Method for producing cell having genome with genetic modification

Abstract

The present disclosure provides a method for producing a cell that has a genetic modification. The present disclosure provides a method for producing, from a cell that has a genome having a target region, a cell that has a genome from which the target region has been deleted. The present disclosure includes, for example, introducing a selectable marker gene into a flanking region of the target region, and inducing deletion of the selectable marker gene and the target region.

Description

Method for producing a cell having a genome with genetic modification

[0001] The present disclosure relates to a method for producing a cell having a genome with genetic modification. In one aspect, the present disclosure comprises introducing a selectable marker gene into flanking regions of a target region, and inducing deletion of at least the selectable marker gene.

[0002] Genome editing technology is used in a wide range of applications in the fields of biology and medicine, such as production of disease model cells, construction of cells for gene therapy, and development of improved organisms for agriculture. In particular, technology for deleting a specific target region present in the genome of a cell is important for analysis of gene function, evaluation of pathogenicity of a specific gene, and inactivation of a target gene for therapeutic applications, etc. (Cong et al., Science, 2013; Mali et al., Science, 2013).

[0003] Conventionally, as a method for deleting a specific region of a genome, methods using a specific site-specific recombinase such as the Cre-loxP system or the FLP-FRT system are known (Sauer, B., Methods in Enzymology, 1993).

[0004] On the other hand, in recent years, new genome editing technologies including CRISPR / Cas9 have emerged, making it possible to edit a target genomic sequence with a high degree of freedom (Jinek et al., Science, 2012).

[0005] The present disclosure provides a method for producing a cell having a genome with genetic modification. The present disclosure comprises introducing a selectable marker gene into flanking regions of a target region, and inducing deletion of at least the selectable marker gene.

[0006] The present disclosure may provide the following invention: (1) A method for producing cells having a genome having a deletion of a target region from cells having a genome having a target region, comprising: (A) inserting a selection marker gene into (i) the left flanking region or (ii) the right flanking region of the target region on the genome of the cell; (B) selecting cells having the insertion of the selection marker gene into the flanking region of the target region; and (C) deleting a contiguous region on the genome containing the selection marker gene and the target region, thereby obtaining cells having a genome having a deletion of a target region without leaving an insertion of the selection marker gene on the genome. (2) The method according to (1), wherein the foreign sequence includes, in the case of (i) above, a left homologous sequence homologous to the left-side flanking sequence of the left flanking region, and in the case of (ii) above, a right homologous sequence homologous to the right-side flanking sequence of the right flanking region, and step (C) is achieved by inducing a cleavage between the right-side flanking sequence and the left-side homologous sequence in the case of (i) above, and by inducing a cleavage between the left-side flanking sequence and the right-side homologous sequence in the case of (ii) above. (3) The method according to (1) or (2), wherein step (A) includes introducing a sequence-specific cleavage into the left flanking region or (ii) the right flanking region, and inducing homologous recombination by applying donor DNA having a left homology arm and a right homology arm homologously recombinable to the left-side and right-side regions of the cleavage, respectively, and containing a selection marker gene between the left homology arm and the right homology arm to the flanking region. (4) The method according to (3) above, wherein the donor DNA is (i) applied to the left flanking region to induce homologous recombination, and the donor DNA contains a unique sequence that is targeted by a sequence-specific cleavage molecule to the left of the selection marker gene. (5) The method according to any one of (1) to (4) above, wherein the selection marker gene includes (i) a positive selection marker gene and a negative selection marker gene; or (ii) a marker gene that can be used for both positive and negative selection.(6) The method according to any one of (1) to (5) above, wherein step (C) includes introducing sequence-specific cuts at both ends of the series of regions to induce deletion of the selection marker gene and the target region, and selecting cells in which the expression of the negative selection marker has disappeared. (7) A method for producing a genetically modified cell, comprising: (A) inserting an exogenous sequence into one flanking region of a target region on the genome of a cell in the presence of donor DNA containing an exogenous homologous sequence and an exogenous sequence containing a selection marker gene, wherein the exogenous homologous sequence is a sequence homologous to the sequence of the other flanking region of the target region (endogenous homologous sequence); (B) selecting cells having the insertion of the exogenous homologous sequence and the selection marker gene into one flanking region of the target region; and (C) deleting the sequence between the exogenous homologous sequence and the endogenous homologous sequence, thereby obtaining a genetically modified cell without leaving an insertion of the selection marker gene on the genome. (8) The method according to (7) above, wherein the donor DNA contains a desired nucleic acid sequence, an exogenous homologous sequence, and a selection marker gene in that order, or contains a selection marker gene, an exogenous homologous sequence, and a desired nucleic acid sequence in that order, and recombination is induced between the exogenous homologous sequence and the endogenous homologous sequence, so that the region containing the selection marker gene is deleted, and the desired nucleic acid sequence remains on the genome. (9) A method for producing genetically modified cells, comprising: (A) inserting an exogenous sequence into a cleavage site on the genome of a cell in the presence of donor DNA containing a desired nucleic acid sequence and an exogenous homologous sequence, wherein the exogenous homologous sequence is homologous to a sequence (endogenous homologous sequence) that exists on the opposite side of the genome from the desired nucleic acid sequence via an arbitrary genomic region {that is, arranged in the order of desired nucleic acid sequence, exogenous homologous sequence, arbitrary genomic region, and endogenous homologous sequence}; (B) selecting cells having the insertion of the exogenous sequence into the cleavage site; and (C) deleting a sequence between the exogenous homologous sequence and the endogenous homologous sequence, thereby obtaining genetically modified cells {preferably a method that does not insert a selection marker gene into the genome}.(10) The method according to (9) above, wherein the donor DNA contains a desired nucleic acid sequence and an exogenous homologous sequence in that order, and the desired nucleic acid sequence remains on the genome by inducing recombination between the exogenous homologous sequence and the endogenous homologous sequence. (11) A method for producing a genetically modified cell, comprising: (A) inserting an exogenous sequence into a cleavage site on the genome of a cell in the presence of donor DNA containing an exogenous sequence containing an exogenous sequence, wherein the exogenous homologous sequence is a sequence homologous to a sequence (endogenous homologous sequence) present through a target region; (B) selecting a cell having the insertion of the exogenous sequence into the target region; and (C) deleting a sequence between the exogenous homologous sequence and the endogenous homologous sequence, thereby obtaining a cell with a deleted target region {preferably a method that does not insert a selection marker gene into the genome}.

