knocking out long genomic regions and knocking in long DNA. During knock-in, donor DNA is typically used, which also constitutes a molecule in the CRISPR-Cas3 system of this invention.

Furthermore, in this specification, when referred to only as "Cas3," it means "Cas3 protein." The same applies to cascade proteins.

The CRISPR-Cas3 system of this invention includes all six subtypes of type I. That is, the proteins constituting the CRISPR-Cas3 system sometimes differ in composition, etc., depending on the subtype (for example, the proteins constituting the cascade may differ), and this invention includes all of these proteins. In fact, this embodiment demonstrates that genome editing can be performed not only in types I-E, but also in types 1-G and I-F (Figure 15).

In type I CRISPR-Cas3 systems, typical type I-E CRISPR-Cas3 systems work together with crRNA, Cas3, and a cascade (Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7) to cut DNA.

In type I-A systems, the cascade consists of Cas8a1, Csa5 (Cas11), Cas5, Cas6, and Cas7. In type I-B, the cascade consists of Cas8b1, Cas5, Cas6, and Cas7. In type I-C, the cascade consists of Cas8c, Cas5, and Cas7. In type I-D, the cascade consists of Cas10d, Csc1 (Cas5), Cas6, and Csc2 (Cas7). In type I-F, the cascade consists of Csy1 (Cas8f), Csy2 (Cas5), Cas6, and Csy3 (Cas7). In type I-G systems, the cascade consists of Cst1 (Cas8a1), Cas5, Cas6, and Cst2 (Cas7). In this invention, Cas3 and the cascade are collectively referred to as the "Cas protein group".

The following explanation uses the I-E type CRISPR-Cas3 system as a representative example. However, for other types of CRISPR-Cas3 systems, it is appropriate to replace the reading with a cascade of the system components.

-Cas protein groups-

In the CRISPR-Cas3 system of the present invention, Cas protein groups can be introduced into eukaryotic cells in the form of proteins, polynucleotides encoding the proteins, or expression vectors containing the polynucleotides. When introducing Cas protein groups into eukaryotic cells in the form of proteins, the amount of each protein can be appropriately adjusted, which is preferable from a processing point of view. Furthermore, considering intracellular cleavage efficiency, a complex of the Cas protein groups can be formed first before introduction into the eukaryotic cells.

In this invention, it is preferable to add nuclear localization signals to the Cas protein groups. The nuclear localization signals can be added to the N-terminal and/or C-terminal sides of the Cas protein groups (the 5' and/or 3' ends of the polynucleotides encoding each Cas protein group). Thus, by adding nuclear localization signals to the Cas protein groups, there is the advantage of promoting nuclear localization within the cell, resulting in efficient DNA editing.

The aforementioned nuclear localization signal is a peptide sequence consisting of several to dozens of basic amino acids. The sequence is not particularly limited as long as it enables protein translocation into the nucleus. Specific examples of such nuclear localization signals are described, for instance, in [Wu J et al. (2009) The Intracellular Mobility of Nuclear Import Receptors and NLS Cargoes, Biophysical Journal, Vol. 96 (Issue 9), pp. 3840-3849]. Any nuclear localization signal commonly used in this technical field can be used in this invention.

Nuclear localization signals can be, for example, PKKKRKV (Sequence No. 52) (encoded by the base sequence CCCAAGAAGAAGCGGAAGGTG (Sequence No. 53)). When using the above nuclear localization signal, it is preferable, for example, to place a polynucleotide consisting of the base sequence of Sequence No. 53 at the 5' end of each polynucleotide encoding the Cas protein group. Alternatively, nuclear localization signals can be, for example, KRTADGSEFESPKKKRKVE (Sequence No. 54) (encoded by the base sequence AAGCGGACTGCTGATGGCAGTGAATTTGAGTCCCCAAAGAAGAAGAGAAAGGTGGAA (Sequence No. 55)). When using the above nuclear localization signal, it is preferable, for example, to place a polynucleotide consisting of the base sequence of Sequence No. 55 at both ends of each polynucleotide encoding the Cas protein group (i.e., using a "bipartite nuclear localization signal (bpNLS)").

Such changes, in conjunction with the use of pre-crRNA described later, are important for enabling the CRISPR-Cas3 system of the present invention to be effectively expressed and function in eukaryotic cells.

A preferred embodiment of the Cas protein group used in this invention is as follows.

Cas3: A protein encoded by a polynucleotide consisting of a sequence of bases represented by Serial No. 1 or Serial No. 7.

Cse1 (Cas8): A protein encoded by a polynucleotide consisting of a sequence of bases represented by Serial Number 2 or Serial Number 8.

Cse2 (Cas11): A protein encoded by a polynucleotide consisting of a sequence of bases indicated by sequence number 3 or 9.

Cas5: A protein encoded by a polynucleotide consisting of a sequence of bases represented by Serial Number 4 or Serial Number 10.

Cas6: A protein encoded by a polynucleotide consisting of the base sequence shown in Serial Number 5 or Serial Number 11.

Cas7: A protein encoded by a polynucleotide consisting of the base sequence shown in Serial Number 6 or Serial Number 12.

The aforementioned Cas protein group consists of (1) proteins with PKKKRKV (Sequence No. 52) added as a nuclear localization signal to the N-terminus of Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7 in wild-type E. coli, or (2) proteins with KRTADGSEFESPKKKRKVE (Sequence No. 54) added as nuclear localization signals to the N-terminus and C-terminus of Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7 in wild-type E. coli. By producing proteins with such amino acid sequences, the aforementioned Cas protein group can be transferred into the nucleus of eukaryotic cells. The Cas protein group transferred into the nucleus then cleaves the target DNA. Furthermore, even in DNA regions with robust structures (such as heterochromatin), which are considered difficult for the CRISPAR-Cas9 system, editing of the target DNA can be achieved.

Another form of the proteins of the Cas protein group used in this invention is a protein encoded by a base sequence having more than 90% sequence identity with the base sequence of the aforementioned Cas protein group. Another form of the proteins of the Cas protein group used in this invention is a protein encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide composed of a polynucleotide consisting of a complementary base sequence of the aforementioned Cas protein group. The aforementioned proteins possess DNA cleavage activity when forming complexes with other proteins constituting the Cas protein group. Furthermore, the meanings of terms such as "sequence identity" and "stringent conditions" will be explained later.

-Polynucleotides encoding the Cas protein group-

The polynucleotides encoding wild-type proteins constituting the I-E type CRISPR-Cas system contain polynucleotides that have been modified for efficient expression in eukaryotic cells. That is, modified polynucleotides encoding the Cas protein group can be used. A preferred approach to modifying the polynucleotides is to alter the base sequence to adapt to expression in eukaryotic cells, for example, by optimizing the codons for expression in eukaryotic cells.

A preferred mode for encoding the polynucleotides of the Cas protein group used in this invention is as follows.

Cas3: A polynucleotide consisting of the base sequence shown in Serial No. 1 or Serial No. 7.

Cse1 (Cas8): A polynucleotide consisting of the base sequence shown in sequence number 2 or sequence number 8.

Cse2 (Cas11): A polynucleotide consisting of the base sequence shown in sequence number 3 or 9.

Cas5: A polynucleotide consisting of the base sequence shown in sequence number 4 or sequence number 10.

Cas6: A polynucleotide consisting of the base sequence shown in sequence number 5 or sequence number 11.

Cas7: A polynucleotide consisting of the base sequence shown in sequence number 6 or sequence number 12.

These are polynucleotides that can be expressed and function in mammalian cells by artificially altering the base sequences of the wild-type Cas protein group (Cas3: sequence number 13, Cse1 (Cas8): sequence number 14, Cse2 (Cas11): sequence number 15, Cas5: sequence number 16, Cas6: sequence number 17, Cas7: sequence number 18) of Escherichia coli.

The aforementioned artificial alterations to the polynucleotides involve modifying the base sequences to be suitable for expression in eukaryotic cells and adding nuclear localization signals. The changes to the base sequences and the addition of nuclear localization signals are described above. Therefore, a more substantial increase in the expression levels and functional enhancement of the Cas protein population can be expected.

Another way to encode the Cas protein group used in this invention is through a polynucleotide whose base sequence has more than 90% sequence identity with the aforementioned Cas protein group, but which modifies the base sequence encoding the wild-type Cas protein group. Proteins expressed by these polynucleotides have DNA-cleaving activity when forming complexes with proteins expressed by other polynucleotides that constitute the Cas protein group.

The sequence identity of the base sequences can be at least 90%, more preferably 95% or more (e.g., 95%, 96%, 97%, 98%, 99% or more) across the entire base sequence (or the region required to encode the function of Cse3). The sequence identity can be determined using procedures such as BLASTN (see [Altschul SF (1990) Basic local alignment search tool, Journal of Molecular Biology, Vol. 215 (Issue 3), pp. 403-410]). As an example of parameters for analyzing base sequences using BLASTN, a setting of score = 100 and word length = 12 can be cited. Specific methods for analysis using BLASTN are known to those skilled in the art. Additions or deletions (gap, etc.) are permitted to arrange the base sequences of the comparison objects in an optimal state.

