WO2023054573A1 - 相同染色体の一方に特異的なｄｎａ欠失を有する細胞を製造する方法 - Google Patents

相同染色体の一方に特異的なｄｎａ欠失を有する細胞を製造する方法 Download PDF

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WO2023054573A1
WO2023054573A1 PCT/JP2022/036403 JP2022036403W WO2023054573A1 WO 2023054573 A1 WO2023054573 A1 WO 2023054573A1 JP 2022036403 W JP2022036403 W JP 2022036403W WO 2023054573 A1 WO2023054573 A1 WO 2023054573A1
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chromosome
dna
site
specific
strand
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慎一郎 中田
亜希子 富田
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University of Osaka NUC
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Priority to EP22876437.9A priority Critical patent/EP4410975A4/en
Priority to CN202280065806.1A priority patent/CN118043456A/zh
Priority to JP2023551836A priority patent/JP7672012B2/ja
Priority to US18/696,121 priority patent/US20250129390A1/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention relates to a method for producing cells with a DNA deletion specific to one of the homologous chromosomes using a site-specific nickase.
  • the present invention also relates to a method for evaluating the homologous recombination function of cells using the DNA deletion as an index.
  • TALENs the genome editing technology of the previous generation, is a fusion protein between a DNA-targeting TALE protein and a DNA-cleaving nuclease (mainly FokI), but similar to the CRISPR-Cas system, the target on the genome Generates a double-strand break in the DNA at the site.
  • nucleotide insertion/deletion occurs, and gene knockout can be performed by frameshifting.
  • gene knock-in can be achieved by homologous recombination between the genome and the donor DNA when donor DNA serving as a repair template is introduced from outside the cell. In this gene knock-in, not only DNA insertion but also substitution or deletion of one to several nucleotides can occur.
  • genome editing using programmable nucleases has major problems.
  • One of them is the occurrence of unintended gene mutation due to DNA double-strand breaks.
  • repair by non-homologous end joining is superior to repair by homologous recombination.
  • ) is likely to occur. Therefore, in the genome-edited cell population, in addition to cells that achieved the desired knock-in, cells that did not undergo any changes in the gene sequence, and cells that had unintended mutations due to non-homologous end joining, It is included to a lesser extent.
  • the single-cell level even if one of the autosomal alleles is knocked in as planned, the other may be mutated unintendedly.
  • programmable nucleases generate DNA double-strand breaks even in DNA sequences (off-targets) that are highly similar to the target sequence, resulting in genomic mutations.
  • Non-Patent Document 1 Non-Patent Document 1
  • the present inventors have further developed this method by using a combination of single nicks in the SNGD method, in which the target genome is nicked at one site and the donor plasmid containing the repair template is nicked at one site.
  • a target gene and donor plasma Patent Document 1.
  • Patent Document 2 a method of genome editing using homologous chromosomes originally present in cells as repair templates.
  • Patent Document 2 a method of genome editing using homologous chromosomes originally present in cells as repair templates.
  • this genome editing when heterozygous mutations or compound heterozygous mutations are present in chromosomes in cells, by inducing homologous recombination between homologous chromosomes, one homologous chromosome where the mutation does not exist can be used as a repair template to repair the mutation present in the other homologous chromosome to the normal sequence.
  • the normal sequence utilizing such homologous recombination Besides repairing to , it is also conceivable to specifically delete the DNA regions containing these mutations.
  • the present invention has been made in view of such circumstances, and an object of the present invention is to efficiently produce homologous chromosomes without double-strand breaks in DNA and without using exogenous donor DNA.
  • the object is to provide a method for producing a DNA deletion specific to one side.
  • Another object of the present invention is to provide a method for evaluating the homologous recombination function of cells using the DNA deletion as an indicator.
  • chromosome A When a heterozygous mutation exists in a gene in a cell, the gene mutation present on one of the homologous chromosomes (this is called “chromosome A”) is transferred to the other of the homologous chromosomes (this is called “chromosome B"). does not exist.
  • chromosome B the gene mutation present on one of the homologous chromosomes
  • the present inventors have introduced DNA single-strand breaks in various ways around the mutation site of the homologous chromosome, and as a result, at multiple locations in the DNA region near the mutation site of chromosome A, on both DNA strands or on the same DNA strand and, on chromosome B, nicking at one location corresponding to the nicking location on chromosome A, thereby effectively deleting a DNA region of more than 100 bp from chromosome A. (Examples of this mode of nick introduction are shown in FIGS. 2 and 4).
  • the present inventors have found that in the above aspect, it is possible to efficiently delete a DNA region exceeding 100 bp from chromosome A even when chromosome B is not nicked (this Examples of the introduction of nicks in embodiments are shown in Figures 3 and 5). Furthermore, the present inventors have found that it is possible to improve the efficiency of DNA region deletion by using cells in which the function of genes involved in homologous recombination (for example, each FANC gene) is suppressed. Found it.
  • the principle of the method of the present invention it is possible to broadly target bases that differ between homologous chromosomes and to delete DNA exceeding 100 bp in only one of the homologous chromosomes.
  • the suppression of the homologous recombination function in cells improves the efficiency of DNA deletion, it is also possible to evaluate the homologous recombination function in cells using the DNA deletion as an indicator.
  • the present invention is based on these findings, and more specifically provides the following.