[0007] Figure 1A shows an example of process (A). Figure 1B shows an example of process (B). Figure 1C shows an example of process (C). Figure 1D shows the deletion of the target gene from the genome of the obtained cells. This is an example of process (C) in Modification 2 of this disclosure. This shows the success and efficiency of editing by the method of this disclosure. This shows the success and efficiency of editing by Modification 2 of this disclosure. This shows the success and efficiency of BMP7 gene deletion by Modification 2 of this disclosure. This shows the success and efficiency of B2M gene deletion by Modification 2 of this disclosure. This shows an example of the editing process in Modification 3 of this disclosure. This shows the results of investigating the lengths of homologous sequences that can be used in Modifications 2 and 3 of this disclosure.

[0008] [Definitions] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotide polymers in which nucleotides are linked by phosphodiester bonds. "Polynucleotides" and "nucleic acids" may be DNA, RNA, or a combination of DNA and RNA. Furthermore, "polynucleotides" and "nucleic acids" may be polymers of natural nucleotides, polymers of natural nucleotides and non-natural nucleotides (analogs of natural nucleotides, nucleotides in which at least one of the base, sugar, and phosphate parts is modified (e.g., phosphorothioate skeletons), etc.), or polymers of non-natural nucleotides.

[0009] The base sequences of "polynucleotides" or "nucleic acids" are generally written using commonly accepted single-letter codes unless otherwise specified. Unless otherwise specified, base sequences are written from the 5' end to the 3' end. The nucleotide residues that make up "polynucleotides" or "nucleic acids" may simply be written as adenine, thymine, cytosine, guanine, or uracil, or by their single-letter codes.

[0010] The term "gene" refers to a polynucleotide containing at least one open reading frame that codes for a specific protein. Genes can contain both exons and introns.

[0011] The terms “polypeptide,” “peptide,” and “protein” are interchangeable and refer to polymers of amino acids linked by amide bonds. A “polypeptide,” “peptide,” or “protein” may be a polymer of natural amino acids, a polymer of natural amino acids and non-natural amino acids (chemical analogues, modified derivatives, etc. of natural amino acids), or a polymer of non-natural amino acids. Unless otherwise specified, amino acid sequences are written from the N-terminus to the C-terminus. The term “cell” refers to the cells of an organism and includes the cells of animals with an immune system (i.e., vertebrates). Cells may be, for example, mammalian cells, such as primate cells such as humans, rodents such as mice and rats, livestock such as cattle, horses, sheep, llamas, camels, goats, and pigs, pets such as dogs and cats, and birds such as chickens.

[0012] The terms "genome modification" and "genome editing" are used interchangeably and refer to inducing mutations at a desired location (target region) on the genome. Genome modification may include the use of sequence-specific nucleic acid cleavage molecules designed to cleave target region DNA. In a preferred embodiment, genome modification may include the use of nucleases engineered to cleave target region DNA. In a preferred embodiment, genome modification may include the use of nucleases engineered to cleave target sequences having a specific base sequence within the target region (e.g., TALEN or ZFN). In a preferred embodiment, genome modification may include the use of restriction enzymes having only one cleavage site in the genome, such as meganucleases (e.g., 16-base sequence-specific restriction enzymes (theoretically 4)) to cleave target sequences having a specific base sequence within the target region. １６ Restriction enzymes with 17-base sequence specificity (theoretically 4) (present at a ratio of one per base) １７ (present at a ratio of one per base), and restriction enzymes with 18-base sequence specificity (theoretically 4 １８In some cases, sequence-specific endonucleases, such as those present at a ratio of one per base, can be used. Typically, the use of site-specific nucleases induces double-strand breaks (DSBs) in the DNA of the target region, after which the genome is repaired by endogenous cellular processes such as homologous directed repair (HDR) and non-homological end-joining repair (NHEJ). NHEJ is a repair method that ligates double-strand breaks without using donor DNA, and insertions and / or deletions (indels) are frequently induced during repair. HDR is a repair mechanism that uses donor DNA, and it is also possible to introduce desired mutations into the target region. As a genome modification technology, the CRISPR / Cas system is a preferred example.

[0013] Examples of meganucleases include I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI , I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-Na nI, I-NclIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-Pg rIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp68031, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I- TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-Mtul, PI-MtuHIP A meganuclease and its cleavage site (or recognition site) selected from the group consisting of PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, and PI-TliII, and functional derivative restriction enzymes thereof, can be used. Preferably, a meganuclease and its cleavage site (or recognition site) that is a restriction enzyme having sequence specificity of 18 bases or more, in particular, a meganuclease and its cleavage site that does not cleave the cell genome at one or more locations can be used.

[0014] The term "target region" refers to the genomic region that is the target of genome modification. "Deletion" includes deletions of one or more bases relative to the reference genome, and deletions of one or more genes. A deletion may be 100 bp or larger, 200 bp or larger, 300 bp or larger, 400 bp or larger, 500 bp or larger, 600 bp or larger, 700 bp or larger, 800 bp or larger, 900 bp or larger, 1 kbp or larger, 10 kbp or larger, 50 kbp or larger, 100 kbp or larger, 200 kbp or larger, 300 kbp or larger, 400 kbp or larger, 500 kbp or larger, or 1 Mbp or larger or smaller. A deletion may be 1 Mbp or smaller. A deletion may be 700 kbp or smaller. The deletion may be less than 600 kbp. The deletion may be less than 500 kbp. The deletion may be between 10 kbp and 600 kbp. The deletion may be between 100 kbp and 600 kbp. The deletion may be between 100 kbp and 500 kbp.

