Improved crisper-cas3 system

Abstract

This study provides a CRISPR-Cas3 system that enables small-scale mutation introduction in eukaryotic cells, compared to existing CRISPR-Cas3 systems. It was discovered that by using a Cas3 protein with a mutation introduced into motif 3 of the SF2 helicase domain as the Cas3 protein in the CRISPR-Cas3 system, mutations can be generated in target DNA in eukaryotic cells.

Description

Technical Field

[0001] This invention relates to a modified CRISPR-Cas3 system, and more specifically, to a CRISPR-Cas3 system capable of small-scale DNA editing by utilizing mutant Cas3 proteins. Background Technology

[0002] Genome editing technologies, utilizing zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9, are techniques that specifically cut target DNA in animal and plant cells and rewrite the target DNA sequence using intrinsic repair mechanisms. Genome editing technology is not only applied in basic research areas such as elucidating the function of target genes and proteins, but its applications are also rapidly expanding to areas such as the improvement of agricultural and livestock products, biomaterial production, regenerative medicine, and genome editing therapy. For example, regarding the improvement of agricultural and livestock products, soybean oil rich in oleic acid and mustard greens with suppressed spiciness have been launched in the United States, while tomatoes with high GABA accumulation and thick-fleshed sea bream have been launched in Japan.

[0003] Alongside the rapid expansion of research and industrial applications of genome editing technology, the development and improvement of genome editing tools are also advancing rapidly. The CRISPR-Cas systems possessed by bacteria and archaea are divided into two categories: those that cleave target sequences through complexes of multiple proteins and those that cleave through single proteins. To date, CRISPR-Cas9 and CRISPR-Cas12a, developed as genome editing tools, are classified into these two categories.

[0004] In Japan, advancements in the development of CRISPR-Cas3, a type 1 type I genome editing technology, have led to the world's first discovery of its potential as a genome editing tool for eukaryotic cells (Non-Patent Document 1, Patent Document 1).

[0005] For example, in the IE-type CRISPR-Cas3 system, a cascade complex formed by 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. Then, Cas3, possessing both helicase and nuclease activity, unwinds the double-stranded DNA structure while simultaneously cleaving the DNA, thereby inducing extensive DNA cleavage upstream of the target sequence (Non-Patent Literature 2). To date, large-scale deletion mutations ranging from hundreds of bp to several kb upstream of the target sequence have been reported in genome editing using the CRISPR-Cas3 system in human, rice, and other cells (Non-Patent Literature 1, 3, 4, 5).

[0006] Thus, the CRISPR-Cas3 system is particularly adept at generating large-scale DNA deletions compared to the CRISPR-Cas9 system, and is therefore suitable for, for example, gene knockout that deletes the entire target gene. On the other hand, genome editing that targets 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 desired.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: International Publication No. 2018 / 225858

[0010] Non-patent literature

[0011] Non-patent literature 1: Morisaka H, ​​et al. Nat Commun. 2019; 10(1): 5302.

[0012] Non-patent literature 2: Yoshimi K, et al. Nat Commun. 2022; 13(1): 4917.

[0013] Non-patent literature 3: Dolan AE, et al. Mol Cell. 2019; 74(5): 936-950.

[0014] Non-patent literature 4: Li Y, et al. Plant Biotechnol J. 2023;21(11): 2196-2208.

[0015] Non-Patent Literature 5: Saiga et al., "Knockout of Intrinsic Genes in Rice Using CRISPR / Cas3", 143rd Lecture of the Japan Society for Rice Breeding, March 2023 Summary of the Invention

[0016] The problem that the invention aims to solve

[0017] The present invention was made in view of the following circumstances, and its object is to provide a CRISPR-Cas3 system that enables the introduction of small-scale mutations in eukaryotic cells compared with existing CRISPR-Cas3 systems.