  • a method for producing a cell having a DNA deletion specific to chromosome A in a homologous chromosome composed of chromosome A and chromosome B introducing a combination of site-specific nickases that cause single-strand breaks in the DNA region near the specific site into cells that have different bases between homologous chromosomes at a specific site on the homologous chromosome;
  • the combination of site-specific nickases causes single-strand breaks at multiple locations in the DNA region adjacent to the specific site, and the single-strand breaks occur in both DNA strands or the same DNA region in the vicinity of the specific site.
  • a single-strand break on the DNA strand, and in chromosome B a single-strand break at one location corresponding to the single-strand break on chromosome A, or a single strand break at the corresponding location A non-breaking method.
  • a kit for use in the method according to (1) In cells having different bases between homologous chromosomes at a specific site of homologous chromosomes, a combination of site-specific nickases that cause single-strand breaks in the DNA region near the specific site, In chromosome A, the combination of site-specific nickases causes single-strand breaks at multiple locations in the DNA region adjacent to the specific site, and the single-strand breaks occur in both DNA strands or the same DNA region in the vicinity of the specific site.
  • a single-strand break on the DNA strand, and in chromosome B a single-strand break at one location corresponding to the single-strand break on chromosome A, or a single-strand at the corresponding location Kit that does not cut.
  • a method for evaluating the homologous recombination function of a cell comprising: A combination of site-specific nickases that cause single-strand breaks in the DNA region near the specific site is introduced into cells that have different bases between the homologous chromosomes at the specific site of the homologous chromosomes composed of chromosome A and chromosome B. , detecting a chromosome A-specific DNA deletion resulting therefrom; In chromosome A, the combination of site-specific nickases causes single-strand breaks at multiple locations in the DNA region adjacent to the specific site, and the single-strand breaks occur in both DNA strands or the same DNA region in the vicinity of the specific site.
  • the present invention it is possible to efficiently generate a specific DNA deletion in one of the homologous chromosomes.
  • double-strand breakage of DNA is not performed, unintended mutations are unlikely to occur in the entire genome including chromosome B.
  • no exogenous donor DNA is used, the problem of random integration of donor DNA does not occur. Therefore, even when the present invention is applied to medical treatment such as gene therapy, the safety is high.
  • the homologous recombination function of cells can be evaluated using DNA deletion specific to one of the homologous chromosomes as an index.
  • the base of target site a on chromosome A and the base of target site b on chromosome B are different bases.
  • one nick is generated on each of both DNA strands
  • a nick is generated at one location corresponding to the location where the nick is generated on chromosome A. give rise to This results in deletion of a DNA fragment in chromosome A.
  • one nick is generated on each of both DNA strands, and on chromosome B, at one location corresponding to the location where the nick is generated on chromosome A
  • a nick is generated at a location corresponding to the location where the nick is generated on chromosome A. It is a figure which shows the example of the aspect (2nd aspect of this invention) which does not allow.
  • FIG. 10 is a diagram showing an example of an aspect (third aspect of the present invention) of generating nicks;
  • two nicks are generated on the same DNA strand in the region near the target site a on chromosome A, and on chromosome B, a nick is generated at a site corresponding to the site where the nick is generated on chromosome A. It is a figure which shows the example of the aspect (4th aspect of this invention) which does not let.
  • FIG. 10 is a diagram showing an example of an aspect (third aspect of the present invention) of generating nicks;
  • FIG. 2 is a diagram showing the positional relationship between PCR products obtained using PCR primer set L-AS15s or PCR primer set L-AS15s and nicks.
  • (a) is the nick of the PCR primer set L-AS15s and sgRNA TKex4_20s and sgRNA AS15
  • (b) is the nick of the PCR primer set L-AS15s and sgRNA TKex4_20s and sgRNA S8
  • c is the PCR primer set L-AS15l and sgRNA TKex4_20s and sgRNA AS15 nicks
  • (d) shows PCR primer set L-AS15l and sgRNA TKex4_20s and sgRNA S8 nicks.
  • Nicks of (a) and (c) are examples of the aspect (first aspect of the present invention) shown in FIG.
  • This is an example of the third aspect of This is an electropherogram when a cell clone with DNA deletion was identified using the PCR primer set L-AS15s.
  • (a) shows the results of nick introduction with sgRNA TKex4_20s and sgRNA AS15
  • (b) shows the results of nick introduction with sgRNA TKex4_20s and sgRNA S8.
  • 4 is a graph showing the ratio of cell clones in which a deletion of 600 bp or more was detected in only one homologous chromosome. Experiments were performed in triplicate with 94 clones for each experiment.
  • FIG. 3 is a diagram showing the positional relationship between the PCR product obtained using the PCR primer set L-TKex57 and the nick.
  • (a) shows nicks of PCR primer set L-TKex57 with sgRNA TKex5_m4s and sgRNA TKex7_105s
  • (b) shows nicks of PCR primer set L-TKex57 with sgRNA TKex5_m4s and sgRNA TKex7_mut121s.
  • the nick in (a) is an example of the aspect (third aspect of the present invention) shown in FIG. 4, and the nick in (b) is an example of the aspect (fourth aspect of the present invention) shown in FIG. be.
  • FIG. 4 is a graph showing the ratio of cell clones in which a deletion of 500 bp or more was detected in only one homologous chromosome. Experiments were performed in triplicate with 94 clones for each experiment.
