Improved crispr-cas3 system

Abstract

The present invention addresses the problem of providing a CRISPR-Cas3 system that, in comparison with conventional CRISPR-Cas3 systems, enables the introduction of small-scale mutations in an eukaryotic cell. The inventors of the present invention found that a mutation can be generated in a target DNA in an eukaryotic cell by utilizing a Cas3 protein with a mutation introduced into a motif 3 of SF2 helicase domain as the Cas3 protein in a CRISPR-Cas3 system.

Description

Improved CRISPR-Cas3 system

[0001] The present invention relates to an improved CRISPR-Cas3 system, and more particularly to a CRISPR-Cas3 system that enables small-scale DNA editing by utilizing a mutant Cas3 protein.

[0002] Genome editing technologies using zinc finger nucleases (ZFNs), TAL effector nucleases (transcription activator-like effector nucleases; TALENs), CRISPR-Cas9, and other technologies specifically cleave target DNA in animal and plant cells and rewrite the sequence of the target DNA by utilizing endogenous repair mechanisms. Genome editing technologies are rapidly expanding in use not only in basic research such as elucidating the functions of target genes and proteins, but also in agricultural and livestock breeding, biomaterial production, regenerative medicine, and genome editing therapy. For example, in the United States, oleic acid-rich soybean oil and reduced-spiciness mustard have been marketed, while in Japan, tomatoes with high GABA accumulation and thick-fleshed sea bream have been marketed.

[0003] In parallel with the rapid expansion of research into genome editing technology and its industrial use, the development and improvement of genome editing tools is progressing at a rapid pace. The CRISPR-Cas systems found in bacteria and archaea are divided into Class 1, which cleaves the target sequence using a complex of multiple proteins, and Class 2, which cleaves using a single protein. CRISPR-Cas9 and CRISPR-Cas12a, which have been developed as genome editing tools to date, are classified as Class 2.

[0004] In Japan, development of the Class 1 Type I CRISPR-Cas3 genome editing technology has progressed, and it has been discovered for the first time in the world that it can be used as a genome editing tool for eukaryotic cells (Non-Patent Document 1, Patent Document 1).

[0005] For example, in the Type I-E CRISPR-Cas3 system, a cascade complex consisting of five proteins (Cas5, Cas6, Cas7, Cas8, and Cas11) and crRNA recognizes and binds to a 3-base PAM sequence and a 27-base target sequence. Cas3, which has helicase and nuclease activities, then unwinds the double-stranded DNA structure and cleaves the DNA, causing extensive DNA cleavage upstream of the target sequence (Non-Patent Document 2). Genome editing using the CRISPR-Cas3 system has previously been reported to result in extensive deletion mutations ranging from several hundred bp to several kb upstream of the target sequence in cells such as humans and rice (Non-Patent Documents 1, 3, 4, 5).

[0006] Thus, compared with the CRISPR-Cas9 system, the CRISPR-Cas3 system is particularly good at causing large-scale DNA deletions, making it suitable for, for example, gene knockout, which involves deleting the entire target gene. On the other hand, genome editing targeting only a specific region of a gene requires the introduction of small-scale mutations, and therefore, improvements to the CRISPR-Cas3 system to enable such genome editing are also desired.

[0007] International Publication No. 2018 / 225858

[0008] Morisaka H, ​​et al. Nat Commun. 2019; 10(1): 5302. Yoshimi K, et al. Nat Commun. 2022; 13(1): 4917. Dolan AE, et al. Mol Cell. 2019; 74(5): 936-950. Li Y, et al. Plant Biotechnol J. 2023; 21(11): 2196-2208. Saiga et al. "Knockout of endogenous genes in rice using CRISPR / Cas3," 143rd Annual Meeting of the Japanese Society of Breeding, March 2023

[0009] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a CRISPR-Cas3 system that enables the introduction of smaller mutations in eukaryotic cells than conventional CRISPR-Cas3 systems.

[0010] It has been reported that the CRISPR-Cas3 system is unable to cleave target DNA in vitro when the following Cas3 proteins, which are its constituent elements, are used: an HD domain mutant (H74A), an SF2 domain motif 1 mutant (K320N), and a double SF2 domain motif 3 mutant (S483A / T485A) (Fig. 14 of Patent Document 1). Therefore, this report proposes fusing these mutant Cas3 proteins with other effector proteins (e.g., transcription factors) and allowing the other effector proteins to function in the CRISPR-Cas3 system.