[0018] Methods for solving problems

[0019] The report describes how the CRISPR-Cas3 system, using mutants of the HD domain (H74A), the SF2 domain motif 1 mutant (K320N), and the SF2 domain motif 3 double mutant (S483A / T485A) as its constituent Cas3 proteins, failed to cleave target DNA in vitro (Figure 14 of Patent Document 1). Therefore, this report proposes fusing these mutant Cas3 proteins with other effector proteins (e.g., transcription factors) to enable these other effector proteins to function within the CRISPR-Cas3 system.

[0020] In order to solve the problem of developing a CRISPR-Cas3 system capable of introducing mutations on a smaller scale than before in eukaryotic cells, the inventors verified the effectiveness of a CRISPR-Cas3 system containing these mutant Cas3 proteins in eukaryotic cells. Unexpectedly, they found that by introducing a mutant Cas3 protein into motif 3 of the SF2 helicase domain in the mutant Cas3 protein, mutations of several to tens of bases could be generated in the genomes of eukaryotic cells such as rice cells and human cells, thus completing the present invention.

[0021] This invention relates to DNA editing in eukaryotic cells using a CRISPR-Cas3 system that incorporates a mutant Cas protein into motif 3 of the SF2 helicase domain, and more specifically includes the following scheme.

[0022] (1) A method for manufacturing eukaryotic cells with a mutation introduced into target DNA, the method comprising introducing a CRISPR-Cas3 system into the eukaryotic cells, the CRISPR-Cas3 system comprising the following (A) to (C), (A) A Cas3 protein whose helicase activity is inhibited 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. (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.

[0023] (2) According to the method described in (1), nuclear localization signals were added to the Cas3 protein and / or cascade proteins.

[0024] (3) A kit for use in the method described in (1) comprising (A) a Cas3 protein whose helicase activity is inhibited 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.

[0025] (4) The kit according to (3) further comprises (B) cascade protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide.

[0026] (5) The kit according to (3) or (4) further comprises (C)crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide.

[0027] (6) The kit described in any one of (3) to (5) is used to add nuclear localization signals to the Cas3 protein and / or cascade proteins.

[0028] The effects of the invention

[0029] This invention provides a CRISPR-Cas3 system capable of generating small-scale DNA mutations in eukaryotic cells. This enables more precise control over the edited regions in genome editing. Attached Figure Description

[0030] Figure 1 This is a graph about DNA editing in eukaryotic cells, showing a comparison between the present invention's method using mutant Cas3 protein and existing methods using wild-type Cas3 protein.

[0031] Figure 2This is a graph showing the results of DNA editing in rice cells performed by the method of the present invention. The rice phytoene desaturase PDS gene was used as the target DNA. A shows the results of electrophoresis after PCR amplification of the region containing the target DNA in callus tissues in which mutant Cas3 protein (mtCas3-h2) and mutant Cas3 protein (Cas3(H74A)) lacking helicase activity and nickase activity were introduced, as well as in callus tissues without Cas3 protein introduction. B is a graph showing the deletion introduction efficiency on the target DNA in callus tissues treated with each method. The deletion introduction efficiency was calculated as the proportion of callus tissues in which PCR products shorter than expected were detected relative to the analyzed callus tissues.

[0032] Figure 3 This indicates the use of wild-type Cas3 protein or various mutant Cas3 proteins (mtCas3-h2, mtCas3-h2-1, or mtCas3-h2-2) to perform [interaction / reaction]. Figure 2 The same experiment was performed, and the results of the mutation introduction efficiency of each Cas3 protein were analyzed graphically. The experiment was conducted independently twice, and the results of each experiment are referred to as "Experiment 1" and "Experiment 2".

[0033] Figure 4 This figure shows the results of reporter gene editing after introducing crRNA-1 targeting the EGFP gene and a CRISPR-Cas3 system containing wild-type Cas3 protein or various mutant Cas3 proteins (mtCas3-h1~h4) into HEK293T reporter cells with mCherry-P2A-EGFP. The top of the figure shows the structure of the reporter vector used and the target site of the crRNA. The lower left of the figure shows the mutation introduction efficiency using each Cas3 protein, and the lower right of the figure shows the mutation patterns introduced through each Cas3 protein. In the figure, "Gnega" indicates GFP negative, "mCnega" indicates mCherry negative, and "Wnega" indicates both GFP negative and mCherry negative.