  • FIG. 2 shows the positional relationship between the PCR product obtained using the PCR primer set L-S14 and the nick. The nicks of the PCR primer set L-S14 and sgRNA S14 and sgRNA TKex4_20s are shown. The nick in this figure is an example of the aspect (third aspect of the present invention) shown in FIG. This is an electropherogram when a cell clone with DNA deletion was identified using the PCR primer set L-S14.
  • FIG. 1 is a graph showing the ratio of cell clones in which a deletion of 1000 bp or more was detected in only one homologous chromosome. Experiments were performed in triplicate with 94 clones for each experiment. This is an electropherogram when a cell clone with DNA deletion was identified using the PCR primer set L-S14.
  • the top panel shows the results of nicking with sgRNA TKex4 — 20s and sgRNA S14 in cells in which FANCS protein was abolished by auxin induction, and the bottom panel shows the results in cells without auxin induction (control).
  • the top panel shows the results of nicking with sgRNA TKex4 — 20s and sgRNA S14 in cells in which FANCD1 protein was abolished by auxin induction, and the bottom panel shows the results in cells without auxin induction (control).
  • 1 is a graph showing the ratio of cell clones in which a deletion of 1000 bp or more was detected in only one homologous chromosome.
  • the present invention provides methods for producing cells with DNA deletions specific to chromosome A in homologous chromosomes composed of chromosome A and chromosome B.
  • a combination of site-specific nickases that cause single-strand breaks in the DNA region near the specific site is introduced into cells having different bases between homologous chromosomes at a specific site on the homologous chromosome.
  • the "bases that differ between homologous chromosomes" at a specific site of homologous chromosomes may be one base or multiple bases (base sequences). Moreover, it may be a mutation or a polymorphism. Mutations include, for example, substitutions, deletions, insertions, or combinations thereof, and polymorphisms include, for example, single nucleotide polymorphisms and microsatellite polymorphisms.
  • the base may exist in the gene coding region or in the gene noncoding region.
  • deletion of DNA containing the mutation or polymorphism can also be caused by deletion of the corresponding DNA in the other chromosome. It can also cause loss.
  • a typical use of the present invention from the viewpoint of medical utility is to delete DNA containing the mutation in human cells in order to treat or prevent human diseases caused by heterozygous mutation.
  • Preferred heterozygous mutations to be treated or prevented are, for example, gain-of-function mutations and dominant-negative mutations.
  • the "diseases caused by heterozygous mutations” include, for example, diseases that develop due to autosomal heterozygous mutations that take an autosomal dominant inheritance form (e.g., OAS1 abnormalities in congenital immunodeficiency diseases, ELANE mutations Severe congenital neutropenia, chronic mucocutaneous candidiasis, hereditary motor/sensory/autonomic neuropathy type IIC, post-synaptic slow channel type myasthenia congenita, Parkinson's disease type 8, tubular aggregates Myopathy, thyroid hormone unresponsiveness, hyper-IgE syndrome, Ehlers-Danlos syndrome, collagen disorders (osteogenesis imperfecta, dystrophic epidermolysis bullosa, type II collagen disorders, etc.), retinitis pigmentosa, achondroplasia, etc.
  • OAS1 abnormalities in congenital immunodeficiency diseases ELANE mutations Severe congenital neutropenia, chronic mucocutaneous candidia
  • autosomal dominant inherited triplet repeat diseases e.g., Huntington's disease, spinocerebellar ataxia, tonic muscular dystrophy, Friedreich's ataxia, oculopharyngeal muscular dystrophy, etc.
  • Huntington's disease spinocerebellar ataxia
  • tonic muscular dystrophy Friedreich's ataxia
  • oculopharyngeal muscular dystrophy etc.
  • the "site-specific nickase” used in the present invention is not limited as long as it can site-specifically cut DNA single-strands on the genome, but the CRISPR-Cas system having a nickase-type Cas protein as a constituent is preferable.
  • Cas proteins usually contain a domain responsible for cleavage of the target strand (RuvC domain) and a domain responsible for cleavage of the non-target strand (HNH domain), but nickase-type Cas proteins typically Mutations in any of the domains abolish its cleavage activity.
  • Such mutations include, in the case of spCas9 protein (Cas9 protein derived from S.
  • pyogenes for example, mutation of the 10th amino acid (aspartic acid) from the N-terminus to alanine (D10A: Mutation in RuvC domain) , mutation of the 840th amino acid (histidine) from the N-terminus to alanine (H840A: mutation within the HNH domain), mutation of the 863rd amino acid (asparagine) from the N-terminus to alanine (N863A: mutation within the HNH domain) , mutation of the 762nd amino acid (glutamic acid) from the N-terminus to alanine (E762A: mutation within the RuvCII domain), mutation of the 986th amino acid (aspartic acid) from the N-terminus to alanine (D986A: mutation within the RuvCIII domain ).
  • Cas9 proteins from various sources are known (eg, WO2014/131833), and their nickase forms can be used.
  • amino acid sequence and nucleotide sequence of the Cas9 protein are published databases, for example, are registered in GenBank (http://www.ncbi.nlm.nih.gov) (e.g., accession number: Q99ZW2.1, etc. ), which can be used in the present invention.
  • Cas proteins other than Cas9 such as Cpf1 (Cas12a), Cas12b, CasX (Cas12e), Cas14, etc. can also be used.