[0011] In order to solve the problem of developing a CRISPR-Cas3 system that enables the introduction of smaller mutations in eukaryotic cells than conventional methods, the present inventors tested the effect of CRISPR-Cas3 systems that include these mutant Cas3 proteins in eukaryotic cells. They unexpectedly found that when mutant Cas3 proteins in which a mutation has been introduced into motif 3 of the SF2 helicase domain are used, it is possible to generate mutations of several to several tens of bases in the genomes of eukaryotic cells such as rice cells and human cells, and thus completed the present invention.

[0012] The present invention relates to DNA editing in eukaryotic cells using a CRISPR-Cas3 system comprising a Cas protein in which a mutation has been introduced into motif 3 of the SF2 helicase domain, and more specifically includes the following aspects.

[0013] (1) A method for producing a eukaryotic cell in which a mutation has been introduced into target DNA, the method comprising introducing a CRISPR-Cas3 system into the eukaryotic cell, wherein the CRISPR-Cas3 system comprises the following (A) to (C):

[0014] (A) A Cas3 protein whose helicase activity is suppressed by a mutation in motif 3 of the superfamily 2 (SF2) helicase domain, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; (B) A Cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; and (C) a crRNA, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide.

[0015] (2) The method according to (1), wherein a nuclear localization signal is added to the Cas3 protein and / or the Cascade protein.

[0016] (3) A kit for use in the method described in (1), comprising: (A) a Cas3 protein whose helicase activity is suppressed by a mutation in motif 3 of the superfamily 2 (SF2) helicase domain; a polynucleotide encoding the protein; or an expression vector containing the polynucleotide.

[0017] (4) The kit according to (3), further comprising (B) a cascade protein, a polynucleotide encoding the protein, or an expression vector containing the polynucleotide.

[0018] (5) The kit according to (3) or (4), further comprising (C) crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[0019] (6) The kit according to any one of (3) to (5), wherein a nuclear localization signal is added to the Cas3 protein and / or the cascade protein.

[0020] The present invention provides a CRISPR-Cas3 system that can generate small-scale DNA mutations in eukaryotic cells, thereby enabling more precise control of the edited region in genome editing.

[0021] This figure shows a comparison of the method of the present invention, which utilizes a mutant Cas3 protein, with a conventional method, which utilizes a wild-type Cas3 protein, regarding DNA editing in eukaryotic cells. This figure shows the results of detecting DNA editing in rice cells using the method of the present invention. The rice phytoene desaturase PDS gene was used as the target DNA. Figure A shows the results of electrophoresis of a region containing the target DNA amplified by PCR in callus into which a mutant Cas3 protein lacking helicase activity (mtCas3-h2) and a mutant Cas3 protein lacking nickase activity (Cas3(H74A)) had been introduced, as well as callus into which no Cas3 protein had been introduced. Figure B shows a graph showing the efficiency of deletion introduction into the target DNA in callus subjected to each treatment. The deletion introduction efficiency was calculated as the percentage of callus in which a PCR product shorter than the expected size was detected, relative to the analyzed callus. 2 using wild-type Cas3 protein or each mutant Cas3 protein (mtCas3-h2, mtCas3-h2-1 or mtCas3-h2-2), and the same experiment as in FIG. 2 was performed, and the results of each experiment are shown as "Experiment 1" and "Experiment 2". The CRISPR-Cas3 system containing crRNA-1 and wild-type Cas3 protein or each mutant Cas3 protein (mtCas3-h1 to h4) targeting the EGFP gene was introduced into reporter HEK293T cells carrying mCherry-P2A-EGFP, and the results of the reporter gene editing were detected. The upper part of the figure shows the structure of the reporter vector used and the target site of the crRNA used, the lower left part shows the mutation introduction efficiency by each Cas3 protein, and the lower right part shows the mutation pattern introduced by each Cas3 protein. In the figure, "Gnega" indicates GFP negative, "mCnega" indicates mCherry negative, and "Wnega" indicates GFP negative and mCherry negative, respectively. This figure shows the results of analyzing mutations introduced into the EGFP gene using crRNA-2, which targets another site on the EGFP gene, as a guide RNA, in an experiment similar to that shown in FIG. 4.