[0034] Figure 5 It uses crRNA-2, which targets other sites on the EGFP gene, as a guide RNA to perform [action / action]. Figure 4 The same experiment was used to analyze the results of the mutations introduced into the EGFP gene. Detailed Implementation

[0035] The present invention provides a method for manufacturing eukaryotic cells with a mutation introduced into target DNA, the method comprising introducing the eukaryotic cells into a CRISPR-Cas3 system, the CRISPR-Cas3 system comprising the following (A) to (C).

[0036] (A) A Cas3 protein whose helicase activity is inhibited by a mutation in the superfamily 2 (SF2) helicase domain, a 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.

[0037] CRISPR-Cas systems of type 1 are classified into type I and type III. Further type I CRISPR-Cas3 systems are classified into six types: type IA, type IB, type IC, type ID, type IE, and type IF, as well as type IG, which is a subtype of type IB (e.g., see [van der Oost J et al. (2014) Unravelling the structural and mechanical basis of CRISPR-Cas systems, Nature Reviews Microbiology, Vol. 12 (No. 7), pp. 479-492], [Jackson RN et al. (2014) Fitting CRISPR-associatedCas3 into the Helicase Family Tree, Current Opinion in Structural Biology, Vol. 24, pp. 106-114]).

[0038] In the CRISPR-Cas3 system of this invention, the use of a Cas3 protein is characterized by the use of a protein whose helicase activity is inhibited by a mutation in the superfamily 2 (SF2) helicase domain.

[0039] It is known that in type IA, Cas3 proteins are divided into Cas3-HD, which has nuclease activity, and Cas3-HEL, which has helicase activity. In this invention, a combination of Cas3-HD and Cas3-HEL with inhibited helicase activity can be used. Examples of Cas3-HEL include, for instance, proteins derived from *Pyrococcus furiosus* having the amino acid sequence described in Serial No. 1, in which the superfamily 2 (SF2) helicase domain is located at positions 18–207 and motif 3 is located at positions 198–200. In this invention, it is preferable that motif 3 has a mutation, more preferably a mutation at positions 198 and / or 200, and particularly preferably mutations at positions 198 and 200.

[0040] Examples of Cas3 proteins in type IB include proteins derived from *Synechocystis* sp. with the amino acid sequence described in Serial No. 2, in which the superfamily 2 (SF2) helicase domain is located at positions 296–467 and motif 3 is located at positions 458–460. In this invention, a mutation in motif 3 is preferred, a mutation at positions 458 and / or 460 is more preferred, and mutations at positions 458 and 460 are particularly preferred.

[0041] Examples of Cas3 proteins in type IC include proteins derived from *Neisseria lactamica* having the amino acid sequence described in sequence number 3, in which the superfamily 2 (SF2) helicase domain is located at positions 260–428 and motif 3 is located at positions 424–426. In this invention, a mutation in motif 3 is preferred, a mutation at positions 424 and / or 426 is more preferred, and mutations at positions 424 and 426 are particularly preferred.

[0042] Examples of Cas3 proteins in the ID type include, for instance, proteins derived from *Microcystis aeruginosa* having the amino acid sequence described in Serial No. 4, in which the superfamily 2 (SF2) helicase domain is located at positions 35–217 and motif 3 is located at positions 212–214. In this invention, a mutation in motif 3 is preferred, a mutation at positions 212 and / or 214 is more preferred, and mutations at positions 212 and 214 are particularly preferred.

[0043] Examples of Cas3 proteins in the IE type include, for instance, proteins derived from *Escherichia coli* having the amino acid sequence described in Serial No. 5, in which the superfamily 2 (SF2) helicase domain is located at positions 309–491 and motif 3 is located at positions 483–485. In this invention, a mutation in motif 3 is preferred, a mutation at positions 433 and / or 435 is more preferred, and mutations at positions 483 and 485 are particularly preferred.