  • Mutations in the nickase-type Cpf1 protein include, for example, mutation of the 1226th amino acid (arginine) from the N-terminus to alanine (R1226A: mutation within the Nuc domain) in AsCpf1 (Cas12).
  • Cpf1 The amino acid sequence of Cpf1 is registered in a public database such as GenBank (http://www.ncbi.nlm.nih.gov) (eg, accession numbers: WP_021736722, WP_035635841, etc.).
  • a protein that constitutes the CRISPR-Cas system for example, one to which a nuclear localization signal has been added may be used.
  • the nickase-type Cas protein binds to the guide RNA to form a complex, is targeted by the target DNA sequence, and single-strand breaks the DNA.
  • the guide RNA includes crRNA and tracrRNA, but in the CRISPR-Cpf1 system no tracrRNA is required.
  • the guide RNA in the CRISPR-Cas9 system may be a single-molecule guide RNA containing crRNA and tracrRNA, or a bimolecular guide RNA consisting of a crRNA fragment and a tracrRNA fragment.
  • a crRNA contains a base sequence complementary to the target DNA sequence.
  • the target DNA sequence is usually a base sequence consisting of 12 to 50 bases, preferably 17 to 30 bases, more preferably 17 to 25 bases, and is selected from the region adjacent to the PAM (proto-spacer adjacent motif) sequence. preferably.
  • PAM proto-spacer adjacent motif
  • site-specific cleavage of DNA occurs at locations dictated by both the base-pairing complementarity between the crRNA and the target DNA sequence and the PAMs present adjacent thereto.
  • crRNA further contains a nucleotide sequence on the 3' side that can interact (hybridize) with a tracrRNA fragment.
  • tracrRNA contains a nucleotide sequence on the 5' side that can interact (hybridize) with a portion of the nucleotide sequence of crRNA.
  • CrRNA/tracrRNA single molecule or two molecules
  • PAM differs depending on the type and origin of the Cas protein. Typical PAM sequences are, for example, S . In the Cas9 protein (type II) from S. pyogenes, it is “5'-NGG"; In the Cas9 protein (I-A1 type) derived from solfataricus, it is “5'-CCN”. In the Cas9 protein (I-A2 type) derived from solfataricus, it is "5'-TCN", and H. In the Cas9 protein from walsbyl (type IB), it is "5'-TTC"; In the Cas9 protein (IE type) derived from E. coli, it is "5'-AWG".
  • meningitidis it is "5'-NNNNGATT.”
  • Cas9 protein from denticola it is "5'-NAAAC”.
  • Cpf1 it is typically '5'-TTN' or '5'-TTTN'.
  • PAM recognition by modifying the protein (for example, introducing mutation) (Benjamin, P. et al., Nature 523, 481-485 (2015), Hirano, S. et al., Molecular Cell 61, 886-894 (2016), Walton, RT et al., Science 368, 290-296 (2020)).
  • site-specific nickases other than the CRISPR-Cas system can also be used.
  • site-specific nickases include, for example, artificial nucleases fused with enzymes having nickase activity.
  • Artificial nucleases that can be used include, for example, TALE (transcription activator-like effector), ZF (zinc finger), and PPR (pentatriceptide repeat).
  • Enzymes that can exhibit nickase activity by fusion with these artificial nucleases include, for example, TevI (Nat Commun. 2013; 4: 1762. doi: 10.1038/ncomms2782).
  • DNA-binding domains constructed by linking modules (peptides) that recognize specific bases (or specific base sequences), and are fused to the DNA-binding domains.
  • a nickase causes a single-strand break in the DNA.
  • a suitable spacer peptide may be introduced between the DNA-binding domain and the nickase in the artificial nuclease.
  • single-strand breaks are made at multiple locations in the DNA region near the above-mentioned specific site (bases that differ between homologous chromosomes).
  • both strands of DNA in the neighboring DNA region are single-strand cut, and in another embodiment, single-strand cleavage is performed at multiple sites on the same DNA strand in the neighboring DNA region.
  • corresponding site one site corresponding to the site where the single-strand break occurs in chromosome A
  • chromosome A the same DNA strand in the above neighboring DNA region is single-strand cut at multiple locations, and in chromosome B, single-strand break is performed at one corresponding location (Fig. 4).
  • the "neighboring DNA region” is usually a region within 100,000 bases (for example, within 10,000 bases, within 5,000 bases, within 2,000 bases, within 1,000 bases, and within 600 bases) from a specific site.
  • the distance between nicks to be introduced is usually 100 bases or more (eg, 200 bases or more, 300 bases or more, 500 bases or more, 700 bases or more, 1000 bases or more, 1500 bases or more).
  • One of the nicks introduced into the chromosome is preferably immediately adjacent to the specific site.
  • the term “closest” is usually within 100 base positions, more preferably within 50 base positions (for example, within 40 bases, within 30 bases, within 20 bases, within 10 bases).
  • one single-strand break is performed on both DNA strands across the above-mentioned specific site (2-1, 2-2, 2-7, and 2 in FIG. 2).
  • single-strand cleavage is performed one by one on the same DNA strand across the above-mentioned specific site (4-3 to 4-6 in FIG. 4), one in both DNA strands on one side of the above-mentioned specific site A mode of single-strand breakage at each point (2-3 to 2-6 in FIG. 2), and a mode of single-strand breakage at two points on the same DNA strand on one side of the specific site (4-1 in FIG. 4, 4-2, 4-7, 4-8).