[0022] The present invention provides a method for producing a eukaryotic cell in which a mutation has been introduced into target DNA, the method comprising introducing a CRISPR-Cas3 system into the eukaryotic cell, wherein the CRISPR-Cas3 system comprises the following (A) to (C):

[0023] (A) A Cas3 protein whose helicase activity is suppressed by a mutation in the superfamily 2 (SF2) helicase domain, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; (B) a Cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide; and (C) a crRNA, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide.

[0024] Class 1 CRISPR-Cas systems are classified into Type I and Type III, and Type I CRISPR-Cas systems are further classified into six types: Type IA, Type IB, Type I-C, Type I-D, Type I-E, and Type I-F, as well as Type I-G, a subtype of Type IB (see, for example, van der Oost J et al. (2014) Unraveling the structural and mechanistic basis of CRISPR-Cas systems, Nature Reviews Microbiology, Vol. 12 (No. 7), pp. 479-492, Jackson RN et al. (2014) Fitting CRISPR-associated Cas3 into the Helicase Family Tree, Current Opinion in Structural Biology, Vol. 24, pp. 106-114).

[0025] The CRISPR-Cas3 system of the present invention is characterized in that a protein whose helicase activity is suppressed by a mutation in the superfamily 2 (SF2) helicase domain is used as the Cas3 protein.

[0026] In Type IA, Cas3 proteins are divided into Cas3-HD, which has nuclease activity, and Cas3-HEL, which has helicase activity. In the present invention, a combination of Cas3-HD and Cas3-HEL, in which helicase activity is suppressed, can be used. Examples of Cas3-HEL include a protein derived from Pyrococcus furiosus having the amino acid sequence set forth in SEQ ID NO: 1. In this amino acid sequence, the superfamily 2 (SF2) helicase domain is located at positions 18 to 207, and motif 3 is located at positions 198 to 200. In the present invention, a mutation in motif 3 is preferred, with mutations at positions 198 and / or 200 being more preferred, and mutations at positions 198 and 200 being particularly preferred.

[0027] An example of a Type I-B Cas3 protein is a protein derived from Synechocystis sp. having the amino acid sequence set forth in SEQ ID NO: 2, in which the superfamily 2 (SF2) helicase domain is located at positions 296 to 467, and motif 3 is located at positions 458 to 460. In the present invention, it is preferable for motif 3 to have a mutation, more preferably at positions 458 and / or 460, and particularly preferably at positions 458 and 460.

[0028] An example of a Type I-C Cas3 protein is a protein derived from Neisseria lactamica having the amino acid sequence set forth in SEQ ID NO: 3, in which the superfamily 2 (SF2) helicase domain is located at positions 260 to 428, and motif 3 is located at positions 424 to 426. In the present invention, it is preferable for motif 3 to have a mutation, more preferably at positions 424 and / or 426, and particularly preferably at positions 424 and 426.

[0029] An example of a Type I-D Cas3 protein is a protein derived from Microcystis aeruginosa having the amino acid sequence set forth in SEQ ID NO: 4, in which the superfamily 2 (SF2) helicase domain is located at positions 35 to 217, and motif 3 is located at positions 212 to 214. In the present invention, it is preferable for motif 3 to have a mutation, more preferably at positions 212 and / or 214, and particularly preferably at positions 212 and 214.

[0030] An example of a Type I-E Cas3 protein is a protein derived from Escherichia coli having the amino acid sequence set forth in SEQ ID NO: 5, in which the superfamily 2 (SF2) helicase domain is located at positions 309 to 491, and motif 3 is located at positions 483 to 485. In the present invention, it is preferable for motif 3 to have a mutation, more preferably at positions 433 and / or 435, and particularly preferably at positions 483 and 485.

[0031] The Cas3 protein in Type I-F is fused with the Cas2 protein. Examples of Cas3 proteins (in the form of a fusion protein of Cas2 and Cas3) include a protein derived from Pseudomonas aeruginosa having the amino acid sequence set forth in SEQ ID NO: 6, in which the superfamily 2 (SF2) helicase domain is located at positions 419 to 611, and motif 3 is located at positions 607 to 609. In the present invention, it is preferable for motif 3 to have a mutation, more preferably at positions 607 and / or 609, and particularly preferably at positions 607 and 609.