In addition, "having DNA cleavage activity" means being able to cleave a polynucleotide chain at at least one location.

The CRISPR-Cas3 system of the present invention preferably specifically recognizes the target sequence and cuts the DNA. Whether the CRISPR-Cas3 system specifically recognizes the target sequence can be determined, for example, by a dual-luciferase assay as described in Example A-1.

Another way to encode the polynucleotides of the Cas protein group used in this invention is through hybridization under stringent conditions with polynucleotides composed of complementary base sequences of the aforementioned Cas protein group. Proteins expressed by these polynucleotides exhibit DNA cleavage activity when forming complexes with proteins expressed by other polynucleotides constituting the Cas protein group.

"Strict conditions" refer to conditions under which two polynucleotide chains form a base sequence-specific double-stranded polynucleotide, but do not form a non-specific double-stranded polynucleotide. "Hybridization under strict conditions" can also be described as conditions within which hybridization can occur between nucleic acids with high sequence identity (e.g., perfectly paired hybrids) at a temperature range from the melting temperature (Tm value) to 15°C below the melting temperature, preferably 10°C below the melting temperature, and more preferably 5°C below the melting temperature.

If a stringent condition is indicated, the procedure is as follows: First, the two polynucleotides are hybridized for 16–24 hours in a buffer solution (pH 7.2) consisting of _0.25 M _Na₂HPO₄ , 7% SDS, 1 mM EDTA, and 1× Denhardt's solution at 60–68°C (preferably 65°C, more preferably 68°C). Then, the mixture is washed twice for 15 minutes each in a buffer solution (pH 7.2) consisting of ₂₀ mM _Na₂HPO₄ , 1% SDS, and 1 mM EDTA at 60–68°C (preferably 65°C, more preferably 68°C).

As another example, the following method is listed. First, in a hybridization solution containing 25% formamide (50% formamide under more stringent conditions), 4×SSC (sodium chloride/sodium citrate), 50mM Hepes (4-hydroxyethylpiperazine ethanesulfonic acid, pH 7.0), 10×Denhardt’s solution, and 20μg/mL denatured salmon sperm DNA, pre-hybridization is performed overnight at 42°C. Then, a labeled probe is added, and the mixture is incubated at 42°C overnight to hybridize the two polynucleotides.

Next, wash under any of the following conditions: Normal conditions: Wash at approximately 37°C using a washing solution of 1×SSC and 0.1% SDS. Strict conditions: Wash at approximately 42°C using a washing solution of 5×SSC and 0.1% SDS. Even more stringent conditions: Wash at approximately 65°C using a washing solution of 0.2×SSC and 0.1% SDS.

Thus, the more stringent the washing conditions for hybridization, the more specific the hybridization. Furthermore, the combination of SSC, SDS, and temperature conditions described above is merely illustrative. The same stringency as described above can be achieved by appropriately combining the above-mentioned elements, or other elements (e.g., probe concentration, probe length, hybridization reaction time, etc.), which determine the stringency of hybridization. This is described, for example, in [Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001].

- An expression vector containing multiple nucleotides encoding the Cas protein group-

In this invention, expression vectors for expressing the Cas protein group can be used. Various vectors commonly used as substrate vectors can be used, and the appropriate vector can be selected based on the cells to which the expression is performed or the method of expression. Specifically, plasmids, bacteriophages, granules, etc., can be used. The specific type of vector is not particularly limited; any vector capable of expression in the host cells can be selected.

Examples of the aforementioned expression vectors include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosome vectors, episome vectors, and virus-derived vectors (bacterial plasmids, phages, yeast episomes, etc.), yeast chromosome elements and viruses (baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, avipoxviruses, pseudorabies viruses, herpesviruses, lentiviruses, retroviruses, etc.), and vectors derived from combinations thereof (plasmids, phage particles, etc.).

The expression vector is further preferably equipped with sites for transcription initiation and termination, and the transcription region contains a ribosome-binding site. The coding portion of the mature transcript in the vector contains the transcription initiation codon AUG at the beginning of the polypeptide to be translated and a stop codon at an appropriate position at the end.

In this invention, the expression vector for expressing the Cas protein group may contain a promoter sequence. The promoter sequence may be appropriately selected based on the type of eukaryotic cell that serves as the host. Additionally, the expression vector may contain sequences for enhancing transcription from DNA, such as enhancer sequences. Examples of enhancers include, for example, the SV40 enhancer (located 100–270 bp downstream of the origin of replication), the cytomegalovirus initial promoter enhancer, a polyomavirus enhancer located downstream of the origin of replication, and an adenovirus enhancer. Furthermore, the expression vector may contain sequences for stabilizing the transcribed RNA, such as polyA addition sequences (polyadenylation sequences, polyA). Examples of polyA addition sequences include polyA addition sequences derived from the growth hormone gene, bovine growth hormone gene, human growth hormone gene, SV40 virus, and human or rabbit β-globin gene.

The number of polynucleotides encoding Cas protein groups inserted into the same vector is not particularly limited, as long as the CRISPR-Cas system can function within the host cell where the expression vector has been introduced. For example, it could be a design where polynucleotides encoding Cas protein groups are carried in one (same) vector, or further, where all or part of the polynucleotides encoding each Cas protein group are carried in different vectors. For example, it could be a design where polynucleotides encoding cascade proteins are carried in one (same) vector, and polynucleotides encoding Cas3 are carried in other vectors. From the viewpoint of expression efficiency, it is preferable to use a method that carries polynucleotides encoding each Cas protein group in six different vectors.

Furthermore, for purposes such as adjusting expression levels, multiple polynucleotides encoding the same protein can be carried in the same vector. For example, a design could be used to place the polynucleotide encoding Cas3 in two locations within one (same) vector.

Alternatively, an expression vector can be used containing multiple base sequences encoding Cas protein groups, with base sequences encoding amino acid sequences (such as 2A peptides) inserted between these multiple base sequences (see, for example, the structure of the vector in Figure 8). If a polynucleotide having such a base sequence is transcribed/translated, a polypeptide chain linked together is expressed intracellularly. Then, through the action of intracellular proteases, the Cas protein groups are separated, forming individual proteins that then form a complex to perform their function. Thus, the ratio of Cas protein groups expressed intracellularly can be adjusted. For example, it can be predicted that "an expression vector containing a base sequence encoding Cas3 and a base sequence encoding Cse1 (Cas8) can be used," with Cas3 and Cse1 (Cas8) expressed in equal amounts. Furthermore, since multiple Cas protein groups can be expressed in a single expression vector, it is advantageous in terms of operability. On the other hand, from the viewpoint of high DNA cleavage activity, it is generally superior to express Cas protein groups using separate expression vectors.

The expression vectors used in this invention can be prepared by known methods. In addition to the methods described in the instruction manual accompanying the vector preparation kit, various other guidelines can be cited as examples. For instance, [Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001] is a general guideline.

-crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide- The CRISPR-Cas3 system of the present invention contains crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide for targeting DNA for genome editing.

crRNA is an RNA that forms part of the CRISPR-Cas system and has a base sequence complementary to the target sequence. The CRISPR-Cas3 system of this invention can specifically recognize and cleave the target sequence using crRNA. In CRISPR-Cas systems, such as the CRISPR-Cas9 system, mature crRNA has been commonly used as cRNA until now. However, while the reasons are unclear, it is clearly unsuitable to use mature crRNA when enabling the CRISPR-Cas3 system to function in eukaryotic cells. Surprisingly, it has been determined that genome editing can be performed efficiently in eukaryotic cells by using pre-crRNA instead of mature crRNA. This fact is evident from comparative experiments of mature crRNA and pre-crRNA (Figure 10). Therefore, pre-crRNA is particularly preferred as the crRNA of this invention.

The pre-crRNA used in this invention typically has a structure of "leader sequence-repetitive sequence-spacer sequence-repetitive sequence (LRSR structure)" or "repetitive sequence-spacer sequence-repetitive sequence (RSR structure)". The leader sequence is an AT-enriched sequence that functions as a promoter for expressing the pre-crRNA. The repetitive sequence is a sequence repeated between spacer sequences, which are sequences designed in this invention as complementary to the target DNA (originally sequences introduced during adaptation from foreign DNA sources). The pre-crRNA is cleaved by proteins that form a cascade (e.g., Cas6 in types I-A, B, D-E, and Cas5 in types I-C) to become mature crRNA.

Typically, the leader sequence is 86 bases long, and the repeat sequence is 29 bases long. The spacer sequence is, for example, 10–60 bases long, preferably 20–50 bases long, more preferably 25–40 bases long, and typically 32–37 bases long. Therefore, the pre-crRNA used in this invention has a chain length of, for example, 154–204 bases long in the case of an LRSR structure, preferably 164–194 bases long, more preferably 169–184 bases long, and typically 176–181 bases long in the case of an RSR structure. Furthermore, in the case of an RSR structure, it is, for example, 68–118 bases long, preferably 78–108 bases long, more preferably 83–98 bases long, and typically 90–95 bases long.