  • DNA containing a plurality of specific sites can be deleted in chromosome A by introducing nicks so as to sandwich a plurality of specific sites.
  • the number of single-strand breakage sites may be three or more.
  • chromosome A and chromosome B when the corresponding sites are simultaneously single-strand cut, the site-specific nickase that binds to the target DNA sequence of chromosome A is linked to chromosome B may be designed so that it also binds to the corresponding DNA sequence of .
  • the target DNA sequence on chromosome A and the corresponding DNA sequence on chromosome B are typically identical DNA sequences.
  • a site-specific nickase that binds to the target DNA sequence of chromosome A is added to the corresponding DNA of chromosome B. It should be designed so that it does not bind to the array.
  • the target DNA sequence on chromosome A and the corresponding DNA sequence on chromosome B are different DNA sequences.
  • the target DNA sequence of a site-specific nickase is set so as to include a base at a specific site on chromosome A (a base that differs between homologous chromosomes), the target DNA sequence differs from the corresponding DNA sequence on chromosome B. DNA sequence.
  • the site-specific nickase is a CRISPR-Cas system
  • the guide RNA can be designed to bind specifically to the target DNA sequence on chromosome A.
  • the DNA binding domain may be designed to have binding specificity to the target DNA sequence of chromosome A.
  • the site to be single-strand cut is usually within 100 base positions, more preferably within 50 base positions from the above-mentioned specific site (bases that differ between homologous chromosomes) ( For example, within 40 bases, within 30 bases, within 20 bases, within 10 bases).
  • the distance of single-strand breaks is Too close can lead to double-strand breaks. Therefore, the distance between single-strand breaks on different DNA strands is usually 100 bases or more, preferably 200 bases or more.
  • the above combination of site-specific nickases is introduced into cells.
  • the "site-specific nickase" introduced into the cell is, in the case of the CRISPR-Cas system, for example, even in the form of a combination of guide RNA and Cas protein, and messenger RNA translated into guide RNA and Cas protein. or a combination of vectors expressing them.
  • the guide RNA may be modified (such as chemical modification) to suppress degradation.
  • an artificial nuclease fused with an enzyme having nickase activity for example, in the form of a protein, in the form of messenger RNA translated into the protein, in the form of a vector expressing the protein may be
  • operably linked means that the DNA is expressably linked to the regulatory element.
  • regulatory elements include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements such as transcription termination signals, such as polyadenylation signals and polyU sequences.
  • Regulatory elements may be those that direct DNA expression only in specific cells, tissues, or organs, depending on the purpose, for example, those that direct constitutive expression of DNA in a variety of host cells. may be In addition, it may be one that directs the expression of DNA only at a specific time, or one that directs the expression of an artificially inducible DNA.
  • promoters examples include pol III promoters (e.g., U6 and H1 promoters), pol II promoters (e.g., retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, ⁇ -actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1 ⁇ promoter), pol I promoter, or combinations thereof.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter cytomegalovirus
  • dihydrofolate reductase promoter promoter
  • ⁇ -actin promoter phosphoglycerol kinase (PGK) promoter
  • PGK phosphoglycerol kinase
  • EF1 ⁇ promoter EF1 ⁇ promoter
  • Cells to which the method of the present invention is applied are not particularly limited as long as they have homologous chromosomes, and various eukaryotic cells can be targeted.
  • Eukaryotic cells include, for example, animal cells, plant cells, algal cells, and fungal cells.
  • Animal cells include, for example, mammalian cells, as well as fish, bird, reptile, amphibian, and insect cells.
  • Animal cells include, for example, cells that make up an individual animal, cells that make up an organ or tissue extracted from an animal, and cultured cells derived from animal tissue. Specifically, for example, embryonic cells of embryos at each stage (e.g., 1-cell stage embryo, 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, 16-cell stage embryo, morula stage embryo, etc.); induction Stem cells such as pluripotent stem (iPS) cells, embryonic stem (ES) cells, hematopoietic stem cells; fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, brain cells, kidney cells, etc.
  • iPS pluripotent stem
  • ES embryonic stem
  • fibroblasts hematopoietic stem cells
  • neurons e.g., muscle cells, bone cells, hepatocytes, pancreatic cells, brain cells, kidney cells, etc.
  • a fertilized oocyte ie, a fertilized egg
  • the fertilized egg is a pronuclear stage embryo.
  • Cryopreserved oocytes can be used after thawing before fertilization.
  • the term "mammal” is a concept that includes both human and non-human mammals.
  • non-human mammals include artiodactyls such as bovines, wild boars, pigs, sheep and goats, peri-hoofed animals such as horses, rodents such as mice, rats, guinea pigs, hamsters and squirrels, and lagomorphs such as rabbits. , dogs, cats, and meats such as ferrets.
  • the non-human mammals described above may be domestic animals or companion animals (pet animals), or may be wild animals.
  • Plant cells include, for example, cells of grains, oil crops, fodder crops, fruits, and vegetables.
  • the term “plant cell” includes, for example, cells constituting an individual plant, cells constituting an organ or tissue isolated from a plant, cultured cells derived from plant tissue, and the like.