[0032] An example of a Type I-G Cas3 protein is a protein derived from Thioalkalivibrio sulfidiphilus having the amino acid sequence set forth in SEQ ID NO: 7, in which the superfamily 2 (SF2) helicase domain is located at positions 33 to 202, and motif 3 is located at positions 198 to 200. In the present invention, it is preferable for motif 3 to have a mutation, more preferably at positions 198 and / or 200, and particularly preferably at positions 198 and 200.

[0033] The SF2 helicase domain and motif 3 in the amino acid sequence of a Cas3 protein derived from a different biological species can be identified as the amino acid sequence corresponding to the SF2 helicase domain and motif 3 in the amino acid sequence by aligning the amino acid sequence with the above amino acid sequence.

[0034] The conserved region of motif 3 has the amino acid sequence "SAT (serine-alanine-threonine)" or "TAT (threonine-alanine-threonine)" (Tuteja N, et al., Eur J Biochem. 2004; 271: 1849-1863).

[0035] The "mutation of motif 3 of the SF2 helicase domain" of the present invention may be any mutation that suppresses the helicase activity of the Cas3 protein, and may include, for example, substitution, deletion, and / or insertion of one or more amino acids in motif 3. Examples of "multiple amino acids" include 2 to 5 amino acids, 2 to 4 amino acids, or 2 or 3 amino acids. The "mutation of motif 3 of the SF2 helicase domain" of the present invention is preferably substitution of at least one amino acid in motif 3, more preferably substitution of the first amino acid and / or the third amino acid in motif 3, and even more preferably substitution of the first amino acid and / or the third amino acid in motif 3 with a neutral amino acid having a hydrocarbon chain (alanine, glycine, valine, leucine, isoleucine, or proline). The amino acids substituted at the first and third positions in motif 3 may be the same or different; however, in the present invention, substitution of the first amino acid of motif 3 with alanine and / or substitution of the third amino acid with alanine is particularly preferred.

[0036] "Suppression of helicase activity" of a Cas3 protein means that the helicase activity of the Cas3 protein is lower than before the mutation, and includes both complete suppression (i.e., abolition) and partial suppression. The helicase activity of a Cas3 protein can be evaluated, for example, by a method in which dissociation of RI-labeled single-stranded DNA is detected by gel electrophoresis (Kim JH and Seo YS Methods Mol Biol. 2009;521:361-379).

[0037] The CRISPR-Cas3 system of the present invention includes, in addition to the mutant Cas3 protein, a cascade protein and crRNA as a guide RNA.

[0038] Cascade proteins typically include Cas5, Cas7, Cas8, and Cas11 in Type I-A, Cas5, Cas6, Cas7, Cas8, and Cas11 in Type I-B, Cas5, Cas7, Cas8, and Cas11 in Type I-C, Cas5, Cas7, Cas8, and Cas11 in Type I-D, Cas5, Cas6, Cas7, Cas10, and Cas11 in Type I-E, Cas5, Cas6, Cas7, Cas8, and Cas11 in Type I-F, and Csb2 (Cas6-like), Cas7, Cas8g, and Cas11 in Type I-G. The component having nuclease activity in the CRISPR-Cas3 system is typically the Cas3 protein, but in Type ID, it is Cas10d.

[0039] It should be understood that even when Cas11 is excluded from the components of the CRISPR-Cas3 system, DNA editing activity can still be exhibited (for example, it is known that Type I-B and Type I-C can exhibit DNA editing activity even when they do not contain Cas11, albeit at a lower level compared to when they contain Cas11, and the inventors have confirmed that this is also true for Type I-D). Therefore, the CRISPR-Cas3 system of the present invention also includes systems that do not contain Cas11, as long as they exhibit DNA editing activity.

[0040] When targeting the chromosomal genome of a eukaryotic cell, it is preferable to add a nuclear localization signal to promote the localization of the Cas3 protein or Cascade protein to the nucleus. The nuclear localization signal can be added to the N-terminus and / or C-terminus of each protein. Similarly, when targeting, for example, the mitochondrial genome and chloroplast genome, it is preferable to add a localization signal that promotes localization thereto.