[0044] In the IF type, the Cas3 protein is fused with the Cas2 protein. Examples of Cas3 proteins (the fusion protein of Cas2 and Cas3) include, for instance, proteins derived from *Pseudomonas aeruginosa* having the amino acid sequence described in Serial No. 6, in which the superfamily 2 (SF2) helicase domain is located at positions 419–611 and motif 3 is located at positions 607–609. In this invention, a mutation in motif 3 is preferred, a mutation at positions 607 and / or 609 is more preferred, and mutations at positions 607 and 609 are particularly preferred.

[0045] Examples of Cas3 proteins in the IG type include proteins derived from *Thioalkalivibrio sulfidiphilus* with the amino acid sequence described in sequence number 7, in which the superfamily 2 (SF2) helicase domain is located at positions 33–202 and motif 3 is located at positions 198–200. In this invention, it is preferable that motif 3 has a mutation, more preferably a mutation at positions 198 and / or 200, and particularly preferably mutations at positions 198 and 200.

[0046] The SF2 helicase domain and motif 3 in the amino acid sequences of different Cas3 proteins from different biological species can be specified as follows: the amino acid sequence is compared with the above-mentioned amino acid sequence and specified as the amino acid sequence corresponding to the SF2 helicase domain and motif 3 in the above-mentioned amino acid sequence.

[0047] Among them, the conserved region of motif 3 has an amino acid sequence of "SAT (serine-alanine-threonine)" or "TAT (threonine-alanine-threonine)" (Tuteja N, et al., Eur J Biochem. 2004; 271: 1849-1863).

[0048] In this invention, the "mutation of motif 3 of the SF2 helicase domain" can be any mutation that inhibits the helicase activity in the Cas3 protein. Examples include, for instance, the substitution, deletion, and / or insertion of one or more amino acids in motif 3. "Multiple amino acids" can be, for example, 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" in this invention preferably involves the substitution of at least one amino acid in motif 3, more preferably the substitution of the amino acid at position 1 and / or position 3, and even more preferably the substitution of the amino acid at position 1 and / or position 3 to a neutral amino acid with a hydrocarbon chain (alanine, glycine, valine, leucine, isoleucine, or proline). The substituted amino acids may be the same or different at positions 1 and 3 of motif 3. In this invention, the substitution of the amino acid at position 1 to alanine and / or position 3 to alanine is particularly preferred.

[0049] "Inhibition of helicase activity" in Cas3 proteins refers to a decrease in helicase activity compared to the pre-mutation state, including both complete inhibition (i.e., disappearance) and partial inhibition. The helicase activity of Cas3 proteins can be evaluated, for example, by detecting the dissociation of RI-labeled single-stranded DNA using gel electrophoresis (Kim JH and Seo YS Methods Mol Biol. 2009; 521: 361-379).

[0050] In addition to the mutant Cas3 protein mentioned above, the CRISPR-Cas3 system in this invention also includes cascade proteins and crRNA as a guide RNA.

[0051] Typically, cascade proteins include Cas5, Cas7, Cas8, and Cas11 in type IA; Cas5, Cas6, Cas7, Cas8, and Cas11 in type IB; Cas5, Cas7, Cas8, and Cas11 in type IC; Cas5, Cas6, Cas7, Cas10, and Cas11 in type ID; Cas5, Cas6, Cas7, Cas8, and Cas11 in type IE; Cas5, Cas6, Cas7, and Cas8 as cascade proteins in type IF; and Csb2 (Cas6-like), Cas7, Cas8g, and Cas11 in type IG. The nuclease-active component in the CRISPR-Cas3 system is typically the Cas3 protein, but in type ID it is Cas10d.

[0052] Furthermore, regarding Cas11, it should be understood that DNA editing activity can still be exhibited even when it is excluded from the components of a CRISPR-Cas3 system (for example, it is known that DNA editing activity can be exhibited in types IB and IC without Cas11, although at a lower level compared to those containing Cas11; the inventors have also confirmed that this is also the case in type ID). Therefore, the CRISPR-Cas3 system of the present invention, as long as it exhibits DNA editing activity, also includes systems that do not contain Cas11.