[0015] The term "donor DNA" refers to DNA used to repair DNA breaks (preferably double-strand breaks) and which is homologous to the DNA surrounding the target region. Donor DNA includes homology arms consisting of the base sequence to the left and the base sequence to the right of the target region (e.g., a base sequence adjacent to the target region). In this specification, a homology arm consisting of the base sequence to the left of the target region (e.g., a base sequence adjacent to the left) may be referred to as the "left homology arm," and a homology arm consisting of the base sequence to the right of the target sequence (e.g., a base sequence adjacent to the right) may be referred to as the "right homology arm." Donor DNA may include a desired base sequence between the left homology arm and the right homology arm. The length of each homology arm is preferably 300 bp or more, and is usually around 500 to 3000 bp. The lengths of the left homology arm and the right homology arm may be the same or different. If homologous recombination is successfully induced between the target region and the donor DNA after sequence-dependent cleavage, the sequence between the base sequence on the left and the base sequence on the right of the target region (sometimes called the "insertion sequence") will be replaced with the sequence from the donor DNA.

[0016] The "left side" of a target region refers to a position or direction in the double-stranded DNA of the target region where the genomic coordinate value is smaller relative to the reference nucleotide strand (especially the coding region). The "right side" of a target region refers to a position or direction where the genomic coordinate value is larger relative to the reference nucleotide strand. The genomic coordinate system is determined by a mechanism in which numerical values ​​are assigned continuously from the tip to the end of the chromosome. Typically, the left tip has telomeres on the short arm (p arm), and the right end has telomeres on the long arm (q arm).

[0017] The term "sequence-specific nucleic acid cleavage molecule" refers to a molecule that recognizes a specific nucleic acid sequence and can cleave the nucleic acid at that specific sequence. A sequence-specific nucleic acid cleavage molecule is a molecule that has the activity to cleave nucleic acids in a sequence-specific manner (sequence-specific nucleic acid cleavage activity). Examples of sequence-specific nucleic acid cleavage molecules include Cas nucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs).

[0018] The term "target sequence" refers to the DNA sequence in the genome that is targeted for cleavage by a sequence-specific nucleic acid cleavage molecule. When the sequence-specific nucleic acid cleavage molecule is a Cas protein, the target sequence refers to the DNA sequence in the genome that is targeted for cleavage by the Cas protein. When using the Cas9 protein as the Cas protein, the target sequence must be a sequence adjacent to the 5' side of the protospacer adjacent motif (PAM). Typically, the target sequence is selected from a sequence of 17 to 30 bases (preferably 18 to 25 bases, more preferably 19 to 22 bases, and even more preferably 20 bases) adjacent to the 5' side of the PAM. Known design tools such as CRISPR DESIGN (crispr.mit.edu / ) can be used to design the target sequence.

[0019] The term "Cas protein" refers to a CRISPR-associated protein. In a preferred embodiment, the Cas protein forms a complex with guide RNA and exhibits endonuclease activity or nickase activity. Examples of Cas proteins are not particularly limited, but include Cas9 protein, Cpf1 protein, MAD7 protein, C2c1 protein, C2c2 protein, and C2c3 protein. Insofar as the Cas protein cooperates with guide RNA to exhibit endonuclease activity or nickase activity, it includes wild-type Cas proteins and their homologs (paralogs and orthologs), as well as their variants. In a preferred embodiment, the Cas protein is involved in a class 2 CRISPR / Cas system, and more preferably in a type II or type V CRISPR / Cas system. A preferred example of a Cas protein is the Cas9 protein. A preferred example of a Cas protein is the Cas3 protein. For the V-type CRISPR / Cas system, those disclosed in Nature Microbiology volume 10, 3346-3361 (2025) that introduce double-strand or single-strand breaks into the dsDNA (e.g., V-A (Cas12a), V-B (Cas12b), V-E (Cas12e / CasX), CasΦ (Cas12j), V-C (Cas12c), V-H (Cas12h), and V-I (Cas12i), etc.) can be used.

[0020] The term "Cas9 protein" refers to the Cas protein involved in the type II CRISPR / Cas system. The Cas9 protein forms a complex with guide RNA and exhibits activity in cooperation with the guide RNA to cleave DNA in a target region. The Cas9 protein includes the wild-type Cas9 protein and its homologs (paralogs and orthologs), as well as their variants, as long as they possess the aforementioned activity. The wild-type Cas9 protein has a RuvC domain and an HNH domain as nuclease domains, but the Cas9 protein as used herein may have either the RuvC domain or the HNH domain inactivated. Cas9 with either the RuvC domain or the HNH domain inactivated introduces single-strand breaks (nicks) into double-stranded DNA. Therefore, when using Cas9 in which either the RuvC domain or the HNH domain is inactivated to cleave double-stranded DNA, a modified system can be configured such that target sequences for Cas9 are set for both the sense strand and the antisense strand, and nicks occur in sufficiently close positions on the sense and antisense strands, thereby inducing double-strand breaks.

[0021] The species from which the Cas9 protein originates is not particularly limited, but bacteria belonging to the genera Streptococcus, Staphylococcus, Neisseria, or Treponema are preferred examples. More specifically, Cas9 proteins derived from S. pyogenes, S. thermophilus, S. aureus, N. meningitidis, or T. denticola are preferred examples. In a preferred embodiment, the Cas9 protein is a Cas9 protein derived from S. pyogenes. The Cas9 protein may also be eSpCas9, SpCas9-HF1, HiFi Cas9, xCas9, SpCas9-NG, or SpRY.