[0041] In the CRISPR-Cas3 system of the present invention, Cas3 and Cascade may be in the form of a protein, a polynucleotide (DNA, RNA) encoding the protein, or a vector expressing the protein. In the polynucleotide form, the base sequence may be modified (e.g., codon optimization) to make it suitable for expression in a host cell.

[0042] In constructing an expression vector, the DNAs encoding the Cas3 protein and the Cascade protein can be designed to be carried on a single (same) vector, or all or part of them can be carried on separate vectors. Alternatively, the DNAs encoding each protein can be linked via DNA encoding an amino acid sequence (such as a 2A peptide) that is cleaved by a protease in cells, allowing them to be expressed as a single protein, which can then be separated into individual proteins by the action of the protease and used.

[0043] Various commonly used vectors can be used as the base vector for the expression vector. The type of expression vector is not particularly limited, and a vector capable of expressing guide RNA in the environment in which it is used (e.g., in host cells) can be appropriately selected. Examples of expression vectors include plasmid vectors, phage vectors, viral vectors, chromosomal vectors, episomal vectors, and virus-derived vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.), yeast chromosomal elements and viruses (baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, avian poxviruses, pseudorabies viruses, herpes viruses, lentiviruses, retroviruses, various plant viruses, etc.), and vectors derived from combinations thereof (cosmids, phagemids, etc.). The expression vector may contain a promoter sequence for inducing transcription or a sequence for enhancing transcription (e.g., an enhancer sequence).

[0044] Expression vectors can be prepared by known techniques. These techniques include those described in the operating manuals included with vector preparation kits, as well as those described in various manuals. For example, Joseph Sambrook & David W. Russell, Molecular cloning: a laboratory manual 3rd Ed., New York: Cold Spring Harbor Laboratory Press, 2001, is a comprehensive manual.

[0045] In the CRISPR-Cas3 system, the guide RNA, crRNA, forms a complex with the mutant Cas3 protein and the cascade protein, and the complex acts on the target DNA. Pre-crRNA is preferably used as the crRNA in the present invention. Pre-crRNA is typically a crRNA in which repeat sequences that undergo cleavage by processing are arranged on both sides of a spacer sequence. However, it is also possible to use a crRNA in which a repeat sequence that undergoes cleavage by processing is arranged only on the 5' side of the spacer sequence (Japanese Patent Application: Patent Application No. 2023-121605). When the CRISPR-Cas3 system is introduced into a eukaryotic cell in the form of a ribonucleoprotein (RNP), the RNP can contain crRNA in the form of a mature crRNA.

[0046] The "repeat sequence" in crRNA is a sequence that is repeated via a spacer sequence in the CRISPR structure of the bacterial genome from which the CRISPR-Cas3 system is derived. The wild-type repeat sequence differs depending on the subtype of the CRISPR-Cas3 system and the type of bacteria from which it is derived. For example, in the Type I-A system derived from Pyrococcus furiosus, it typically consists of the 30-base chain sequence set forth in SEQ ID NO: 8, and in Synechocystis sp. The type I-B system derived from Neisseria lactamica typically consists of the nucleotide sequence set forth in SEQ ID NO: 9, which has a chain length of 36 bases; the type I-C system derived from Neisseria lactamica typically consists of the nucleotide sequence set forth in SEQ ID NO: 10, which has a chain length of 32 bases; the type I-D system derived from Microcystis aeruginosa typically consists of the nucleotide sequence set forth in SEQ ID NO: 11, which has a chain length of 37 bases; the type I-E system derived from Escherichia coli typically consists of the nucleotide sequence set forth in SEQ ID NO: 12, which has a chain length of 29 bases; the type I-F system derived from Pseudomonas aeruginosa typically consists of the nucleotide sequence set forth in SEQ ID NO: 13, which has a chain length of 28 bases; The type IG system derived from S. sulfidiphilus typically consists of the nucleotide sequence shown in SEQ ID NO: 14, which has a chain length of 36 bases.

[0047] In a typical crRNA processing process, the repeat sequence forms a loop structure, which is cleaved by the action of a specific Cas (e.g., Cas6, Cas5, etc.). The cleavage site is typically between the 22nd and 23rd bases of the repeat sequence in Type I-A, between the 28th and 29th bases of the repeat sequence in Type I-B, between the 19th and 20th bases of the repeat sequence in Type I-C, between the 31st and 32nd bases of the repeat sequence in Type I-D, between the 21st and 22nd bases of the repeat sequence in Type I-E, between the 20th and 21st bases of the repeat sequence in Type I-F, and between the 28th and 29th bases of the repeat sequence in Type I-G.