  • Plant organs and tissues include, for example, leaves, stems, shoot apexes (growing points), roots, tubers, tuberous roots, seeds, and callus. Examples of plants include rice, corn, bananas, peanuts, sunflowers, tomatoes, rape, tobacco, wheat, barley, potatoes, soybeans, cotton, carnations, and the like.
  • the FANC gene includes family genes such as the FANCA gene, the FANCB gene, the FANCC gene, the FANCD1 (BRCA2) gene, the FANCD2 gene, and the FANCS (BRCA1) gene.
  • the base sequence of a typical human-derived FANCA gene and the amino acid sequence of the protein encoded by the gene are found in the database (Accession numbers: NM — 000135, NP — 000126).
  • the amino acid sequence of the encoded protein is in the database (Accession number: NM_001018113, NP_001018123), and the base sequence of a typical human-derived FANCC gene and the amino acid sequence of the protein encoded by the gene are in the database (Accession number: NM_000136 , NP_000127), the base sequence of a typical human-derived FANCD1 gene and the amino acid sequence of the protein encoded by the gene are listed in the database (accession number: NM_000059, NP_000050), the base of a typical human-derived FANCD2 gene
  • the sequence and the amino acid sequence of the protein encoded by the gene are available in the database (accession numbers: NM_001018115, NP_001018125), and the base sequence of a typical human-derived FANCS gene and the amino acid sequence of the protein encoded by the gene are available in the database ( Accession numbers: NM_007294, NP_009225), respectively.
  • the mechanism of "suppression of the function of the FANC gene" in the present invention may be suppression of the activity of the translation product (protein) of the gene or suppression of the expression of the gene. Suppression of the activity of the translation product (protein) of a gene can be performed, for example, by introducing mutation (deletion, substitution, insertion, etc.) into the gene or treatment with an inhibitor.
  • Mutations can be introduced into the FANC gene, for example, using a genome editing system.
  • genome editing systems include CRISPR-Cas targeting the FANC gene (eg, CRISPR-Cas9), TALENs, site-specific artificial nucleases such as ZFNs, and the like.
  • a gene is cleaved at a target site by the action of the genome editing system, and mutation (deletion, insertion, etc. of a base) is introduced into the gene via the DNA repair mechanism of the cell.
  • mutation deletion, insertion, etc. of a base
  • the deletion may be a deletion of the entire gene or a partial deletion.
  • FANC inhibitors include molecules that inhibit FANC's single-strand annealing activity and strand exchange activity (see, for example, Benitez, A. et al., Mol Cell 16; 71(4), 621-628 (2016)).
  • “Suppression of the function of the FANC gene” can also be performed by suppressing the expression of the gene.
  • Suppression of FANC gene expression may be suppression of gene translation or transcription.
  • Suppression of translation of the FANC gene can be performed, for example, using double-stranded RNA (dsRNA) that binds to the FANC gene transcript.
  • double-stranded RNA include siRNA, shRNA (short haipin RNA), miRNA and the like.
  • Suppression of FANC gene transcription can be performed, for example, by introducing a mutation into the expression control region of the FANC gene.
  • the above-described genome editing system targeting the expression control region can be used to introduce the mutation.
  • Whether or not the expression of the FANC gene is suppressed can be determined at the translation level, for example, by an immunoassay using an antibody that binds to the translation product, and at the transcription level, for example, RT-PCR or Northern Each can be evaluated by a blotting method.
  • These molecules for suppressing the function of the FANC gene may be introduced into cells themselves, and when the molecules are nucleic acids, DNA encoding the molecules may be used to express them in cells. (typically, a vector into which the DNA has been inserted) may be introduced into cells.
  • Introduction of site-specific nickases or molecules that suppress the function of the FANC gene into cells can be performed, for example, by electroporation, microinjection, DEAE-dextran method, lipofection method, nanoparticle-mediated transfection method, virus-mediated nucleic acid delivery. It can be carried out by a known method such as the method.
  • high efficiency is preferably 3% or higher, more preferably 5% or higher, and particularly preferably 10% or higher (eg, 20% or higher, 30% or higher, 35% or higher).
  • kits for use in the method of the present invention comprising the combination of the site-specific nickases.
  • kits may further comprise one or more additional reagents such as dilution buffers, reconstitution solutions, wash buffers, nucleic acid transfer reagents, protein transfer reagents, control reagents. (eg, control guide RNA).
  • additional reagents such as dilution buffers, reconstitution solutions, wash buffers, nucleic acid transfer reagents, protein transfer reagents, control reagents. (eg, control guide RNA).
  • the kit may contain instructions for carrying out the methods of the invention.
  • the present invention also provides methods for assessing homologous recombination function of cells.
  • a cell having different bases between homologous chromosomes at a specific site of homologous chromosomes composed of chromosome A and chromosome B is added to DNA near the specific site. It involves introducing a combination of site-specific nickases that produce single-strand breaks at the region and detecting the resulting chromosome A-specific DNA deletion.
  • the site-specific nickase combination causes single-strand breaks at multiple locations in the DNA region near the specific site on chromosome A, and the single-strand breaks occur in both DNA strands or the same DNA region in the vicinity of the specific site.
  • the combination of site-specific nickases causes single-strand breaks at multiple locations in the DNA region near the specific site on chromosome A, and the single-strand breaks occur at one point on the same DNA strand in the neighboring DNA region.