It is considered important that the repetitive sequence of pre-crRNA is cleaved by the proteins constituting the cascade in order for the CRISPR-Cas3 system of the present invention to function in eukaryotic cells. Therefore, it should be understood that the repetitive sequence can be shorter or longer than the aforementioned chain length, provided such cleavage occurs. That is, pre-crRNA can be described as crRNA with sequences sufficiently cleaved by the proteins constituting the cascade added to both ends of the mature crRNA described later. A preferred embodiment of the method of the present invention includes the step of cleaving the crRNA by the proteins constituting the cascade after the CRISPR-Cas3 system is introduced into eukaryotic cells.

On the other hand, mature crRNA, generated by cleaving pre-crRNA, has a structure of "5' handle sequence - spacer sequence - 3' handle sequence". Typically, the 5' handle sequence consists of 8 bases from positions 22 to 29 of the repeat sequence and is held by Cas5. Additionally, the 3' handle sequence typically consists of 21 bases from positions 1 to 21 of the repeat sequence, forming a stem-loop structure with bases from positions 6 to 21, and is held by Cas6. Therefore, the chain length of mature crRNA is usually 61–66 bases. However, depending on the type of CRISPR-Cas3 system, there are also mature crRNAs without a 3' handle sequence, in which case the chain length becomes shorter, down to 21 bases.

Furthermore, the RNA sequence can be appropriately designed based on the target sequence for which DNA editing is desired. Additionally, RNA synthesis can be performed using any method known in the field.

-Eukaryotic cells-

Examples of "eukaryotic cells" in this invention include, for example, animal cells, plant cells, algal cells, and fungal cells. Examples of animal cells include, for example, mammalian cells, and cells of fish, birds, reptiles, amphibians, and insects.

"Animal cells" include, for example, cells that constitute an individual animal, cells that constitute organs or tissues removed from an animal, and cultured cells derived from animal tissues. Specifically, examples include, for instance, germ cells such as oocytes and sperm; embryonic cells at various stages of the embryo (e.g., 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, and morula stages); stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs); and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, brain cells, and kidney cells. Oocytes used in the creation of genome-edited animals can be pre-fertilized or post-fertilized oocytes, but post-fertilized oocytes, i.e., zygotes, are preferred. Pronuclear embryos are particularly preferred. Oocytes can be thawed from cryopreservation for use.

In this invention, "mammal" encompasses both human and non-human mammals. Examples of non-human mammals include even-toed ungulates such as cattle, wild boars, domestic pigs, sheep, and goats; odd-toed ungulates such as horses; rodents such as mice, rats, guinea pigs, hamsters, and squirrels; lagomorphs such as rabbits; and carnivores such as dogs, cats, and weasels. These non-human mammals can be domesticated animals or companion animals (pets), or they can be wild animals.

Examples of "plant cells" include, for example, the cells of cereals, oil crops, forage crops, fruits, and vegetables. "Plant cells" also include, for example, the cells that constitute an individual plant, the cells that constitute organs or tissues separated from a plant, and cultured cells derived from plant tissues. Examples of plant organs or tissues include, for example, leaves, stems, shoot tips (growing points), roots, tubers, and callus tissue. Examples of plants include, for example, rice, corn, bananas, peanuts, sunflowers, tomatoes, Arabidopsis thaliana, tobacco, wheat, barley, potatoes, soybeans, cotton, and carnations, as well as their propagation materials (e.g., seeds, tubers, rhizomes, etc.).

-DNA Editing-

In this invention, "editing eukaryotic cell DNA" can refer to a process of editing eukaryotic cell DNA in vivo or in vitro. Furthermore, "editing DNA" refers to operations exemplified in the following types (including combinations thereof).

Furthermore, the DNA used in this specification under the above provisions is not limited to DNA existing in the cell nucleus, but also includes DNA existing outside the cell nucleus, such as mitochondrial DNA, and exogenous DNA.

1. Cut the DNA strands in the target area.

2. Cause the deletion of bases in the DNA strand at the target site.

3. Insert bases into the DNA strand at the target site.

4. Replace the bases in the DNA strand at the target site.

5. Modify the bases of the DNA strand in the target site.

6. Regulate the transcription of DNA (genes) in the target region.

In one embodiment of the CRISPR-Cas3 system of the present invention, a protein having enzymatic activity that modifies target DNA is utilized by means other than DNA cutting. This embodiment can be achieved, for example, by fusing Cas3 or a cascade with a heterologous protein having the desired enzymatic activity to create a chimeric protein. Therefore, the terms "Cas3" and "cascade" in the present invention also include such fused proteins. Examples of enzymatic activities of the fused protein include, but are not limited to, deaminase activity (e.g., cytidine deaminase activity, adenosine deaminase activity), methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, photorepair enzyme activity, and glycosylation enzyme activity. At this point, the nuclease and helicase activities of Cas3 are not necessarily required. Therefore, as Cas3, mutants that have partially or completely lost these activities can be used (e.g., mutants of the D domain H74A (dnCas3), mutants of the SF2 domain motif 1 K320N (dhCas3), and double mutants of the SF2 domain motif 3 S483A/T485A (dh2Cas3)). For example, by using a fusion protein of a mutant that has partially or completely lost the nuclease activity of Cas3 with a deaminase as a component of the CRISPR-Cas3 system of the present invention, precise genome editing can be performed without producing large deletions or substitutions in the target site. The method of applying deaminase to the CRISPR-Cas system is well known (Nishida K. et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems, Science, DOI:10.1126/science.aaf8729, (2016)), and can be applied to the CRISPR-Cas3 system of this invention.

In other embodiments of the CRISPR-Cas3 system of the present invention, transcription of genes at the binding site of the system is regulated without cleaving the DNA. This can be achieved, for example, by fusing Cas3 or a cascade with a desired transcriptional regulatory protein to create a chimeric protein. Therefore, the terms "Cas3" and "cascade" in the present invention also include such fused proteins. Examples of transcriptional regulatory proteins include, but are not limited to, photoinducible transcriptional control factors, small molecule/drug-responsive transcriptional control factors, transcription factors, transcriptional repressors, etc. In this case, nuclease activity and helicase activity of Cas3 are not necessarily required; therefore, mutants lacking some or all of these activities can be used as Cas3 (e.g., mutants of the D domain H74A (dnCas3), mutants of the SF2 domain motif 1 K320N (dhCas3), and double mutants of the SF2 domain motif 3 S483A/T485A (dh2Cas3)). Methods for applying transcriptional regulatory proteins to the CRISPR-Cas system are well known to those skilled in the art.

Furthermore, in the CRISPR-Cas3 system of the present invention, for example, in the case of using a mutant that partially or completely omits the nuclease activity of Cas3, other proteins with nuclease activity can also be fused with Cas3 or in a cascade. Such methods are also included in the present invention.

Furthermore, in the CRISPR-Cas3 system of the present invention, when using mutants that partially or completely delete the nuclease activity of Cas3 to utilize the activity of other proteins in DNA editing, the term "DNA cutting activity" in this specification may be appropriately replaced with the various activities possessed by those other proteins.

Furthermore, DNA editing can be performed on the DNA contained in specific cells within an individual. For example, such DNA editing can target specific cells within the cells that make up an individual of an animal or plant.

The method for introducing the molecules constituting the CRISPR-Cas3 system of this invention into eukaryotic cells in the form of polynucleotides or expression vectors containing such polynucleotides is not particularly limited. Examples include electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, cationic liposome-mediated transfection, electroporation, transduction, infection using viral vectors, etc. Such methods are described in numerous standard laboratory manuals, such as "Leonard G. Daviset al., Basic methods in molecular biology, New York: Elsevier, 1986".

The method for introducing molecules into eukaryotic cells in the form of proteins using the CRISPR-Cas3 system of the present invention is not particularly limited. For example, electroporation, cationic liposome-mediated transfection, microinjection, etc.

The DNA editing techniques employed in this invention can be applied in a variety of fields. These applications include, for example, gene therapy, breed improvement, the creation of transgenic animals or cells, the production of useful substances, and life science research.

Known methods can be used as a means of creating non-human individuals from cells. In creating non-human individuals from cells in animals, germ cells or pluripotent stem cells are typically used. For example, molecules constituting the CRISPR-Cas3 system of this invention are introduced into oocytes, and the resulting oocytes are then transplanted into the uterus of a female non-human mammal in a pseudopregnancy state, resulting in offspring. Transplantation can be performed using fertilized eggs from 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, or morula stages. Oocytes are cultured under appropriate conditions as needed until transplantation. Oocyte transplantation and culture are based on existing known methods (Nagy A. et al., Manipulating the Mouse Embryo. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press, 2003). From the resulting non-human individual, desired DNA-edited offspring or clones can also be obtained.

Furthermore, plants have been known since ancient times to possess the totipotency of their somatic cells, and methods for regenerating plant bodies from plant cells have been established in various plants. Therefore, for example, by introducing the molecules constituting the CRISPR-Cas3 system of this invention into plant cells, and regenerating plant bodies from the resulting plant cells, it is possible to obtain plant bodies with the desired DNA knocked in. From the obtained plant bodies, it is also possible to obtain offspring, clones, or propagation materials with the desired DNA edited. As a method for obtaining individuals by redifferentiating plant tissues through tissue culture, the methods established in this technical field can be utilized (Form and Material Transformation Program [Plant Edition], edited by Yutaka Tabei, Chemical Writers, pp. 340-347 (2012)).