[0053] When targeting the chromosomal genome of a eukaryotic cell, it is preferable to add nuclear transfer signals to promote the localization of Cas3 proteins and cascade proteins to the nucleus. These nuclear transfer signals can be added to the N-terminus and / or C-terminus of each protein. Similarly, for example, when targeting the mitochondrial genome and chloroplast genome, it is preferable to add transfer signals that promote localization to them.

[0054] In the CRISPR-Cas3 system of this invention, Cas3 and the cascade can be in the form of a protein, a polynucleotide (DNA, RNA) encoding the protein, or a vector expressing the protein. In the form of a polynucleotide, modifications to the base sequence suitable for expression within the host cell can also be performed (e.g., codon optimization).

[0055] In the construction of expression vectors, the DNA encoding Cas3 proteins and cascade proteins can be designed to be carried in one (same) vector, or all or part of it can be carried in separate vectors. Alternatively, the DNA encoding each protein can be combined with DNA encoding an amino acid sequence (such as a 2A peptide) that can be cleaved by intracellular proteases, thereby expressing it as a protein, and then it can be separated into individual proteins by the action of proteases for use.

[0056] Expression vectors, as the base vector, can be any type of commonly used vector. The type of expression vector is not particularly limited; a suitable vector can be selected that can express guide RNA in its environment of use (e.g., host cells). Examples of expression vectors include, for example, plasmid vectors, phage vectors, viral 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, various plant viruses, etc.), and vectors derived from combinations thereof (plasmids, phage particles, etc.). Expression vectors may also contain promoter sequences for inducing transcription and sequences for enhancing transcription (e.g., enhancer sequences).

[0057] Expression vectors can be prepared using well-known methods. In addition to the methods described in the instruction manual accompanying the vector preparation kit, various 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.

[0058] In the CRISPR-Cas3 system, the crRNA serving as the guide RNA forms a complex with the aforementioned mutant Cas3 protein and cascade proteins, which acts on the target DNA. As the crRNA in this invention, a precursor crRNA is preferred. The precursor crRNA is typically crRNA with processing-induced cleaved repeat sequences positioned on both sides of the spacer sequence, but crRNA with processing-induced cleaved repeat sequences positioned only on the 5' side of the spacer sequence can also be used (Japanese Patent Application: 2023-121605). Furthermore, when the CRISPR-Cas3 system is introduced into eukaryotic cells in the form of a ribonucleoprotein (RNP), the RNP may contain crRNA in the form of mature crRNA.

[0059] "Repetitive sequences" in crRNA are sequences that appear repeatedly in the CRISPR structure of bacterial genomes derived from the CRISPR-Cas3 system, separated by spacer sequences. The wild-type repetitive sequences vary depending on the CRISPR-Cas3 system subtype and the species of bacteria from which they originate. For example, in the type IA system derived from *Pyrococcus furiosus*, they typically consist of the sequence described in sequence number 8, which has a chain length of 30 bases; in the type IB system derived from *Synechocystis* sp., they typically consist of the sequence described in sequence number 9, which has a chain length of 36 bases; in the type IC system derived from *Neisseria lactamica*, they typically consist of the sequence described in sequence number 10, which has a chain length of 32 bases; in the type ID system derived from *Microcystis aeruginosa*, they typically consist of the sequence described in sequence number 11, which has a chain length of 37 bases; and in the type I system derived from *Escherichia coli*, they typically consist of the repetitive sequences described in sequence number 11, which has a chain length of 37 bases. In the IE system derived from *Pseudomonas aeruginosa*, the sequence typically consists of the 29-base sequence described in sequence number 12. In the IF system derived from *Pseudomonas aeruginosa*, the sequence typically consists of the 28-base sequence described in sequence number 13. In the IG system derived from *Thioalkalivibrio sulfidiphilus*, the sequence typically consists of the 36-base sequence described in sequence number 14.