[0048] The "spacer sequence" in crRNA is a sequence designed as a sequence complementary to the target DNA. The target DNA may be endogenous DNA or exogenous DNA. Endogenous DNA includes, for example, genomic DNA in chromosomes, mitochondria, and chloroplasts. Exogenous DNA includes, for example, reporter genes, marker genes, genes of viruses, bacteria, protozoa, etc. that infect hosts.

[0049] In the CRISPR-Cas3 system of the present invention, the crRNA may be in the form of RNA, a polynucleotide (DNA) encoding the RNA, or a vector that expresses the crRNA. The expression vector may be any of the above-mentioned commonly used vectors.

[0050] The "mutations" introduced by the CRISPR-Cas3 system of the present invention include DNA deletions, insertions, substitutions, and combinations thereof at the target site, and the CRISPR-Cas3 system can cause small-scale DNA deletions more frequently than when wild-type Cas3 proteins are used. Here, "small-scale" refers to, for example, 100 bases or less, preferably 50 bases or less (e.g., 30 bases or less, 20 bases or less, 10 bases or less, 5 bases or less, 3 bases or less, or 2 bases or less).

[0051] In the present invention, a desired nucleotide sequence can be knocked into a target DNA region by adding a donor DNA as a component of the CRISPR-Cas3 system. The donor DNA typically has homology arms on both sides of the desired nucleotide sequence to be knocked in, and the desired nucleotide sequence is inserted into the target DNA region via a homologous recombination repair mechanism or the like.

[0052] Examples of "eukaryotic cells" used to introduce mutations into target DNA include animal cells, plant cells, algae cells, and fungal cells. Examples of animal cells include mammalian cells as well as fish, bird, reptile, amphibian, and insect cells. Examples of animal cells include cells constituting individual animals, cells constituting organs and tissues removed from animals, and cultured cells derived from animal tissues. Specific examples include germ cells such as oocytes and sperm; embryonic cells of various stages of embryos (e.g., 1-cell embryo, 2-cell embryo, 4-cell embryo, 8-cell embryo, 16-cell embryo, morula, etc.); stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, brain cells, and kidney cells. The oocytes used to create genome-edited animals can be either pre- or post-fertilization oocytes, but are preferably fertilized oocytes, i.e., fertilized eggs. The fertilized eggs are particularly preferably pronuclear stage embryos. The oocytes can be thawed after being cryopreserved.

[0053] The term "mammal" encompasses both humans and non-human mammals. Examples of non-human mammals include ungulates such as cattle, boars, pigs, sheep, and goats, perissodactyls such as horses, rodents such as mice, rats, guinea pigs, hamsters, and squirrels, lagomorphs such as rabbits, and carnivores such as dogs, cats, and ferrets. The non-human mammals may be livestock or companion animals (pets), or wild animals.

[0054] Known methods can be used to create non-human individuals from cells. When creating non-human individuals from cells in animals, germ cells or pluripotent stem cells are usually used. For example, molecules constituting the CRISPR-Cas3 system of the present invention are introduced into oocytes, and the resulting oocytes are then implanted into the uterus of a pseudopregnant female non-human mammal, followed by the production of offspring. Implantation can be performed using fertilized eggs at the 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, or morula stage. If necessary, oocytes can be cultured under appropriate conditions until implantation. The transplantation and culture of oocytes can be carried out according to known techniques (Nagy A. et al., Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2003). From the obtained non-human individual, offspring or clones in which the desired DNA has been edited can also be obtained.

[0055] Examples of plant cells include cells of grains, oil crops, forage crops, fruits, and vegetables. Examples of plant cells include cells constituting individual plants, cells constituting organs or tissues isolated from plants, and cultured cells derived from plant tissue. Examples of plant organs and tissues include leaves, stems, shoot tips (growing points), roots, tubers, and calli. Examples of plants include rice, corn, banana, peanut, sunflower, tomato, rapeseed, tobacco, wheat, barley, potato, soybean, cotton, and carnation, as well as their propagation materials (e.g., seeds, tuberous roots, tubers, etc.).