[2] Reagent kits used in the CRISPR-Cas3 system

The kit used in the CRISPR-Cas3 system of the present invention comprises the following (A) and (B).

(A) Cas3 protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide, and

(B) Cascade protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide.

It may also contain crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

The components of the kit of the present invention can be all or partly mixed, or they can be independent of each other.

The kit of the present invention can be used in fields such as pharmaceuticals, food, animal products, aquatic products, industry, bioengineering, and life science research.

The following describes the contemplated pharmaceutical preparations (reagents) for the kit of the present invention. Furthermore, when using the above-described kit in fields such as animal husbandry, bioengineering, and life science research, the following description can be adapted by making appropriate substitutions based on common technical knowledge in those fields.

Pharmaceuticals for editing DNA containing human animal cells using the CRISPR-Cas3 system of the present invention can be prepared by conventional methods. More specifically, they can be prepared by blending the molecules constituting the CRISPR-Cas3 system of the present invention described above with, for example, pharmaceutical additives.

"Pharmaceutical additives" refer to substances in a pharmaceutical product other than the active ingredient. Pharmaceutical additives are substances included in a pharmaceutical product to facilitate formulation, stabilize quality, and improve efficacy. For example, these pharmaceutical additives may include excipients, binders, disintegrants, lubricants, flow agents (anti-caking agents), colorants, capsule coatings, coating agents, plasticizers, flavoring agents, sweeteners, aroma enhancers, solvents, solubilizers, emulsifiers, suspending agents (binders), thickeners, pH adjusters (acidifiers, alkalizers, buffers), humectants (solvents), antibacterial preservatives, chelating agents, suppository bases, ointment bases, curing agents, softeners, medical water, propellants, stabilizers, and preservatives. These pharmaceutical additives can be readily selected by those skilled in the art according to the desired dosage form and route of administration, and standard pharmaceutical practices.

Furthermore, the pharmaceutical preparations used for editing the DNA of animal cells using the CRISPR-Cas3 system of the present invention may contain further active ingredients. These further active ingredients are not particularly limited and can be suitably designed by those skilled in the art.

Specific examples of the active ingredients and drug additives described above can be obtained from the standards set by the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Japanese Ministry of Health, Labour and Welfare.

Methods for delivering drugs to desired cells include, for example, using viral vectors targeting those cells (adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, Sendai virus vectors, etc.) or antibodies that specifically recognize those cells. Drugs can be in any dosage form depending on the purpose. Furthermore, the aforementioned drugs should be appropriately prescribed by a physician or medical practitioner.

The kit of the present invention is further preferably equipped with an instruction manual.

Example

The present invention will be described in more detail below through embodiments, but the present invention is not limited to the following embodiments.

A. Establishment of the CRISPR-Cas3 system in eukaryotic cells

〔Materials and methods〕

[1] Production of a report carrier containing the target sequence

The target sequences are the human CCR5 gene sequence (Sequence No. 19) and the CRISPR spacer sequence of E. coli (Sequence No. 22).

To insert the target sequence into the vector, a synthetic polynucleotide (SEQ ID NO. 20) containing the target sequence (SEQ ID NO. 19) derived from the human CCR5 gene, and a synthetic polynucleotide (SEQ ID NO. 21) containing the complementary sequence of the aforementioned target sequence (SEQ ID NO. 19) were prepared. Similarly, a synthetic polynucleotide (SEQ ID NO. 23) containing the target sequence (SEQ ID NO. 22) derived from the CRISPR spacer sequence of E. coli, and a synthetic polynucleotide (SEQ ID NO. 24) containing the complementary sequence of the aforementioned target sequence (SEQ ID NO. 22) were prepared. All of the above synthetic polynucleotides were obtained from Hokkaido System Cyence Co., Ltd.

The aforementioned polynucleotides were inserted into a reporter vector using the method described in [Sakuma T et al. (2013) Efficient TALEN construction and evaluation methods for human cell and animal applications, Genes to Cells, Vol. 18 (Issue 4), pp. 315-326]. A summary is as follows: First, polynucleotides with complementary sequences (polynucleotides 20 and 21; polynucleotides 23 and 24) were heated at 95°C for 5 minutes and then cooled to room temperature to allow hybridization. A block incubator (BI-515A, Actions) was used in the above process. Next, the hybridized polynucleotides, forming a double-stranded structure, were inserted into a substrate vector to create a reporter vector.

The sequences of the prepared reporter vectors are shown in sequence number 31 (report vector containing the target sequence derived from the human CCR5 gene) and sequence number 32 (report vector containing the target sequence derived from the CRISPR spacer sequence of E. coli). The structures of the reporter vectors are also shown in Figure 4(d).

[2] Preparation of Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, Cas7 and crRNA expression vectors

[Amplification and Preparation of Insert Fragments]

Regarding the polynucleotides encoding Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7 with altered base sequences (sequence numbers 2, 3, 4, 5, and 6, respectively), firstly, polynucleotides linked in the order of sequence number 2-sequence number 3-sequence number 6-sequence number 4-sequence number 5 (polynucleotides encoding their respective base sequences linked sequentially in the order of Cse1 (Cas8)-Cse2 (Cas11)-Cas7-Cas5-Cas6) were manufactured by Genes Script Co., Ltd. The base sequences of each protein encoding Cse1 (Cas8)-Cse2 (Cas11)-Cas7-Cas5-Cas6 were linked together using 2A peptides (amino acid sequence: GSGATNFSLLKQAGDVEENPGP (sequence number 58)).

Furthermore, the base sequence encoding the 2A peptide varies in several ways depending on the respective Cas protein linker, as follows: Sequence between Cse1 (Cas8) and Cse2 (Cas11): GGAAGCGGAGCAACCAACTTCAGCCTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCAGGCCCC (Sequence No. 59). Sequence between Cse2 (Cas11) and Cas7: GGCTCCGGCGCCACCAATTTTTCTCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAACCCAGGACCT (Sequence No. 60). Sequence between Cas7 and Cas5: GGATCTGGAGCCACCAATTTCAGCCTGCTGAAGCAAGCAGGCGACGTGGAAGAAAACCCAGGACCA (Sequence No. 61). Sequence between Cas5 and Cas6: GGATCTGGGGCTACTAATTTTTCTCTGCTGAAGCAAGCCGGCGACGTGGAAGAGAATCCAGGACCG (Sequence No. 62).

Next, amplify each polynucleotide using the PCR conditions (primers and time process) listed in the table below. PCR was performed using a 2720 Thermal Cycler (applied biosystems).

Table 1

As polynucleotides having a base sequence for expressing crRNA, the following polynucleotides with complementary sequences were obtained.

1. Polynucleotides (Sequence numbers 25 and 26, obtained from Hokkaido System Sciences) for expressing crRNA corresponding to sequences derived from the human CCR5 gene.

2. Polynucleotides (Sequence numbers 27 and 28, obtained from Hokkaido System Cyence) used to express crRNA corresponding to the CRISPR spacer sequence of Escherichia coli.

3. Polynucleotides (Sequence numbers 29 and 30, obtained from Fasmac) used to express crRNA corresponding to the sequence derived from the human EMX1 gene.

[Connection and Transformation]

pPB-CAG-EBNXN (provided by Sanger Center) was used as the base plasmid. 1.6 μg of the base plasmid was mixed with 1 μl of restriction enzymes BglII (New England Biolabs) and XhoI (New England Biolabs) in NEB buffer and reacted at 37°C for 2 hours. The cleaved base plasmid was purified using a gel extraction kit (Qiagen).

The prepared matrix particles and the inserted fragments were ligated using the Gibson Assembly system. During ligation, the ratio of matrix particles to inserted fragments was 1:1, and the process was performed according to the Gibson Assembly system protocol (25 minutes at 50°C, total reaction volume: 8 μL).

Next, using 6 μL of the plasmid solution (ligation reaction solution) obtained above and competent cells (prepared by Takeda Laboratory), transformation was performed using standard methods.

Then, the plasmid vector was purified from the transformed *E. coli* using an alkaline preparation method. In short, the plasmid vector was recovered using the QIAprepSpin Miniprep Kit (Qiagen), purified by ethanol precipitation, and then prepared to a concentration of 1 μg/μL in TE buffer.

The structures of each plasmid vector are shown in Figures 4(a)–(c). Additionally, the base sequences of the pre-crRNA expression vectors are shown in sequence number 33 (expression vector expressing crRNA corresponding to the sequence derived from the human CCR5 gene), sequence number 34 (expression vector expressing crRNA corresponding to the spacer sequence of E. coli CRISPR), and sequence number 35 (expression vector expressing crRNA corresponding to the sequence derived from the human EMX1 gene).

[3] Preparation of Cas3 expression vectors

The polynucleotide encoding Cas3 with the altered base sequence (Sequence No. 1) was obtained from Genscript. Specifically, the pUC57 vector containing the aforementioned polynucleotide was obtained from Genscript.