[0060] In typical crRNA processing, repetitive sequences form loop structures, which are then cleaved by specific Cas sequences (e.g., Cas6, Cas5, etc.). The cleavage sites are typically between bases 22 and 23 of the repetition in type IA, between bases 28 and 29 in type IB, between bases 19 and 20 in type IC, between bases 31 and 32 in type ID, between bases 21 and 22 in type IE, between bases 20 and 21 in type IF, and between bases 28 and 29 in type IG.

[0061] The "spacer sequence" in crRNA is a sequence designed to be complementary to the target DNA. The target DNA can be either endogenous or exogenous. Examples of endogenous DNA include genomic DNA found in chromosomes, mitochondria, and chloroplasts. Examples of exogenous DNA include reporter genes, biomarker genes, and genes from viruses, bacteria, protozoa, etc., that infect the host.

[0062] In the CRISPR-Cas3 system of this invention, the crRNA can be in the form of RNA, the form of a polynucleotide (DNA) encoding the RNA, or the form of a vector expressing the crRNA. The expression vector, as the substrate vector, can be any of the commonly used vectors described above.

[0063] The "mutations" introduced by the CRISPR-Cas3 system of the present invention include deletions, insertions, substitutions, and combinations thereof in the target DNA region. However, the CRISPR-Cas3 system can generate small-scale DNA deletions at a high frequency compared to the use of wild-type Cas3 proteins. Here, "small-scale" means, for example, less than 100 bases, preferably less than 50 bases (e.g., less than 30 bases, less than 20 bases, less than 10 bases, less than 5 bases, less than 3 bases, less than 2 bases).

[0064] In this invention, by adding donor DNA as a component of the CRISPR-Cas3 system, a desired base sequence can be knocked into the target DNA region. The donor DNA typically has homologous arms on both sides of the desired base sequence to be knocked in, and the desired base sequence is inserted into the target DNA region through homologous recombination repair mechanisms, etc.

[0065] Examples of "eukaryotic cells" used in introducing mutations into target DNA include, for example, animal cells, plant cells, algal cells, and fungal cells. Examples of animal cells include, for example, mammalian cells, as well as cells from fish, birds, reptiles, amphibians, and insects. Animal cells include, for example, cells that constitute an individual animal, cells that constitute organs / tissues removed from an animal, and cultured cells derived from animal tissues. Specifically, examples include, for example, germ cells such as oocytes and sperm; embryonic cells at various stages of the embryo (e.g., single-cell embryos, 2-cell embryos, 4-cell embryos, 8-cell embryos, 16-cell embryos, morula embryos, etc.); stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, brain cells, and kidney cells. As for the oocytes used in the creation of genome-edited animals, oocytes before and after fertilization can be used, but oocytes after fertilization, i.e., zygotes, are preferred. The fertilized eggs are especially preferred as they are pronuclear embryos. The oocytes can be frozen and conserved before thawing and use.

[0066] Mammals are a concept that includes both humans and non-human mammals. Examples of non-human mammals include, for instance, even-toed ungulates such as cattle, wild boars, 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 ferrets. These non-human mammals can be domesticated animals or companion animals (pets), or they can be wild animals.

[0067] Known methods can be used as a means of creating non-human individuals from cells. In animals, germ cells or pluripotent stem cells are typically used when creating non-human individuals from cells. 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 non-human female mammal in a pseudopregnant state, resulting in offspring. Transplantation can be performed using fertilized eggs from single-cell, two-cell, four-cell, eight-cell, sixteen-cell, or morula stages. Oocytes can be cultured under appropriate conditions for transplantation as needed. Oocyte transplantation and culture can be performed based on 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 individuals, desired DNA-edited offspring or clones can also be obtained.

[0068] Examples of plant cells include, for example, the cells of cereals, oil crops, forage crops, fruits, and vegetables. Plant cells include, for example, the cells that constitute an individual plant, the cells that constitute organs / tissues separated from a plant, and cultured cells derived from plant tissues. Examples of plant organs / 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.).