[0056] It has long been known that somatic cells of plants possess totipotency, and methods for regenerating plants from plant cells have been established for various plants. Therefore, for example, by introducing molecules constituting the CRISPR-Cas3 system of the present invention into plant cells and regenerating plants from the resulting plant cells, a plant with a desired DNA edited can be obtained. From the resulting plant, progeny, clones, or propagation materials with the desired DNA edited can also be obtained. Methods established in the technical field can be used to regenerate plant tissues by tissue culture to obtain individuals (Transformation Protocols [Plant Edition], edited by Yutaka Tabei, Kagaku Dojin, pp. 340-347 (2012)).

[0057] The method for introducing the CRISPR-Cas3 system of the present invention into eukaryotic cells is not particularly limited. Examples include electroporation, calcium phosphate method, liposome method, DEAE-dextran method, microinjection method, cationic lipid-mediated transfection, electroporation, transduction, and infection with a viral vector. Such methods are described in many standard laboratory manuals, such as "Leonard G. Davis et al., Basic methods in molecular biology, New York: Elsevier, 1986."

[0058] The present invention also provides a kit for use in the above-mentioned method, comprising (A) a Cas3 protein whose helicase activity has been suppressed by a mutation in the superfamily 2 (SF2) helicase domain, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide. The kit of the present invention may further comprise (B) a Cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide. The kit of the present invention may further comprise (C) a crRNA, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide. The Cas3 protein and / or Cascade protein in the kit of the present invention may be tagged with a nuclear localization signal.

[0059] The components of the kit of the present invention may be in a form in which all or some of them are mixed together, or each component may be independent. The kit of the present invention can be used in a wide range of fields, such as pharmaceuticals, food, livestock, fisheries, industry, bioengineering, and life science research.

[0060] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0061] The mutant Cas3 proteins used in this example have the following mutations compared to the wild-type Cas3 protein (SEQ ID NO: 6): mtCas3-h1: K320N mtCas3-h2: S483A & T485A mtCas3-h3: V454S mtCas3-h4: R665A mtCas3-h2-1: S483A mtCas3-h2-2: T485A.

[0062] Example 1: Mutation introduction using mtCas3-h2 in cultured rice cells. Mutations were introduced into the rice phytoene desaturase (PDS) gene using mtCas3-h2. Simian Virus 40 (SV40)-derived nuclear localization signals (NLS) were added to both termini, and Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11 were synthesized with rice-optimized codons. Expression cassettes were constructed for these six genes using the promoter of the maize ubiquitin gene and the 5' untranslated region (translation enhancer) of rice-derived alcohol dehydrogenase. Furthermore, crRNA (SEQ ID NO: 15) recognizing the 27-nucleotide target sequence of the PDS gene was prepared, and a vector for expression using the rice U6-2 snRNA promoter was constructed. The expression cassettes for mtCas3-h2, Cas5, and Cas7 were introduced into a binary vector with a pPZP202 backbone, carrying a hygromycin resistance gene for selection of transformed rice. The expression cassettes for Cas6, Cas8, and Cas11 were introduced into a binary vector with a pPZP202 backbone, carrying a kanamycin resistance gene for selection of transformed rice. These vectors were introduced into Agrobacterium strain EHA105, and rice callus was transformed. Rice callus co-infected with Agrobacterium harboring each binary vector was cultured in a medium containing hygromycin and G418 for 6 weeks, and transformed callus was selected. DNA extracted from the resulting transformed callus was used as a template for PCR amplification of a 328-bp region containing the target sequence, and electrophoresis was performed using a Multi-NA (Shimadzu Corporation).

[0063] The results showed that when mtCas3-h2 with a mutation in motif 3 of the SF2 helicase domain was transformed, a PCR product several tens to hundreds of bases shorter than 328 bp was amplified (Figure 2). On the other hand, when mtCas3-h2 was not transformed, or when a mutant Cas3 with an amino acid substitution (H74A) in the nickase domain and lacking nickase activity was introduced, such a short PCR product was not detected (Figure 2).

[0064] Furthermore, similar to the double mutant (mtCas3-h2 (S483A and T485A)), single mutants (mtCas3-h2-1 (S483A) or mtCas3-h2-2 (T485A)) were used to introduce mutations into the rice phytoene desaturase PDS gene, and the efficiency of deletion introduction using these mutant Cas3 proteins was examined.