[0022] Information on the amino acid sequences and coding sequences of various Cas proteins can be obtained from various databases such as GenBank, UniProt, and Addgene. For example, the amino acid sequence of the Cas9 protein of S. pyogenes can be used, as it is registered in Addgene as plasmid number 42230. An example of the amino acid sequence of the Cas9 protein of S. pyogenes is shown in Sequence ID No. 1.

[0023] The terms “guide RNA” and “gRNA” are used interchangeably and refer to RNA that can form a complex with a Cas protein and guide the Cas protein to a target region. In a preferred embodiment, the guide RNA includes CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). crRNA is involved in binding to a target region on the genome, and tracrRNA is involved in binding to the Cas protein. In a preferred embodiment, crRNA includes a spacer sequence and a repeat sequence, the spacer sequence binding to the complementary strand of the target sequence in the target region. In a preferred embodiment, tracrRNA includes an anti-repeat sequence and a 3' tail sequence. The anti-repeat sequence has a sequence complementary to the repeat sequence of crRNA and forms base pairs with the repeat sequence, and the 3' tail sequence typically forms three stem-loops. The guide RNA may be a single guide RNA (sgRNA) formed by ligating the 5' end of tracrRNA to the 3' end of crRNA, or crRNA and tracrRNA may be separate RNA molecules with base pairings formed by repeat and anti-repeat sequences. In a preferred embodiment, the guide RNA is sgRNA.

[0024] The repeat sequence of crRNA and the sequence of tracrRNA can be appropriately selected depending on the type of Cas protein, and those derived from the same bacterial species as the Cas protein can be used. For example, when using Cas9 protein derived from S. pyogenes, the length of sgRNA can be about 50 to 220 nucleotides (nt), preferably about 60 to 180 nt, and more preferably about 80 to 120 nt. The length of crRNA, including the spacer sequence, can be about 25 to 70 bases, preferably about 25 to 50 nt. The length of tracrRNA can be about 10 to 130 nt, preferably about 30 to 80 nt.

[0025] The repeat sequence of crRNA may be the same as that in the bacterial species from which the Cas protein originates, or it may have a portion of its 3' end removed. TracrRNA may have the same sequence as the mature tracrRNA in the bacterial species from which the Cas protein originates, or it may be a truncated form obtained by cutting the 5' and / or 3' ends of the mature tracrRNA. For example, tracrRNA may be a truncated form obtained by removing about 1 to 40 nucleotide residues from the 3' end of mature tracrRNA. Alternatively, tracrRNA may be a truncated form obtained by removing about 1 to 80 nucleotide residues from the 5' end of mature tracrRNA. Furthermore, tracrRNA may be a truncated form obtained by removing, for example, about 1 to 20 nucleotide residues from the 5' end and about 1 to 40 nucleotide residues from the 3' end.

[0026] Various crRNA repeat sequences and tracrRNA sequences for sgRNA design have been proposed, and those skilled in the art can design sgRNAs based on known techniques (e.g., Jinek et al. (2012) Science, 337, 816-21; Mali et al. (2013) Science, 339: 6121, 823-6; Cong et al. (2013) Science, 339: 6121, 819-23; Hwang et al. (2013) Nat. Biotechnol. 31: 3, 227-9; Jinek et al. (2013) eLife, 2, e00471).

[0027] The terms "protospacer adjacency motif" and "PAM" are used interchangeably and refer to sequences recognized by Cas proteins during DNA cleavage by Cas proteins. The sequence and position of the PAM vary depending on the type of Cas protein. For example, in the case of the Cas9 protein, the PAM must be adjacent to the target sequence immediately after the 3' end. The sequence of the PAM corresponding to the Cas9 protein varies depending on the bacterial species from which the Cas9 protein originates. For example, the PAM corresponding to the Cas9 protein of S. pyogenes is "NGG", the PAM corresponding to the Cas9 protein of S. thermophilus is "NNAGAA", the PAM corresponding to the Cas9 protein of S. aureus is "NNGRRT" or "NNGRR(N)", the PAM corresponding to the Cas9 protein of N. meningitidis is "NNNNGATT", and T. This corresponds to the Cas9 protein of denticola as "NAAAAAC" (where "R" is A or G; "N" is A, T, G, or C).

[0028] The terms “spacer sequence” and “guide sequence” are used interchangeably and refer to sequences included in the guide RNA that can bind to the complementary strand of the target sequence. Typically, the spacer sequence is identical to the target sequence (where T in the target sequence becomes U in the spacer sequence). In embodiments of this disclosure, the spacer sequence may contain one or more nucleotide mismatches with respect to the target sequence. If it contains multiple nucleotide mismatches, the mismatches may be adjacent or distant. In a preferred embodiment, the spacer sequence may contain one to five nucleotide mismatches with respect to the target sequence. In a particularly preferred embodiment, the spacer sequence may contain one nucleotide mismatch with respect to the target sequence. In the guide RNA, the spacer sequence is located at the 5' end of the crRNA.

[0029] The term "functionally linked" as used in relation to polynucleotides means that the first base sequence is positioned close enough to the second base sequence that the first base sequence can influence the second base sequence or a region under the control of the second base sequence. For example, functionally linked polynucleotides to a promoter mean that the polynucleotide is linked in such a way that it is expressed under the control of the promoter.

[0030] The term "expression-capable" refers to a state in which a polynucleotide can be transcribed within a cell into which it has been introduced. The term "expression vector" refers to a vector containing a target polynucleotide that has a system to make the target polynucleotide expressible within the cell into which it has been introduced. For example, "Cas protein expression vector" means a vector that can express the Cas protein within the cell into which it has been introduced. Also, for example, "guide RNA expression vector" means a vector that can express guide RNA within the cell into which it has been introduced.