[0069] In plants, the totipotency of somatic cells has been known since ancient times, and methods for regenerating plant bodies from plant cells have been established in a wide variety of plants. Therefore, for example, by introducing the molecules constituting the CRISPR-Cas3 system of the present invention into plant cells, and regenerating plant bodies from the resulting plant cells, it is possible to obtain plant bodies with the desired DNA edited. 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, methods already established in this technical field can be utilized (Transformation Protocol [Plant Section] (Transformation Procedure [Plant Section]) edited by Yutaka Tabei / Chemical Doujin pp. 340-347 (2012)).

[0070] The method for introducing the CRISPR-Cas3 system of the present invention into cells is not limited. Examples include electroporation, calcium phosphate method, liposome method, DEAE-dextrose method, microinjection, cationic lipid-mediated transfection, electroporation, transduction, and infection using viral vectors. Such methods are described in several standard laboratory manuals, such as "Leonard G. Davis et al., Basic methods in molecular biology, New York: Elsevier, 1986".

[0071] The present invention also provides a kit for use in the above-described methods, the kit comprising (A) a Cas3 protein whose helicase activity is inhibited by a mutation in the superfamily 2 (SF2) helicase domain, a polynucleotide encoding the protein, or an expression vector containing the polynucleotide. The kit of the present invention may further comprise (B) a cascade protein, a polynucleotide encoding the protein, or an expression vector containing the polynucleotide. The kit of the present invention may further comprise (C) crRNA, a polynucleotide encoding the crRNA, or an expression vector containing the polynucleotide. Nuclear localization signals may also be added to the Cas3 protein and / or cascade protein in the kit of the present invention.

[0072] The components of the kit of the present invention can be all or some of the components combined, or they can be independent of each other. The kit of the present invention can be used in a wide range of fields, such as pharmaceuticals, food, animal products, aquaculture, industry, bioengineering, and life science research.

[0073] Example

[0074] The invention is illustrated in more detail by way of examples, but the invention is not limited to the following examples.

[0075] Furthermore, the mutant Cas3 protein used in this embodiment has the following mutations compared to the wild-type Cas3 protein (Sequence No. 6).

[0076] mtCas3-h1: K320N

[0077] mtCas3-h2: S483A & T485A

[0078] mtCas3-h3: V454S

[0079] mtCas3-h4: R665A

[0080] mtCas3-h2-1: S483A

[0081] mtCas3-h2-2:T485A.

[0082] [Example 1] Mutation introduction of mtCas3-h2 in rice cultured cells

[0083] The rice phytoene desaturase PDS gene was mutated using mtCas3-h2. Nuclear localization signals (NLS) derived from Simian Virus40 (SV40) were added to both ends, and Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11 codons optimized for rice were synthesized. Expression cassettes using the promoter of a maize-derived ubiquitin gene and the 5' untranslated region (translation enhancer) of an rice-derived alcohol dehydrogenase were constructed for these six genes. Additionally, a crRNA (serial number 15) recognizing the target 27-nucleotide sequence of the PDS gene was prepared, and a vector expressing this gene using the promoter of rice U6-2 snRNA was constructed. Expression cassettes for mtCas3-h2, Cas5, and Cas7, containing hygromycin resistance genes for screening transformable rice, were introduced into binary vectors with pPZP202 as the backbone. Expression cassettes for Cas6, Cas8, and Cas11, containing kanamycin resistance genes for screening transformable rice, were also introduced into binary vectors with pPZP202 as the backbone. These vectors were introduced into Agrobacterium EHA105 strain for transformation of rice callus. Rice callus co-infected with Agrobacterium using the respective binary vectors was cultured for 6 weeks in a medium containing hygromycin and G418, and transformed callus was screened. Using DNA extracted from the transformed callus as a template, the region containing the target sequence (328 bp) was amplified by PCR, and electrophoresis was performed using Multi-NA (Shimadzu Corporation).