The vector described above was cleaved using the restriction enzyme NotI. Next, the fragment fragment was smoothed using 2 U of Clenno fragment (Takarabio) and 1 μL of a 2.5 mM dNTP mixture (Takarabio). Then, the fragment was purified using gel extraction (Qiagen). The purified fragment was further cleaved using the restriction enzyme XhoI and purified using gel extraction (Qiagen).

The purified fragments were ligated using the base plasmid (pTL2-CAG-IRES-NEO vector, prepared by Takeda Lab) and a ligation kit (Mighty Mix, Takarabiyo). Then, transformation and purification were performed using the same procedure as in [2]. The recovered plasmid vector was prepared to a concentration of 1 μg/μL in TE buffer.

[4] Preparation of plasmid vectors containing BPNLS

Expression vectors for BPNLS with Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6 and Cas7 connected to the 5' and 3' ends were prepared (see Figure 7).

The creation of insert fragments for each Cas protein group containing BPNLS at both ends was commissioned to Thermo Fisher Scientific. The specific sequences of these insert fragments are (AGATCTTAATACGACTCACTATAGGGAGAGCCGCCACCATGGCC: sequence number 56) - (any one of sequences 7-12) - (TAATATCCTCGAG: sequence number 57). Sequence number 56 is the sequence containing a BgIII cleavage site. Sequence number 57 is the sequence containing an XhoI cleavage site.

The pMK vector containing the above sequence was cleaved with restriction enzymes BgIII and XhoI and purified using gel extraction (Qiagen). The purified fragment was ligated using the base plasmid (pPB-CAG-EBNXN, provided by Sanger Center) and a ligation kit (Mighty Mix, Takarabiyo). Then, transformation and purification were performed using the same procedure as in [2]. The recovered plasmid vector was prepared to a concentration of 1 μg/μL in TE buffer.

[5] Fabrication of plasmid vectors containing cascades (2A)

Expression vectors were prepared with the base sequences of Cse1 (Cas8), Cse2 (Cas11), Cas7, Cas5, and Cas6 linked sequentially. More specifically, expression vectors were prepared in the order of (NLS-Cse1 (Cas8): sequence number 2)-2A-(NLS-Cse2 (Cas11): sequence number 3)-2A-(NLS-Cas7: sequence number 6)-2A-(NLS-Cas5: sequence number 4)-2A-(NLS-Cas6: sequence number 5) (see Figure 8). Furthermore, the amino acid sequence of NLS is PKKKRKV (sequence number 52), and the base sequence is CCCAAGAAGAAGCGGAAGGTG (sequence number 53). Additionally, the amino acid sequence of the 2A peptide is GSGATNFSLLKQAGDVEENPGP (sequence number 58) (corresponding to base sequences 59–62).

The polypeptide with the above-mentioned base sequence was obtained from GenScript. The pUC57 vector with the above sequence inserted was cleaved with the restriction enzyme EcoRI-HF and purified using gel extraction (Qiagen). The purified fragment was ligated using the base plasmid (pTL2-CAG-IRES-Puro vector, made by Takeda Laboratory) and a ligation kit (Mighty Mix, Takarabiyo). Then, transformation and purification were performed using the same procedure as in [2]. The recovered plasmid vector was prepared to a concentration of 1 μg/μL in TE buffer.

[Example A-1]

We evaluated the cleavage activity of exogenous DNA target sequences by expressing Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7 with altered base sequences and added nuclear localization signals, along with crRNA, in HEK (human embryonic kidney) 293T cells.

Prior to transfection, HEK293T cells were cultured in 10 cm culture dishes. HEK293T cell culture was performed in EF medium (GIBCO) at 37°C under a 5% _CO2 atmosphere. The density of HEK293T cells in EF medium was prepared to be 3 × ^10⁴ /100 μL.

Additionally, 100 ng of the aforementioned reporter vector; 200 ng each of the Cas3 plasmid, Cse1 (Cas8) plasmid, Cse2 (Cas11) plasmid, Cas5 plasmid, Cas6 plasmid, Cas7 plasmid, and crRNA plasmid; 60 ng of the pRL-TK vector (capable of expressing René luciferase, Promega); and 300 ng of the pBluecscriptII KS(+) vector (Agilent Technologies) were mixed in 25 μL of Opti-MEM (Thermo Fisher Scientific). The conditions for using a reporter vector with a target sequence derived from CCR5 are equivalent to Figure 1, 1; and the conditions for using a reporter vector with a spacer sequence from E. coli CRISPR are equivalent to Figure 1, 10.

Mix 1.5 μL of Lipofectamine 2000 (Thermo Fisher Scientific) with 25 μL of OptiMEM (Thermo Fisher Scientific) and incubate at room temperature for 5 minutes. Then, mix the above plasmid + OptiMEM mixture with Lipofectamine 2000 + OptiMEM mixture and incubate at room temperature for 20 minutes. Mix the resulting mixture with 1 mL of the above EF medium containing HEK293T cells and seed in 96-well plates (one well for each vector combination, for a total of 12 wells).

After incubation at 37°C and 5% _CO2 for 24 hours, dual-luciferase assays were performed according to the procedures of the Dual-Glo Luciferase assay system (Promega). Luciferase and Renilla luciferase were measured using a CentroXS ³ LB 960 (BERTHOLD TECHNOLOGIES).

As a control experiment, the same experiment was conducted under the following conditions.

1. Instead of any one of the Cas3 plasmid, Cse1 (Cas8) plasmid, Cse2 (Cas11) plasmid, Cas5 plasmid, Cas6 plasmid or Cas7 plasmid, mix an equal amount of pBluecscriptII KS(+) vector (Agilent Technologies) and express it (Figure 1, 2-7).

2. Instead of the crRNA plasmid used in the above steps, use a plasmid that co-expresses crRNA that is not complementary to the target sequence. That is, for the target sequence derived from the CCR5 gene, co-express a plasmid that co-expresses crRNA corresponding to the spacer sequence of E. coli CRISPR (Figure 1, 8). When the target is the spacer sequence of E. coli CRISPR, co-express a plasmid that co-expresses crRNA corresponding to the sequence derived from the CCR5 gene to enable its expression (Figure 1, 11).

3. As a negative control, only reporter vectors with the target sequence derived from CCR5 (Figure 1, 9) and reporter vectors with the spacer sequence of E. coli CRISPR (Figure 1, 12) were expressed.

(result)

The results of the dual-luciferase assay are shown in the coordinate graph above Figure 1, and the experimental conditions are shown in the table below Figure 1. In Figure 1(b), “CCR5-target” and “spacer-target” represent the target sequence derived from CCR5 and the spacer sequence of E. coli CRISPR, respectively. In addition, “CCR5-crRNA” and “spacer-crRNA” represent the sequences complementary to the CCR5-target and the spacer-target, respectively.

Figure 1 shows a system incorporating all of the Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7 plasmids, along with a crRNA plasmid complementary to the target sequence. This system exhibits high cleavage activity compared to other systems (comparing 1 with 2–8, and 10 with 11, respectively). Therefore, it can be seen that by using the expression vector according to an embodiment of the present invention, Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7 can be expressed in human cells.

Additionally, it can be noted that by introducing the above expression vector into human cells, a complex of Cas3, cascade, and crRNA is formed in human cells, thereby cleaving the target sequence.

Furthermore, comparing 8 and 9 and 11 and 12 in Figure 1, the cleavage activity in the system expressing crRNA that is not complementary to the target sequence is at the same level as the negative control. This suggests that the CRISPR-Cas3 system of the present invention can specifically cleave sequences complementary to crRNA in mammalian cells.

[Example A-2]

The same method as in Example A-1 was used to conduct an experiment to evaluate whether the endogenous DNA of human cells could be cut by the type I CRISPR-Cas system.

Specifically, in human cells, Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7, with altered base sequences and added nuclear localization signals, were expressed along with pre-crRNA to evaluate whether the endogenous CCR5 gene sequence in the above cells was cleaved.

HEK239T cells, identical to those in Example A-1, were seeded in 24-well plates at a density of 1× ^10⁵ cells/well and cultured for 24 hours.

Mix 1 μg of Cas3 plasmid, 1.3 μg of Cse1 (Cas8) plasmid, 1.3 μg of Cse2 (Cas11) plasmid, 1.1 μg of Cas5 plasmid, 0.8 μg of Cas6 plasmid, 0.3 μg of Cas7 plasmid, and 1 μg of crRNA plasmid in 50 μL of Opti-MEM (Thermo Fisher Scientific). Next, add a mixture of 5 μL of Lipofectamine 2000 (Thermo Fisher Scientific), 50 μL of Opti-MEM (Thermo Fisher Scientific), and 1 mL of EF medium to the DNA mixture. Then, add 1 mL of the resulting mixture to a 24-well plate.

After culturing at 37°C and 5% _CO2 for 24 hours, the medium was exchanged with 1 mL of EF medium. 48 hours after transfection (24 hours after medium exchange), the cells were recovered and adjusted to a concentration of 1 × ^10⁴ cells/5 μL in PBS.

The cells were heated at 95°C for 10 minutes. Next, 10 mg of proteinase K was added, and the cells were incubated at 55°C for 70 minutes. Then, the cells were heated at 95°C for 10 minutes, and the product was used as a template for PCR.