[0065] As a result, it was confirmed that, although the efficiency was lower than that of the double mutant, short deletions could be introduced into target sequences in rice cells even when using Cas3 with a single mutation in motif 3 of the SF2 helicase domain (Figure 3).

[0066] Example 2: Measurement of genome cleavage activity in human cultured HEK293T cells using mutant Cas3. Using reporter HEK293T cells carrying mCherry-P2A-EGFP, the efficiency of mutation introduction in human cells was examined for wild-type Cas3 and mtCas3-h1 to -h4, which have mutations in the helicase domain. Two target sites (GFP-crRNA-1, 2) were designed on GFP. Expression plasmids were prepared, and reporter HEK293T cells were transfected with plasmids encoding crRNA-1 (SEQ ID NO: 16), six Cas (3, 5, 6, 7, 8, 11) effectors, and a puromycin resistance gene using Lipofectamine 2000 (Thermo Fisher Scientific). The cells were cultured in 1 μg / ml puromycin-supplemented medium at 37°C in 5% CO for 5 days, after which all cells were collected and subjected to FACS analysis using AriaIIIu (BD) to measure the GFP and mCherry positivity. The knockout efficiency was calculated from the number of GFP-negative cells.

[0067] As a result, in addition to wild-type Cas3, GFP knockout was observed even when a mutant form of mtCas3-h2 was used (Figure 4). Furthermore, when GFP was negative, we also examined whether the upstream mCherry gene was positive or negative. When wild-type Cas3 was used, most cells (over 95%) lacked expression of both GFP and mCherry, suggesting that a large-scale deletion had occurred. On the other hand, with mtCas3-h2, 38.6% were mCherry positive, suggesting that there were more short mutation patterns in which only GFP was deleted compared to wild-type Cas3.

[0068] To confirm that the short deletion mutation had actually been introduced, a similar experiment was performed using GFP-crRNA-2 (SEQ ID NO: 17). GFP-negative cells were sorted and total DNA was extracted using the Tissue XS kit (Takara Bio). The target locus was amplified using Gflex (Takara Bio), followed by TA cloning to confirm the sequence. TA cloning was performed according to the protocol for the pCR4Blunt-TOPO plasmid vector (Life Technologies).

[0069] As a result, when mtCas3-h2 was used, multiple mutations ranging from a few to several dozen bases were observed, which were not observed when wild-type Cas3 was used (Figure 5). This demonstrated that mtCas3-h2, which has a mutation in motif 3 of the SF2 helicase domain, can induce small-scale deletion mutations even in cultured human cells.

[0070] The CRISPR-Cas3 system of the present invention enables smaller-scale genome editing in eukaryotic cells, including plants and humans, compared to conventional CRISPR-Cas3 systems, which can contribute significantly not only to basic research but also to medical applications that require safety and precise control, as well as industrial applications such as the development of agricultural and livestock products.

Claims

1. A method for producing a eukaryotic cell in which a mutation has been introduced into target DNA, comprising introducing a CRISPR-Cas3 system into the eukaryotic cell, wherein the CRISPR-Cas3 system comprises the following (A) to (C): (A) a Cas3 protein whose helicase activity has been suppressed by a mutation in motif 3 of the superfamily 2 (SF2) helicase domain, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide, (B) a Cascade protein, a polynucleotide encoding the protein, or an expression vector comprising the polynucleotide, and (C) a crRNA, a polynucleotide encoding the crRNA, or an expression vector comprising the polynucleotide.

2. The method according to claim 1, wherein a nuclear localization signal is added to the Cas3 protein and / or the Cascade protein.

3. A kit for use in the method of claim 1, comprising: (A) a Cas3 protein whose helicase activity has been suppressed by a mutation in motif 3 of the superfamily 2 (SF2) helicase domain, a polynucleotide encoding said protein, or an expression vector containing said polynucleotide.

4. The kit according to claim 3, further comprising (B) a Cascade protein, a polynucleotide encoding said protein, or an expression vector containing said polynucleotide.

5. The kit of claim 3 or 4, further comprising (C) a crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

6. The kit according to claim 3 or 4, wherein a nuclear localization signal is added to the Cas3 protein and / or the Cascade protein.

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