  • a preferred embodiment is one in which single-strand breakage is not caused at one site corresponding to the site where single-strand breakage occurs in chromosome A (fourth embodiment, FIG. 5).
  • single-strand breaks are made at multiple locations on the same DNA strand of chromosome A, and in chromosome B can also be carried out in such a manner that single-strand breaks occur at a plurality of sites corresponding to the single-strand break sites on chromosome A.
  • the single-strand break does not necessarily have to be introduced into the DNA region near the base that differs between the homologous chromosomes, and can be introduced into any DNA region.
  • the frequency of occurrence of DNA deletion specific to chromosome A when the frequency of occurrence of DNA deletion specific to chromosome A is higher than in the control, it is evaluated that the homologous recombination function of the cell is suppressed.
  • “higher than the control” means a frequency that is preferably 2 times or more, more preferably 5 times or more, particularly preferably 10 times or more (for example, 20 times or more) than the control.
  • the frequency is preferably 4% or more, more preferably 10% or more, and particularly preferably 20% or more (e.g., 40% or more) (Example (2) (b), FIG. 9B checking).
  • the frequency in cells in which the homologous recombination function is not suppressed can be used.
  • Suppression of the function of homologous recombination can occur, for example, by suppressing the function of genes involved in homologous recombination (eg, gene mutation). Therefore, it is suspected that the function of genes involved in homologous recombination is suppressed in cells evaluated to have suppressed homologous recombination function according to the present invention.
  • Genes involved in homologous recombination include, for example, the FANC gene described above.
  • the method of the present invention can be applied to various cancer cells (e.g., breast cancer, ovarian cancer, prostate cancer, pancreatic cancer). cells such as cells) can be targeted.
  • cancer cells e.g., breast cancer, ovarian cancer, prostate cancer, pancreatic cancer. cells such as cells
  • FANCS/FANCD1 (BRCA1/BRCA2) is a gene responsible for hereditary juvenile breast cancer and ovarian cancer. Abnormalities in function and decreased expression of FANCS/FANCD1 are often observed. Functional abnormalities and decreased expression of FANCS/FANCD1 are caused not only by gene mutations but also by suppression of expression by promoter methylation, etc., so genetic testing alone is insufficient.
  • the present invention can evaluate whether or not the homologous recombination function is suppressed regardless of the cause of the suppression of the homologous recombination function, and is superior to conventional genetic tests in this respect.
  • an evaluation system using a reporter gene can be used to evaluate suppression of homologous recombination function, but it is difficult to implement such an evaluation system with patient-derived cancer cells.
  • the method of the present invention is also excellent in that patient-derived cancer cells can be used as they are without requiring a reporter gene.
  • MyChoice which combines measurement of the frequency of genomic alterations characteristic of homologous recombination deletion and mutation testing of the FANC gene (BRCA gene), has been put into practical use. Based on this, the use of drugs (PARP inhibitors) is judged. Therefore, the evaluation of homologous recombination function in cancer cells by the method of the present invention can also be used to evaluate the efficacy of anticancer agents in patients.
  • kits for use in the method of the present invention comprising the combination of the site-specific nickases.
  • kits may further comprise one or more additional reagents such as dilution buffers, reconstitution solutions, wash buffers, nucleic acid transfer reagents, protein transfer reagents, control reagents. (eg, control guide RNA).
  • additional reagents such as dilution buffers, reconstitution solutions, wash buffers, nucleic acid transfer reagents, protein transfer reagents, control reagents. (eg, control guide RNA).
  • the kit may contain instructions for carrying out the methods of the invention.
  • TK6261 cells TK6 cell-derived lymphoblastoid cells. Allele A (chromosome A) with a loss-of-function frameshift due to a 1-bp insertion on exon 4 of the thymidine kinase 1 gene (chromosome 17) and a loss-of-function frameshift due to an 8-bp deletion on exon 5 and exon 7 that does not affect function Allele B (chromosome B) with a 1 bp insertion above is included.
  • FANCA Mutant TK6261 Cells TK6261 cells are cells in which both alleles of FANCA gene exon 27 have been mutated to lose FANCA function.
  • TSCER2 TIR cells TK6-derived cells in which the thymidine kinase 1 (TK1) gene is a compound heterozygote. The TK1 exon4 mutation is shared with TK6261 cells. Addition of auxin causes TIR1 to be expressed, and the AID-tagged protein is degraded.
  • FANCS (BRCA1) protein expression can be abolished by addition of auxin (AUX).
  • AUX auxin
  • FANCD1 protein-depleted TSCER2 (TIR) cells TSCER2 (TIR)-FANCD1 AID/AID knocks in the auxin-induced degron (AID) tag at the C-terminus of both alleles of the FANCD1 (BRCA2) gene and expresses FANCD1-AID. It is a cell modified as follows. FANCD1 (BRCA2) protein expression can be abolished by addition of auxin (AUX).