Amplify 10 μL of the template by performing a 2-step PCR for 35 cycles. Primers with sequence numbers 47 and 48 were used for the PCR. KOD FX (Toyobosha) was used as the DNA polymerase, and the 2-step PCR was performed according to the instructions provided with KOD FX. The amplified product was purified using the QIAquick PCR Purification Kit (QIAGEN). The specific steps were performed according to the instructions provided with the kit.

Using rTaq DNA polymerase (Toyobo), dA was added to the 3' end of the purified DNA. The purified DNA was then electrophoresed on a 2% agarose gel. After excising a band of approximately 500–700 bp, the DNA was extracted and purified from the excised gel using a gel extraction kit (QIAGEN). Next, TA cloning was performed using the pGEM-T Easy vector system (Promega) to clone the DNA. Finally, the cloned DNA was extracted using an alkaline preparation method and analyzed by Sanger sequencing. Analysis was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and an Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific).

Figure 2 illustrates a summary of the endogenous CCR5 gene sequence that serves as the target of the CRISPR-Cas system in this embodiment. Furthermore, in Figure 2, exons are represented by uppercase letters, and introns by lowercase letters.

In this embodiment, the target sequence is the CCR5 gene located in the region (P)21 of the short arm of chromosome 3 (Figure 2: Serial number 46 shows the full length of the CCR5 base sequence). Specifically, the target sequence is the sequence in exon 3 of the CCR5 gene. As a control, the target sequence for Cas9 is also positioned in almost the same location. That is, the entire underlined sequence is the target sequence of the type I CRISPR-Cas system (AAG and the following 32 bases), and the double-underlined sequence is the target sequence for Cas9 (CGG and the preceding 20 bases). The crRNA sequence was designed to enable guidance to the target sequence (AAG and the following 32 bases) of the type I CRISPR-Cas system.

(result)

The results of the above experiments yielded clones 1 (401 bp deletion), 2 (341 bp deletion), 3 (268 bp deletion), and 4 (344 bp deletion) compared to the original base sequences (Figures 3A-D). This demonstrates that the CRISPR-Cas3 system of this invention can induce deletion of endogenous DNA in human cells. In other words, the CRISPR-Cas system indicates that DNA editing in human cells is possible.

In this embodiment, clones with missing base pairs were observed. This fact supports the ability of the CRISPR-Cas3 system according to the present invention to generate DNA cuts at multiple sites.

The CRISPR-Cas3 system of this invention enables the deletion of hundreds of base pairs (268–401 bp) of DNA. This is a much wider range of deletions than those obtained using the CRISPR-Cas system with Cas9, which typically involves only one cut in the DNA.

[Example A-3]

The same method as in Example A-1 was used to conduct experiments to evaluate whether the CRISPR-Cas3 system could cut endogenous DNA in human cells.

Specifically, by expressing Cas3, Cse1 (Cas8), Cse2 (Cas11), Cas5, Cas6, and Cas7 with altered base sequences and added nuclear localization signals, along with pre-crRNA, in human cells, the endogenous EMX1 gene sequence in the above cells was evaluated to determine whether it was cleaved.

HEK293T cells, identical to those in Example A-1, were seeded in 24-well plates at a density of 1× ^10⁵ cells/well and cultured for 24 hours.

Mix 500 ng of Cas3 plasmid, 500 ng of Cse1 (Cas8) plasmid, 1 μg of Cse2 (Cas11) plasmid, 1 μg of Cas5 plasmid, 1 μg of Cas6 plasmid, 3 μg of Cas7 plasmid, and 500 μg of crRNA plasmid in 50 μL of Opti-MEM (Thermo Fisher Scientific). Further add 4 μL of Lipofectamine 2000 (Thermo Fisher Scientific) and 50 μL of Opti-MEM (Thermo Fisher Scientific) to the above mixture and mix. Incubate the resulting mixture at room temperature for 20 minutes, then add it to the HEK293T cells.

The structure of the expression vector for the Cas protein group used in Examples A-3 is shown in Figure 7. As shown in Figure 7, the expression vector consists of bipartite NLS (BPNLS) sandwiching the sequence encoding the Cas protein group (see [Suzuki K et al. (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration, Nature, Vol. 540 (Issue 7631), pp. 144-149]). The amino acid sequence of the BPNLS is KRTADGSEFESPKKKRKVE (Sequence No. 54), and the base sequence is AAGCGGACTGCTGATGGCAGTGAATTTGAGTCCCCAAAGAAGAAGAGAAAGGTGGAA (Sequence No. 55).

After culturing HEK293T cells at 37°C and 5% _CO2 for 24 hours, the medium was exchanged with 1 mL of EF medium (1 mL per well). 48 hours after transfection (24 hours after medium exchange), the cells were recovered and adjusted to a concentration of 1 × ^10⁴ cells/5 μL in PBS.

The cells were heated at 95°C for 10 minutes. Next, 10 mg of proteinase K was added, and the cells were incubated at 55°C for 70 minutes. The cells were then further heated at 95°C for 10 minutes, and the resulting product was used as a template for PCR.

10 μL of the template was amplified by performing 40 cycles of 3-step PCR. Primers with sequence numbers 50 and 51 were used for the PCR. Hotstartaq (QIAGEN) was used as the DNA polymerase, and the 3-step PCR was performed according to the instructions provided with Hotstartaq. The PCR product was electrophoresed on a 2% agarose gel. After excising a band of approximately 900–1100 bp, DNA was extracted and purified from the excised gel using a gel extraction kit (QIAGEN). The specific steps were performed according to the instructions provided with the kit.

Next, TA cloning was performed using the pGEM-T Easy vector system (Promega) to clone the aforementioned DNA. Finally, the cloned DNA was extracted using the alkaline preparation method and analyzed by Sengge sequencing. Analysis was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and the Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific).

Figure 5 illustrates a summary of the endogenous EMX1 gene sequence that became the target of the CRISPR-Cas3 system in Examples A-3. Furthermore, in Figure 5, exons are represented by uppercase letters, and introns by lowercase letters.

In Example A-3, the target sequence was the EMX1 gene located in the P13 region of the short arm of chromosome 2 (Figure 5: Serial number 49 shows the full length of the EMX1 base sequence). Specifically, the target sequence was the sequence within exon 3 of the EMX1 gene. As a control, the target sequence for Cas9 was located in essentially the same position. That is, the underlined sequence further upstream is the target sequence for the type I CRISPR-Cas system (AAG and the following 32 bases), and the underlined sequence further downstream is the target sequence for Cas9 (TGG and the preceding 20 bases). The crRNA sequence used in Example A-3 was designed to guide the target sequence (AAG and the following 32 bases) to the CRISPR-Cas3 system.

(result)

Compared with the original base sequence, the above experiments yielded clone 1 with deletions of 513 bp and 363 bp, and clone 2 with a deletion of 694 bp (Figures 6A and 6B). These results also demonstrate that the CRISPR-Cas3 system of this invention can induce deletion of endogenous DNA in human cells. This suggests that the CRISPR-Cas3 system can be used for DNA editing in human cells.

Furthermore, the occurrence of cuts at more than two sites in the double-stranded DNA and the deletion of hundreds of base pairs of DNA were also observed as in Example A-2. Therefore, the results of Example A-3 more strongly support the findings obtained from Example A-2.

[Example A-4]

The cleavage activity of a CRISPR-Cas3 system, which modifies the base sequence and then links the base sequence encoding a cascade protein, was evaluated in HEK293T cells to assess the cleavage activity of a target sequence of exogenous DNA.

In Examples A-4, 100 ng of the reporter vector; 200 ng each of the Cas3 plasmid, the cascade (2A) plasmid, and the crRNA plasmid; 60 ng of the pRL-TK vector (expressing René luciferase, Promega); and 300 ng of the pBluecscriptII KS(+) vector (Agilent Technologies) were mixed in 25 μL of Opti-MEM (Thermo Fisher Scientific). As the reporter vector, the conditions for using a reporter vector with a target sequence derived from CCR5 correspond to 1 in Figure 9(b), and the conditions for using a reporter vector with a spacer sequence from E. coli CRISPR correspond to 6 in Figure 9(b).

The aforementioned reporter vector uses two reporter vectors prepared in [1] of the [manufacturing example] (i.e., the vector with the structure shown in FIG4(d)). In addition, the aforementioned cascade (2A) plasmid uses the expression vector prepared in [4] of the [manufacturing example] (i.e., the vector with the structure shown in FIG8).

In addition to using the expression vector described above, dual-luciferase assays were performed using the same method as in Example A-1.

In addition, as a control experiment, the same experiment was conducted under the following conditions.

1. Instead of either the Cas3 plasmid or the cascade (2A) plasmid, mix equal amounts of the pBluscriptII KS(+) vector (Agilent Technologies) and express it (Figure 9, 2 and 3).

2. Instead of the crRNA plasmid used in the above steps, use a plasmid that co-expresses crRNA that is not complementary to the target sequence. That is, for the target sequence derived from the CCR5 gene, co-express a plasmid that co-expresses crRNA corresponding to the spacer sequence of E. coli CRISPR (Figure 9, 4); when using the spacer sequence of E. coli CRISPR as the target, co-express a plasmid that co-expresses gRNA corresponding to the sequence derived from the CCR5 gene, and express it (Figure 9, 7).