[0031] The term "unique sequence" refers to a sequence that is unique to the cleavage site and does not have any similar sequences other than the cleavage site(s). Specifically, it refers to a sequence in which the Identity value obtained by BLAT search for its total length is 80% or more and does not exist anywhere else on the genome except at the cleavage site(s). It is more preferable that the unique sequence has an Identity value obtained by BLAT search of 75% or more and does not exist anywhere else on the genome except at the cleavage site(s). It is even more preferable that the sequence has an Identity value obtained by BLAT search of 70% or more and does not exist anywhere else on the genome except at the cleavage site(s). It is still preferable that the sequence has an Identity value obtained by BLAT search of 65% or more and does not exist anywhere else on the genome except at the cleavage site(s). It is also preferable that the sequence has an Identity value obtained by BLAT search of 60% or more and does not exist anywhere else on the genome except at the cleavage site(s). The term "unique sequence" can be used interchangeably with "specific sequence."

[0032] A "selection marker" refers to a protein that can be used to select cells based on whether or not it is expressed. A selection marker gene is a gene that codes for a selection marker. In a cell population containing both cells expressing and not expressing a selection marker, when selecting cells that express the selection marker, the selection marker is called a "positive selection marker" or "selection marker for positive selection." In a cell population containing both cells expressing and not expressing a selection marker, when selecting cells that do not express the selection marker, the selection marker is called a "negative selection marker" or "selection marker for negative selection."

[0033] To say that selection markers are different from each other means that they are distinguishable from each other (for example, distinguishably different), meaning that they are at least distinguishable from each other in physiological properties such as the drug resistance properties or other physicochemical properties conferred to cells into which the selection markers are introduced. In other words, to say that selection markers are different from each other means that multiple different selection markers can be detected distinguishably from other selection markers, or that drugs can be selected distinguishably from other selection markers. Furthermore, to say that the selection marker gene is unique to each type of selection marker donor DNA means that the selection marker gene present in one type of selection marker donor DNA is not present in other types of selection marker donor DNA, or, if present in multiple types of donor DNA, it is configured so that it is not expressed simultaneously from two or more types of donor DNA. In this case, the two or more types of donor DNA may be identical except for the selection marker, and may differ in the sequence and / or composition other than the selection marker.

[0034] The cells of this disclosure may preferably be human cells in the context of transplantation into humans. In the context of transplantation into non-human animals, they may preferably be non-human animal cells.

[0035] The cells disclosed herein may be pluripotent stem cells, and preferably human pluripotent stem cells from the viewpoint of transplantation into humans. The cells disclosed herein may, for example, be embryonic stem cells (ES cells). The cells disclosed herein may, for example, be induced pluripotent stem cells (iPS cells). Induced pluripotent stem cells are pluripotent stem cells induced from adult cells such as somatic cells. The cells disclosed herein may be human cell-derived iPS cells (human iPS cells).

[0036] The cells may be, for example, primary cells (e.g., primary immune cells). The cells may be, for example, cell lines (e.g., immune cell lines). The cells may be non-cancer cells. These cells may be autologous or allogeneic to the target to which the cells are administered. From the perspective of use in regenerative medicine, it is preferable that the cells are non-cancer cells, preferably non-cell lines, and preferably primary cells.

[0037] Examples of cells, though not particularly limited, include chondrocytes, osteoblasts, myoblasts, skeletal muscle cells, tendon cells, ligament cells, and adipocytes, as well as their stem cells or progenitor cells; nervous system cells such as dopamine-producing neurons, motor neurons, retinal pigment epithelial cells, and oligodendrocyte progenitor cells, as well as their stem cells or progenitor cells; cardiovascular cells such as cardiomyocytes and vascular endothelial cells, as well as their stem cells or progenitor cells; endocrine system cells such as pancreatic β-cells, as well as their stem cells or progenitor cells; immune system cells such as T cells, NK cells, and dendritic cells; hepatocytes (or hepatic parenchymal cells), and gastrointestinal epithelial cells, as well as their stem cells or progenitor cells, as well as their stem cells or progenitor cells; and hematopoietic cells such as erythrocytes, platelets, megakaryocytes, and hematopoietic stem progenitor cells, as well as their stem cells or progenitor cells.

[0038] Traditionally, regenerative medicine has used cells from the patient themselves (autologous cells) or cells from a donor with a matched immune type (allogeneic cells). However, autologous cells require significant time and financial burden to prepare for each patient, and allogeneic cells have the challenge of relying on immunocompatibility (such as HLA type) to suppress immune rejection. The cells disclosed herein are not particularly limited, but may include, for example, low immunogenic cells. Low immunogenic cells may be useful as a technique for producing off-the-shelf cells that can be administered to any patient. Techniques for conferring low immunogenicity to cells include, for example, HLA class I and class II knockout, β2 microglobulin knockout, and CIITA knockout. Other techniques for conferring low immunogenicity to cells include, for example, enhanced CD47 expression (to avoid phagocytosis by macrophages), enhanced PD-L1 expression, and enhanced HLA-G expression (to avoid attack by T cells and NK cells). Another technique for conferring low immunogenicity to cells is to express, for example, HLA-C, HLA-E, etc.

[0039] The cells may also express endogenous or exogenous CD3. Furthermore, the cells may express, or may not express, one or more endogenous or exogenous factors selected from the group consisting of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, and TCR. In one embodiment, the cells have nucleic acids encoding one or more endogenous or exogenous factors selected from the group consisting of HLA-E, HLA-G, CD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, and TCR, the nucleic acids being operably linked to a control sequence. In some embodiments of cells (e.g., immune cells or non-immune cells), NLRC5 may also be knocked out, which may suppress the development of graft-versus-host disease (GVHD), for example. In some embodiments of cells (e.g., immune cells or non-immune cells), a factor selected from the group consisting of PD1, CD52, CTLA4, dCK, GGH, HPRT, and β2-microglobulin may express CAR or TCR. In some preferred embodiments, the cells of the disclosure have functional β2-microglobulin and / or functional CIITA. The cells of the disclosure may express, for example, IL-15:IL15Rα fusion protein.

[0040] <Method of this Disclosure> According to this disclosure, a method is provided for producing cells having a genome having a deletion of the target region from cells having a genome having the target region.