  • TKex4_20s CGTCTCGGAGCAGGCAGGCG[GGG]/SEQ ID NO: 1
  • S8 AGCTTCCCATCTATACCTCC [TGG]/SEQ ID NO: 2
  • AS15 GAGGTAGGTTGATCTTGTGT[GGG]/SEQ ID NO: 3
  • S14 TCCCACCGAAGGCCACACGC[CGG]/SEQ ID NO: 4
  • TKex5_mut4s CTCGCAGAACTCCAGGGAAC[TGG]/SEQ ID NO: 5
  • TKex7_105s CCCCTGGCTTTCCTGGCACT[GGG]/SEQ ID NO: 6
  • TKex7_mut121s TGGCAGCCACGGCTTCCCCC[TGG]/SEQ ID NO: 7
  • TKex4_20s is an sgRNA that targets only exon4 on allele A
  • S8 is an sgRNA that targets intron 4 of
  • the cells were cultured in 10% horse serum + 200 ⁇ g/ml Sodium Pyruvate/RPMI1640 medium at 37°C, 5% CO 2 overnight, followed by 5% horse serum + 200 ⁇ g/ml Sodium Pyruvate/RPMI1640 medium ( basal medium) at 37° C., 5% CO 2 for 1 week.
  • the cultured cells were sorted into a 96-well plate in which 200 ⁇ L of basal medium was dispensed per well so that there would be 1 cell/well. After culturing for about one week, 94 clones derived from the proliferated single cells were collected, and genomic DNA was extracted using a simple DNA extraction kit Version 2 (Kaneka). Separately from this, genomic DNA was extracted from cells that had been continued to be cultured without sorting using cultured cell genomic DNA extraction kit VI (ANIMOS).
  • ANIMOS cultured cell genomic DNA extraction kit VI
  • PCR was performed using KOD FX Neo (TOYOBO) using DNA extracted from a single cell-derived clone as a template.
  • the target sequences of sgRNA TKex4_20s, sgRNA S8 and sgRNA AS15 were included in the PCR product (total length of about 2.1 kbp)
  • the target sequences of the primer set L-AS15s, sgRNA TKex4_20s, sgRNA S8 and sgRNA AS15 were included in the PCR product.
  • PCR designed to include the target sequences of the primer set L-AS15l, sgRNA TKex4_20s and sgRNA S14 in the PCR product (total length of about 5.4 kbp)
  • Primer set L-S14 and primer set L-TKex57 were used in PCR designed so that the target sequences of sgRNA TKex5_mut4s, sgRNA TKex7_105s and sgRNA TKex7_mut121s were included in the PCR product (total length of about 1.9 kbp).
  • the sequences of the primers used for PCR are as follows.
  • L-AS15s (Forward): GCCTTTCCCCATAGGTGCTAACT/SEQ ID NO: 8 L-AS15s (Reverse): AACAAAACACACTCTGGAAGATGGAACC/SEQ ID NO: 9 L-AS151 (Forward): CACAAGGCACAGGACACACTGTTAG/SEQ ID NO: 10 L-AS151 (Reverse): AACAAAACACACTCTGGAAGATGGAACC/SEQ ID NO: 11 L-S14 (Forward): GAGTACTCGGGTTCGTGAACTT/SEQ ID NO: 12 L-S14 (Reverse): GCAGCTTCCCCATCTATACCTCC/SEQ ID NO: 13 L-TKex57 (Forward): AAGCCCCTCACGTCTCAATAACC/SEQ ID NO: 14 L-TKex57 (Reverse): GCTTTAAGCAGACCAGTGGGTA/SEQ ID NO: 15 The PCR product was subjected to electrophoresis in 0.9% TAE gel to visualize the base length of the
  • a part of the PCR product showing a deletion of several 100 bp or more was excised from the gel and purified, and DNA sequence analysis of the exon4 region was performed.
  • PCR was performed using KOD plus neo (TOYOBO) using DNA extracted from a single-cell-derived clone as a template.
  • the primer set S-AS15 was used for each.
  • S-EX4 (Forward): TTTTCTGGACGAGGGGCCTTTC/SEQ ID NO: 16 S-EX4 (Reverse): CTTCCAAGTCAGCGAGGGAAAA/SEQ ID NO: 17 S-AS15 (Forward): CCTCATGGTTCCTTTTGCTTG/SEQ ID NO: 18 S-AS15 (Reverse): GGTGGGAAATCGCTTGAAT/SEQ ID NO: 19 Subsequently, the DNA sequence of the PCR product was analyzed by the Sanger sequencing method. The sequences of the sequencing primers are as follows.
  • Exon 4 region TGAACACTGAGCCTGCTT/SEQ ID NO: 20
  • S8/AS15 region CGTTTATTTTCTTTGTTG/SEQ ID NO: 21
  • TK6261 cells a combination of sgRNA TKex5_mut4s and sgRNA TKex7_mut121s and Cas9D10A were used to create two nicks in tandem on the sense strand of allele A, and allele B was not nicked, resulting in a deletion of 500 bp or more.
  • TSCER2(TIR) cells capable of auxin-inducibly depleting FANCS protein TSCER2(TIR)-FANCS AID/AID
  • TSCER2(TIR) cells capable of auxin-inducibly depleting FANCD1 protein TSCER2(TIR)-FANCD1 AID/AID
  • DNA containing a specific site is deleted from one of the homologous chromosomes by utilizing site-specific nickase-induced single-strand breaks. be able to.
  • the present invention which does not use double-strand breaks or exogenous donor DNA, can greatly contribute to gene therapy, particularly for diseases caused by heterozygous mutations, due to its high safety.
  • the homologous recombination function of cells can be evaluated using the DNA deletion specific to one of the homologous chromosomes as an indicator, which can greatly contribute to the diagnosis of diseases such as cancer. .

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