3. As negative controls, a reporter vector expressing only the target sequence derived from CCR5 (Figure 9, 5) and a reporter vector expressing only the spacer sequence of E. coli CRISPR (Figure 9, 8) were used.

(result)

The results of the dual-luciferase assay are shown in the coordinate graph above Figure 9, and the experimental conditions are shown in the table below Figure 9. In Figure 9, "CCR5-target" and "spacer-target" represent the target sequence derived from CCR5 and the spacer sequence of E. coli CRISPR, respectively. In addition, "CCR5-crRNA" and "spacer-crRNA" represent the sequences complementary to the CCR5-target and the spacer-target, respectively.

As shown in Figure 9, the system incorporating both sides of the Cas3 plasmid and the cascade (2A) plasmid, and the crRNA plasmid complementary to the target sequence, exhibited significantly higher cleavage activity compared to other systems (comparing 1 with 2–5, and 6 with 7–8, respectively). This suggests that, in systems that link and express base sequences encoding cascade proteins, the CRISPR-Cas system according to an embodiment of the present invention can specifically cleave sequences complementary to crRNA within mammalian cells.

B. Verification of factors influencing genome editing, such as the CRISPR-Cas3 system in eukaryotic cells.

〔Materials and methods〕

[1] Composition of Cas gene and crRNA

The design involved adding bpNLS to the Cas3 gene and its cascaded constituent genes (Cse1, Cse2, Cas5, Cas6, Cas7) derived from *E. coli* strain K-12, respectively, on the 5' and 3' sides. Codon optimization was performed on mammalian cells, and the genes were cloned via gene synthesis. These genes were then subcloned downstream of the CAG promoter of the pPB-CAG.EBNXN plasmid, a gift from the Sanger Institute. Cas3 mutants such as H74A (dead nickase; dn), K320N (dead helicase; dh), and a double mutant of S483A and T485A (dead helicase ver. 2; dh2) were created by self-ligation of PrimeSTAR MAX PCR products. For the crRNA expression plasmid, the sequence of crRNA with two BbsI restriction enzyme sites was synthesized at the spacer position below the U6 promoter. The entire crRNA expression plasmid was created by inserting a 32-base-pair double-stranded oligonucleotide of the target sequence at the BbsI restriction enzyme site.

The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, used as the Cas9-sgRNA expression plasmid, was obtained from Addgene. For gRNA design, CRISPR web tool, CRISPR design tool, and/or CRISPRdirect were used to predict unique target sites in the human genome. The target sequence was cloned into the pX330 sgRNA scaffold according to the procedures of the Feng Zhang Institute.

An SSA reporter plasmid containing two BsaI restriction enzyme sites was donated by Professor Taku Yamamoto of Hiroshima University. The target sequence of the genomic region was inserted into the BsaI site. pRL-TK (Promega) was obtained as a Renalis luciferase vector. All plasmids were prepared using the PureLink HiPure Plasmid Purification Kit (Thermo Fisher Scientific) via either mid-iprep or max-iprep methods.

[2] Evaluation of DNA cleavage activity in HEK293T cells

To detect DNA cleavage activity in mammalian cells, the SSA assay was performed in the same manner as in Example A. HEK293T cells were cultured at 37°C and 5% _CO2 in high-glucose Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. 0.5 × ^10⁴ cells were seeded into the wells of 96-well plates. After 24 hours, HEK293T cells were transfected with Cas3, Cse1, Cse2, Cas7, Cas5, Cas6, crRNA expression plasmids (100 ng each), SSA reporter vector (100 ng), and René luciferase vector (60 ng) using lipofectamine 2000 and OptiMEM (Life Technologies) according to a slightly modified protocol. 24 hours after transfection, a dual-luciferase assay was performed using the Dual-Luciferase assay system (Promega) according to the prescribed protocol.

[3] Detection of gain and loss sites in HEK293T cells

Four hours after seeding 2.5 x ^10⁴ cells into the wells of a 24-well plate, HEK293T cells were transfected with Cas3, Cse1, Cse2, Cas7, Cas5, Cas6, and crRNA expression plasmids (250 ng each) using lipofectamine 2000 and OptiMEM (Life Technologies) according to a slightly modified protocol. Two days after transfection, total DNA was extracted from the recovered cells using the Tissue XS kit (Takara-bio) according to protocol. Target loci were amplified using Gflex (Takara-bio) or QuickTaq HS DyeMix (TOYOBO) and electrophoresed on agarose gels. To detect small insertion/deletion mutations in the PCR products, the SURVEYOR Mutation Detection Kit (Integrated DNA Technologies) was used according to protocol. For TA clones, the pCR4Blunt-TOPO plasmid vector (Life Technologies) was used according to protocol. Sequencing analysis was performed using the BigDye Terminator Cycle Sequencing Kit and the ABI PRISM 3130 GeneticAnalyzer (Life Technologies).

To detect rare mutations, DNA libraries containing PCR amplification products were prepared using the TruSeq Nano DNA Library Prep Kit (Illumina), and amplicon sequencing was performed using MiSeq (2 x 150 bp) following Macrogen's standard procedures. Raw sequencing data from each sample were mapped to hg38 of the human genome using BWA-MEM. Coverage data were visualized using Integrative Genomics Viewer (IGV), and histograms were extracted from the target regions.

HEK293T reporter cells with mCherry-P2A-EGFP c321C>G, used for detecting SNP-KI (single base polymorphism knock-in) in mammalian cells, were donated by Professor Shinichiro Nakata. The reporter cells were cultured in 1 μg/ml puromycin. 500 ng of donor plasmid or single-stranded DNA was co-introduced with CRISPR-Cas3 using the method described above. Total cells were recovered 5 days after transfection and analyzed using AriaIIIu (BD) for FACS. GFP-positive cells were cleaned, and total DNA was extracted using the method described above. SNP exchanges in the genome were detected by PCR amplification using HiDi DNA polymerase (myPOLS Biotec).

[4] Detection of candidate off-target sites

Off-target candidates for type I-E CRISPR were detected in the human genome hg38 using GGGenome in two distinct steps. As PAM candidate sequences, AAG, ATG, AGG, GAG, TAG, and AAC were selected according to prior reports (Leenay, R.T., et al. Mol. Cell 62, 137-147 (2016), Jung, et al. Mol. Cell. 2017, Jung et al., Cell 170, 35-47 (2017)). Because positions not previously identified as target sites (Kunne et al., Molecular Cell 63, 1-13 (2016)) were reported, in the initial convergence, the 32-base pairs of target sequences with fewer mismatches were selected. In the subsequent convergence, regions perfectly paired with the 5' end of the PAM side of the target sequence were ranked higher.

[5] Deep sequencing for off-target analysis

In whole-genome sequencing, genomic DNA was extracted from transfected HEK293T cells and cut using a Covaris sonicator. DNA libraries were prepared using the TruSeq DNA PCR-Free LT Library Prep Kit (Illumina), and genome sequencing was performed using HiSeq X (2 × 150 bp) following standard Takarabio procedures. Raw reads from each sample were mapped to hg38 of the human genome using BWA-MEM and filtered using the Trimmomatic program. Discordant read pairs and split reads were excluded using samtools and Lumpy-sv, respectively. To detect only large deletions on the same chromosome, BadMateFilter in the Genome Analysis Toolkit was used to remove leader pairs mapped to different chromosomes. The total number of incompatible sequencing pairs or split sequencing data in each 100kb region was calculated using Bedtools, and the error rate compared to the negative control was determined. To enrich off-target candidates before sequencing, SureSelectXT custom DNA probes were designed under moderately stringent conditions using SureDesign and manufactured by Agilent Technologies. Target regions were selected as follows: Probes near the target region covered 800kb upstream and 200kb downstream of the PAM. Near the off-target region of CRISPR-Cas3, probes covered 9kb upstream and 1kb downstream of the PAM candidate. Near the off-target region of CRISPR-Cas9, probes covered 1kb upstream and 1kb downstream of the PAM. After preparing the DNA library using the SureSelectXT kit and custom probe cassette, genome sequencing was performed using a HiSeq 2500 (2×150bp) according to the standard procedures of Takara Bio. Incompatible sequencing pairs and split sequencing data within the same chromosome were excluded using the methods described above. Use Bedtools to calculate the total number of incompatible sequencing pairs or split sequencing data in each 10kb region, and calculate the error rate compared to the negative control.

[Example B-1] The Influence of the Types of crRNA and Nuclear Localization Signals on DNA Cutting Activity

In Example A, genome editing in eukaryotic cells was successfully performed incidentally using a CRISPR-Cas3 system containing pre-crRNA (LRSR; leader sequence-repetitive sequence-spacer sequence-repetitive sequence) as crRNA. It is believed that the reason the inventors have not been able to successfully perform genome editing in eukaryotic cells using the CRISPR-Cas3 system for so long may be due to the use of mature crRNA as crRNA. Therefore, in addition to pre-crRNA (LRSR), pre-crRNA (RSR; repetitive sequence-spacer sequence-repetitive sequence) and mature crRNA (5’ handle sequence-spacer sequence-3’ handle sequence) were prepared as crRNA, and the genome editing efficiency was verified using the reporter system of Example A (Figures 10A and 10B). Furthermore, the base sequences of pre-crRNA (LRSR), pre-crRNA (RSR), and mature crRNA are shown in sequence numbers 63, 64, and 65, respectively.