[0041] In one embodiment, the present disclosure provides a method for producing cells having a genome having a deletion of a target region from cells having a genome having a target region, the method comprising: (A) inserting a selection marker gene into a flanking region of the target region on the genome of the cells; (B) selecting cells having the insertion of the selection marker gene into the flanking region of the target region; and (C) deleting a contiguous region on the genome that includes the selection marker gene and the target region.

[0042] The following explains each step in detail.

[0043] Step (A) Step (A) is a step of inserting a selectable marker gene into (i) the left flanking region or (ii) the right flanking region of a target region on the genome of a cell (see, for example, FIG. 1A). The size of the flanking region is not particularly limited, but is set to a size that allows deletion of a series of regions in step (C). For example, the size of the series of regions is up to 30 Mbp, up to 20 Mbp, up to 10 Mbp, up to 9 Mbp, up to 8 Mbp, up to 7 Mbp, up to 6 Mbp, up to 5 Mbp, up to 4 Mbp, up to 3 Mbp, up to 2 Mbp, up to 1 Mbp, up to 500 kbp, up to 400 kbp, up to 300 kbp, up to 200 kbp, or up to 100 kbp. In this regard, there has been reported an example of inducing a 30 Mbp deletion from the genome of a human cell by genome editing (see Genome Res., 24(12): 2059-2065, 2014).

[0044] Such step (A) includes, for example, introducing a cleavage (preferably sequence-specific cleavage) into (i) the left flanking region or (ii) the right flanking region, and allowing a donor DNA having a left homology arm and a right homology arm that are respectively capable of homologous recombination with the left region and the right region of the cleavage, wherein the donor DNA comprises a selectable marker gene between the left homology arm and the right homology arm, to act on the flanking region to induce homologous recombination. In this homologous recombination, the sequence between the left region and the right region on the genome is replaced with the sequence between the left homology arm and the right homology arm in the donor DNA. This makes it possible to freely alter the sequence between the left region and the right region on the genome. Cleavage (preferably sequence-specific cleavage) can be achieved by a sequence-specific cleavage molecule. The cleavage is preferably double-strand cleavage.

[0045] The left region and the right region relative to said cleavage are present (i) within the left flanking region when the cleavage is introduced into the left flanking region, and (ii) within the right flanking region when the cleavage is introduced into the right flanking region. This enables introduction of a region comprising a selectable marker gene between the left homology arm and the right homology arm derived from the donor DNA (also referred to as the "intervening region") into (i) the left flanking region or (ii) the right flanking region.

[0046] The selectable marker gene may comprise (1) a positive selectable marker gene and a negative selectable marker gene, or (2) a positive-negative selectable marker gene that functions as both a positive selectable marker and a negative selectable marker.

[0047] The positive selectable marker is not particularly limited as long as it allows selection of cells expressing the same. Examples of the positive selectable marker gene include drug resistance genes, fluorescent protein genes, luminescent enzyme genes, chromogenic enzyme genes, and the like.

[0048] The negative selectable marker is not particularly limited as long as it allows selection of cells that do not express the same. Examples of the negative selectable marker gene include suicide genes (e.g., thymidine kinase), fluorescent protein genes, luminescent enzyme genes, chromogenic enzyme genes, and the like. When the negative selectable marker gene is a gene that negatively affects cell survival (e.g., a suicide gene), the negative selectable marker gene can be operably linked to an inducible promoter. By being operably linked to an inducible promoter, the negative selectable marker gene can be expressed only when it is desired to eliminate cells harboring the negative selectable marker gene. When the negative selectable marker gene is an optically detectable marker gene such as a fluorescence, luminescence or chromogenesis marker gene (visible marker gene) and thus has little negative effect on cell survival, it may be constitutively expressed.

[0049] Examples of drug resistance genes include, but are not limited to, puromycin resistance genes, blastisidin resistance genes, geneticin resistance genes, neomycin resistance genes, tetracycline resistance genes, kanamycin resistance genes, zeosin resistance genes, hygromycin resistance genes, and chloramphenicol resistance genes.

[0050] Examples of fluorescent protein genes include, but are not limited to, the green fluorescent protein (GFP) gene, the yellow fluorescent protein (YFP) gene, and the red fluorescent protein (RFP) gene. Fluorescent protein genes are an example of optically detectable marker genes (visualization marker genes).

[0051] Examples of luminescent enzyme genes include, but are not limited to, the luciferase gene. Examples of chromogenic enzyme genes include, but are not limited to, the β-galactosidase gene, β-glucuronidase gene, and alkaline phosphatase gene.

[0052] Examples of suicide genes include, but are not limited to, the herpes simplex virus thymidine kinase (HSV-TK) and inductive caspase 9.

[0053] The selection marker gene present in the donor DNA is preferably a positive selection marker gene. That is, cells expressing the selection marker can be selected as cells in which the selection marker gene has been knocked in.

[0054] The donor DNA may contain a positive selection marker gene and a negative selection marker gene between the left homology arm and the right homology arm. The positional relationship between the positive selection marker gene and the negative selection marker gene is not particularly limited; the positive selection marker gene may be to the left of the negative selection marker gene, or vice versa. When the selection marker donor DNA contains a positive selection marker gene and a negative selection marker gene, a nucleotide sequence encoding a self-cleaving peptide or an IRES (internal ribozyme entry site) sequence may be interposed between the positive selection marker gene and the negative selection marker gene. By interposing these sequences, the positive selection marker gene and the negative selection marker gene can be expressed independently from a single promoter. Examples of 2A peptides include 2A peptide derived from foot-and-mouth disease virus (FMDV) (F2A), 2A peptide derived from equine rhinitis A virus (ERAV) (E2A), 2A peptide derived from Porcine teschovirus (PTV-1) (P2A), and 2A peptide derived from Thosea assigna virus (TaV) (T2A).