[0084] The results showed that, when mtCas3-h2 with a mutated 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, without conversion of mtCas3-h2, or with the introduction of a mutant Cas3 containing a nickase domain with an amino acid substitution (H74A) and lack of nickase activity, such a short PCR product could not be detected. Figure 2 ).

[0085] In addition, similar to the aforementioned double mutants (mtCas3-h2 (S483A and T485A), single-point mutants (mtCas3-h2-1 (S483A) or mtCas3-h2-2 (T485A)) were used to introduce mutations into the rice phytoene desaturase PDS gene, and the deletion introduction efficiency generated by these mutant Cas3 proteins was studied.

[0086] The results confirmed that, although less efficient than the double mutant, it was still possible to introduce a short deletion into the target sequence using Cas3 with a single-point mutation in motif 3 of the SF2 helicase domain in rice cells. Figure 3 ).

[0087] [Example 2] Assay of genome cleavage activity in HEK293T human culture cells using mutant Cas3

[0088] The efficiency of mutant introduction of wild-type Cas3 and helicase domain mutants mtCas3-h1–h4 into human cells was investigated using HEK293T reporter cells with mCherry-P2A-EGFP. Two target sites were designed on GFP (GFP-crRNA-1 and 2). Expression plasmids were prepared, and in HEK293T reporter cells, plasmids encoding crRNA-1 (serial number 16), six Cas effectors (3, 5, 6, 7, 8, 11), and a puromycin resistance gene were introduced via liposome transfection using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were cultured in medium supplemented with 1 μg / ml puromycin at 37°C and 5% CO2 for 5 days. Total cells were then recovered, and FACS analysis was performed using AriaIIIu (BD) to determine the positivity rates of GFP and mCherry. Knockout efficiency was calculated from the number of GFP-negative cells.

[0089] As a result, GFP knockout was also observed in the case of using the mtCas3-h2 mutant, in addition to wild-type Cas3. Figure 4 Furthermore, for GFP-negative cases, the upstream mCherry gene was also investigated for positivity or negativity. The results showed that with wild-type Cas3, most cells (over 95%) exhibited loss of expression for both GFP and mCherry, indicating a large-scale deletion. On the other hand, with mtCas3-h2, 38.6% showed mCherry positivity, suggesting a higher prevalence of short mutations with only GFP deletion compared to wild-type Cas3.

[0090] To confirm the actual introduction of the short deletion mutation, the same experiment was performed using GFP-crRNA-2 (sequence number 17). GFP-negative cells were selected, 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 followed the pCR4Blunt-TOPOplasmid vector (Life Technologies) protocol.

[0091] The results showed that, when using mtCas3-h2, several mutations ranging from a few to tens of bases were observed that were not seen when using wild-type Cas. Figure 5 This indicates that the mutated mtCas3-h2 motif 3 of the SF2 helicase domain can also induce small-scale deletion mutations in human cultured cells.

[0092] Industry availability

[0093] The CRISPR-Cas3 system of the present invention enables genome editing on a smaller scale compared to existing CRISPR-Cas3 systems in eukaryotic cells, including plants and humans. Therefore, it can make a significant contribution not only to basic research but also to industrial applications requiring safety and precise control, such as medical applications and the development of agricultural and livestock products.

Claims

1. A method for manufacturing eukaryotic cells by introducing a mutation into target DNA, the method comprising introducing a CRISPR-Cas3 system into the eukaryotic cells, the CRISPR-Cas3 system comprising the following (A) to (C), (A) A Cas3 protein whose helicase activity is inhibited 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. (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. The method according to claim 1, wherein nuclear localization signals are added to the Cas3 protein and / or cascade proteins.

3. A kit for use in the method of claim 1, the kit comprising (A) a Cas3 protein whose helicase activity is inhibited 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.

4. The kit according to claim 3 further comprises (B) a cascade protein, a polynucleotide encoding the protein, or an expression vector containing the polynucleotide.

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

6. The kit according to claim 3 or 4, wherein nuclear localization signals are added to the Cas3 protein and / or cascade proteins.

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