The results showed that no target DNA cleavage activity was confirmed in the CRISPR-Cas3 system using mature crRNA. Surprisingly, however, very high target DNA cleavage activity was confirmed using pre-crRNA (LRSR, RSR). This result in the CRISPR-Cas3 system is compared to the CRISPR-Cas9 system, which showed high DNA cleavage activity using mature crRNA. Furthermore, this fact suggests that one of the main reasons why genome editing in eukaryotic cells has not been successfully performed using the CRISPR-Cas3 system to date is the reliance on mature crRNA.

In addition, the nuclear localization signal added to Cas3 was validated using both SV40 and bipartite nuclear localization signals (Figure 11). The results showed that using the bipartite nuclear localization signal confirmed higher target DNA cleavage activity.

Therefore, in subsequent experiments, pre-crRNA (LRSR) was used as crRNA, and bipartite nuclear localization signal was used as nuclear localization signal.

[Example B-2] Effect of PAM sequence on DNA cleavage activity

To confirm the target specificity of the CRISPR-Cas3 system, the effects of various PAM sequences on DNA cleavage activity were investigated (Figure 12). In SSA assays, DNA cleavage activity yielded various results depending on the different PAM sequences. 5’-AAG PAM showed the highest activity, while AGG, GAG, TAC, ATG, and TAG also showed remarkable activity.

[Example B-3] The effect of mismatched crRNA and spacer sequences on DNA cleavage activity

Previous studies of the cascaded crystal structure of *E. coli* have shown that a 5-base region of heterozygous double strands forms between crRNA and spacer DNA due to base pairing failure at position 6 via the CSM element of the Cas7 effector (Fig. 13). The effect of mismatches between crRNA and spacer sequences on DNA cleavage activity was evaluated (Fig. 1g). Any single mismatch in the seed region (positions 1–8), except for the unrecognized base (position 6), resulted in a sharp decrease in cleavage activity.

[Example B-4] Verification of the necessity of each domain of Cas3 in DNA cleavage activity

In the in vitro characterization of the enzymatic properties of the Cas3 protein, it was determined that the N-terminal HD nuclease domain cleaves the single-stranded region of the DNA substrate, followed by the C-terminal SF2 helicase domain, which ATP-dependently unwinds the target DNA from the 3' to the 5' direction. Three Cas3 mutants were created: the HD domain H74A mutant (dnCas3), the SF2 domain motif 1 K320N mutant (dhCas3), and the SF2 domain motif 3 S483A/T485A double mutant (dh2Cas3) to verify whether the Cas3 domain is necessary for DNA cleavage (Figure 14). The results showed that DNA cleavage activity was completely lost in all three Cas3 protein mutants, confirming that Cas3 can cleave target DNA through the HD nuclease domain and the SF2 helicase domain.

[Examples B-5] Validation of DNA cleavage activity of various types of CRISPR-Cas3 systems

The CRISPR-Cas3 system type 1 is highly diverse (7 types, A through G). In the examples above, the DNA cleavage activity of CRISPR-Cas3 systems type I-E in eukaryotic cells was verified. However, in this example, the DNA cleavage activity of other CRISPR-Cas3 systems type 1 (types I-F and I-G) was verified. Specifically, codon optimization and cloning were performed on Cas3 and Cas5-7 of *Shewanella putrefaciens* (types I-F) and Cas5-8 of *Pyrococcus furiosus* (types I-G) (Figure 15). Although the strength of DNA cleavage activity varied, DNA cleavage activity was also confirmed in SSA assays using 293T cells with these CRISPR-Cas3 systems.

[Example B-6] Verification of mutations introduced into endogenous genes via the CRISPR-Cas3 system

Mutations introduced into endogenous genes via the CRISPR-Cas3 system were validated using the I-E system. The EMX1 and CCR5 genes were selected as target genes, and pre-crRNA (LRSR) plasmids were prepared. Liposome transfection of plasmids encoding pre-crRNA and six Cas (3, 5-8, 11) effectors into 293T cells revealed that deletions of hundreds to thousands of base pairs via CRISPR-Cas3 primarily occurred upstream of the 5' PAM of the spacer sequence in the target region (Figure 16). Microhomology of 5-10 base pairs in the repaired join sites was confirmed, likely through annealing of the complementary strand using an anneal-dependent repair pathway. Furthermore, no genome editing was found in the EMX1 and CCR5 regions in the mature crRNA plasmids.

To further identify the characteristics of Cas3-based genome editing through TA cloning of PCR products and Sengge sequencing, 96 TA clones were extracted and compared with wild-type EMX1 sequences (Figure 17). In 24 of the 49 clones where the insertion could be confirmed, deletions ranging from a minimum of 596 base pairs to a maximum of 1447 base pairs (average 985 base pairs) were identified (46.3% efficiency). Half of the clones (n=12) contained PAM and spacer sequences, resulting in large deletions, while the other half had deletions upstream of the PAM.

Next-generation sequencing of PCR amplification products using primer sets in broader regions, such as 3.8 kb of the EMX1 gene and 9.7 kb of CCR5, was used to further characterize Cas3. Additionally, the targeting of multiple PAM sites (AAG, ATG, TTT) in type I-E CRISPR was validated. In amplicon sequencing, AAG was 38.2%, ATG was 56.4%, and TTT was 86.4%, representing a significant reduction in coverage across a wide genomic region upstream of the PAM sites compared to Cas9's 86.4% targeting EMX1. This reduction in coverage was also observed when targeting the CCR5 region. In contrast, Cas9 induced small insertions and deletions (gains and losses) at the target site, while Cas3 showed no small gains or losses mutations in the PAM sites or target sites. These results suggest that the CRISPR-Cas3 system induces deletions in a broad region upstream of the target site in human cells.

Considering the limitations of PCR analysis, such as the strong bias favoring amplifications smaller than 10 kb and shorter PCR fragments, microarray-based capture sequencing was used within 1000 kb or more surrounding the target EMX1 and CCR5 loci (Figures 18A and 18B). Deletions up to 24 kb were confirmed at the EMX1 locus and up to 43 kb at the CCR5 locus, but 90% of the mutations in EMX1 and 95% of those in CCR5 were smaller than 10 kb. These results suggest that the CRISPR-Cas3 system can also possess strong nuclease and helicase activities in eukaryotic genomes.

Furthermore, as demonstrated in the CRISPR-Cas9 system, the ability to induce undesirable off-target mutations in non-target genomic regions is a particularly significant concern for clinical applications, but no significant off-target effects have been observed in the CRISPR-Cas3 system.

Industry availability:

The CRISPR-Cas3 system of this invention is capable of editing the DNA of eukaryotic cells, and therefore can be widely used in fields that require genome editing, such as medicine, agriculture, forestry and fisheries, industry, life sciences, bioengineering, gene therapy and other fields.

Claims (8)

1. A method for manufacturing eukaryotic cells with endogenous DNA edited, the method comprising the step of introducing a type I-E CRISPR-Cas3 system into the eukaryotic cells, the type I-E CRISPR-Cas3 system comprising the following (A) to (C), (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and (C) a pre-crRNA targeting endogenous DNA, a polynucleotide encoding the pre-crRNA, or an expression vector containing the polynucleotide. 2. A method for manufacturing animals or plants with edited endogenous DNA, including the step of introducing a type I-E CRISPR-Cas3 system into the animal or plant, wherein the type I-E CRISPR-Cas3 system comprises the following (A) to (C). (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and (C) pre-crRNA targeting endogenous DNA, polynucleotide encoding the pre-crRNA, or expression vector containing the polynucleotide. The animals referred to are animals other than humans. 3. The method according to claim 1 or 2, comprising the step of cleaving pre-crRNA targeting endogenous DNA by means of proteins constituting a cascade protein after introducing a type I-E CRISPR-Cas3 system into eukaryotic cells. 4. The method according to claim 1 or 2, wherein nuclear localization signals are added to the Cas3 protein and/or cascade proteins. 5. The method according to claim 4, wherein the nuclear positioning signal is a bifurcation nuclear positioning signal. 6. A kit for use in the method of any one of claims 1 to 5, comprising the following (A) to (C), (A) Cas3 protein, the polynucleotide encoding the protein, or an expression vector containing the polynucleotide. (B) cascade proteins, polynucleotides encoding those proteins, or expression vectors containing those polynucleotides, and (C) a pre-crRNA targeting endogenous DNA, a polynucleotide encoding the pre-crRNA, or an expression vector containing the polynucleotide. 7. The kit according to claim 6, wherein nuclear localization signals are added to the Cas3 protein and/or cascade proteins. 8. The kit according to claim 7, wherein the nuclear localization signal is a bifid nuclear localization signal.