[0055] The donor DNA may include two types of donor DNA, namely a first donor DNA and a second donor DNA. In one embodiment, the first donor DNA and the second donor DNA may each contain (1) a positive selection marker gene or (2) a positive and negative selection marker gene, and the selection marker genes on the first donor DNA and the second donor DNA are different from each other. This configuration is advantageous when a cell has two alleles, and a cell in which the first donor DNA homologously recombinates with one of the two alleles and the second donor DNA homologously recombinates with the other will show expression of two different (1) positive selection marker genes or (2) positive and negative selection marker genes. Therefore, by selecting cells that show expression of two different (1) positive selection marker genes or (2) positive and negative selection marker genes, it is advantageous to obtain cells in which each allele has been edited.

[0056] In one preferred embodiment, the donor DNA comprises two types of donor DNA, including a first donor DNA and a second donor DNA, and the cells obtained by the method of this disclosure have a target gene deleted from each of the two alleles.

[0057] If there are three or more alleles, the donor DNA may contain three or more types of donor DNA, and each donor DNA may contain (1) a positive selection marker gene or (2) a positive and negative selection marker gene, and the selection marker genes on each donor DNA are different from each other. By selecting cells that show expression of three or more different (1) positive selection marker genes or (2) positive and negative selection marker genes, it is advantageous to obtain cells in which each allele has been edited.

[0058] In one embodiment, in addition to the selection marker gene, further sequences may be included between the left homology arm and the right homology arm in the donor DNA.

[0059] In one embodiment, the further sequence may include, for example, a recognition sequence for a sequence-specific cleavage molecule. Specific cleavage is possible if the recognition sequence for the sequence-specific cleavage molecule included in the further sequence is a sequence unique to the genome. (i) When introducing a cleavage to the left flanking region, the intervening region has, for example, the recognition sequence for the sequence-specific cleavage molecule to the left of the selection marker gene, and (ii) When introducing a cleavage to the right flanking region, the intervening region has, for example, the recognition sequence for the sequence-specific cleavage molecule to the right of the selection marker gene.

[0060] In one embodiment, the unique sequence may be the same as, or different from, the recognition sequence of the sequence-specific cleavage molecule involved in the other cleavage in step (C) (see, for example, Figure 1C). When the two cleavage sites in step (C) are the same sequence, it may be advantageous that two cleavages can be induced by a single type of sequence-specific cleavage molecule.

[0061] In some embodiments, the further sequence may include sequences other than the recognition sequence of the sequence-specific cleavage molecule. The sequences other than the recognition sequence of the sequence-specific cleavage molecule may be located, for example, (a) to the left of the recognition sequence and the selection marker gene, (b) to the right of the recognition sequence and to the left of the selection marker, or (c) to the right of the recognition sequence and the selection marker gene. In some embodiments, (i) when a cleavage is introduced in the left flanking region, the sequences other than the recognition sequence of the sequence-specific cleavage molecule may be located, for example, (a) to the left of the recognition sequence and the selection marker gene, (b) to the right of the recognition sequence and to the left of the selection marker, or (c) to the right of the recognition sequence and the selection marker gene. In some embodiments, (i) when a cleavage is introduced in the left flanking region, the sequences other than the recognition sequence of the sequence-specific cleavage molecule may be located, for example, to the left of the recognition sequence and the selection marker gene. In one embodiment, (ii) when a cleavage is introduced in the right flanking region, sequences other than the recognition sequence of the sequence-specific cleavage molecule may be located, for example, (a) to the left of the recognition sequence and the selection marker gene, (b) to the right of the recognition sequence and to the left of the selection marker, or (c) to the right of the recognition sequence and the selection marker gene. In one embodiment, (ii) when a cleavage is introduced in the right flanking region, sequences other than the recognition sequence of the sequence-specific cleavage molecule may be located, for example, (c) to the right of the recognition sequence and the selection marker gene. Sequences other than the recognition sequence of the sequence-specific cleavage molecule may include, for example, an inactive sequence (e.g., a linker sequence) or a coding region of a functional molecule.

[0062] Step (B) In Step (B), cells having an insertion of the selection marker gene into the flanking region of the target region are selected based on the presence or absence of the selection marker gene introduced between the left and right regions of the genome in Step (A). This selection can preferably be performed based on the expression of a positive selection marker, or the expression of both a positive and negative selection marker. If the selection marker gene is a drug resistance gene, cells having an insertion of the selection marker gene into the flanking region of the target region can be selected by removing cells other than those expressing the selection marker gene in the presence of the corresponding drug. If the selection marker gene is a visualization marker gene, cells having an insertion of the selection marker gene into the flanking region of the target region can be selected by selecting cells that emit a specific fluorescence in the presence of the corresponding excitation light, for example, by a method such as flow cytometry. This selection can be appropriately carried out by a person skilled in the art based on the type and properties of the selection marker gene.

[0063] Step (B) may include sequencing the target region of cells expressing the selected marker gene to confirm that the flanking region of the target region has the expected insertion of the selected marker gene. Step (B) may involve obtaining a cell suspension containing such cells and concentrating and / or purifying the cells, or obtaining edited cells in the cell suspension without concentrating and / or purifying the cells.

[0064] Step (C) In step (C), a contiguous region on the genome containing the selected marker gene and the target region is deleted. Such a deletion can be formed by introducing one or two cuts in the genome so as to flank the contiguous region. Introducing two cuts may be more effective when the contiguous region to be deleted is large. Such a deletion of a genomic region can be produced by a person skilled in the art using a sequence-specific nucleic acid cleavage molecule as appropriate. The cleavage is preferably a double-strand break.

[0065] Step (C) can also select cells having a genome in which the aforementioned series of regions are deleted. This selection can be achieved, for example, by the non-expression of negative selection markers. Examples of negative selection marker genes that can be used here include, but are not limited to, visualization marker genes and suicide genes. If these markers remain in the genome, the cells will, under appropriate conditions, fluoresce or emit light based on the expression of the visualization marker gene, or die by expressing the suicide gene. Therefore, in step (C), cells that do not express negative selection markers can be selected based on the non-expression of the negative selection marker gene.

[0066] Step (C) may include sequencing the target region of cells expressing the selected marker gene to confirm that the flanking region of the target region contains the expected insertion of the selected marker gene.

[0067] In this way, according to the present disclosure, cells having a genome lacking the target region can be produced from cells having a genome having the target region.

[0068] In a modified version of step (C), a transgene can also be introduced by inducing a cut in the genome in the presence of a second donor DNA. For this purpose, the second donor DNA may have a left homology arm and a right homology arm, and may contain a transgene between the left homology arm and the right homology arm. This makes it possible to delete a target gene from a cell and to introduce a desired nucleic acid sequence, such as a desired gene, into the cell. By introducing a nucleic acid sequence, such as a gene encoding a functional molecule, as the desired gene, it becomes possible to obtain a functional cell.

[0069] In one embodiment, a method for producing genetically modified cells is provided, comprising: (A) inserting a sequence (insertion sequence) between a left homology arm and a right homology arm into the flanking region of a target region on the genome of a cell in the presence of donor DNA containing a left homology arm, a desired nucleic acid sequence, a selection marker gene, and a right homology arm, wherein the left homology arm has a sequence (homologous sequence) homologous to the left region of the insertion site, and the right homology arm has a sequence (homologous sequence) homologous to the right region of the insertion site; (B) selecting cells having the insertion of the desired nucleic acid sequence and the selection marker gene into the flanking region of the target region; and (C) deleting a series of regions on the genome (contiguous region) containing the selection marker gene and the target region, thereby providing a method for obtaining genetically modified cells without leaving an insertion of the selection marker gene on the genome. The cells preferably contain the desired nucleic acid sequence.

[0070] One modified example (Modified Example 1) provides a method for producing genetically modified cells, comprising: (A) inserting a sequence (insertion sequence) between a left homology arm and a right homology arm into an insertion site in the flanking region of a target region on the genome of a cell, in the presence of donor DNA containing a left homology arm, a desired nucleic acid sequence, a selection marker gene, and a right homology arm, wherein the left homology arm has a sequence (homologous sequence) homologous to the left region of the insertion site, and the right homology arm has a sequence (homologous sequence) homologous to the right region of the insertion site; (B) selecting cells having an insertion of a desired nucleic acid sequence and a selection marker gene in the flanking region of the target region; and (C) deleting the selection marker gene {the desired nucleic acid sequence and target region may be left on the genome, or the desired nucleic acid sequence may be left on the genome and the target region may be deleted from the genome together with the selection marker}, thereby providing a method for obtaining genetically modified cells without leaving an insertion of the selection marker gene on the genome. The cells preferably contain the desired nucleic acid sequence.

[0071] The insertion in step (A) preferably involves cutting near the insertion site, thereby facilitating the insertion. Step (B) is the same as step (B). Those skilled in the art will understand that the area to be removed in step (C) can be freely adjusted by changing the cutting location in Figure 1C.

[0072] In one modified example (Modified Example 2), for example, as shown in Figure 1E, an exogenous homologous sequence is introduced, and a deletion is created between the exogenous homologous sequence and the endogenous homologous sequence. Specifically, in one modified example (Modified Example 2), the method includes: (A1) inserting a sequence (insertion sequence) between the left homology arm and the right homology arm into one flanking region of a target region on the genome of a cell in the presence of donor DNA containing a left homology arm, an exogenous homologous sequence, a selection marker gene, and a right homology arm, wherein the exogenous homologous sequence is a sequence homologous to the sequence (endogenous homologous sequence) of the other flanking region of the target region, and the left homology arm has a sequence (homologous sequence) homologous to the left region of the insertion site, and the right homology arm has a sequence (homologous sequence) homologous to the right region of the insertion site; (B1) selecting cells having the insertion of the exogenous homologous sequence and the selection marker gene into one flanking region of the target region; and (C1) deleting the sequence between the exogenous homologous sequence and the endogenous homologous sequence. This provides a method for obtaining genetically modified cells without leaving an insertion of the selection marker gene on the genome. Modification 2 also provides a method for producing genetically modified cells, comprising: (A1') inserting an exogenous sequence into one flanking region of a target region on the genome of a cell in the presence of donor DNA containing an exogenous homologous sequence and an exogenous sequence containing a selection marker gene, wherein the exogenous homologous sequence is homologous to the sequence of the other flanking region of the target region (endogenous homologous sequence); (B1') selecting cells having the insertion of the exogenous homologous sequence and the selection marker gene into one flanking region of the target region; and (C1') deleting the sequence between the exogenous homologous sequence and the endogenous homologous sequence, thereby providing a method for obtaining genetically modified cells without leaving an insertion of the selection marker gene on the genome.

[0073] In steps (A1) and (A1'), the donor DNA may further contain the desired nucleic acid sequence, preferably comprising the left homology arm, the desired nucleic acid sequence, the exogenous homologous sequence, the selected marker sequence, and the right homology arm in that order, or comprising the left homology arm, the selected marker sequence, the exogenous homologous sequence, the desired nucleic acid sequence, and the right homology arm in that order. The region sandwiched between the endogenous homologous sequence and the exogenous homologous sequence is removed by recombination, but the rest, i.e., the sequence outside the endogenous homologous sequence and the exogenous homologous sequence, remains in the genome of the genetically modified cell. This mechanism makes it possible to remove endogenous genes or to remove endogenous genes and then introduce the desired nucleic acid sequence. This mechanism utilizing homologous recombination can increase the efficiency of obtaining modified cells. Steps (A1) and (A1') can be carried out in basically the same way as step (A).

[0074] Processes (B1) and (B1') are the same as process (B) described above.

[0075] Steps (C1) and (C1') can preferably be carried out by introducing a sequence-specific cleavage in the region between the endogenous homologous sequence and the exogenous homologous sequence. This cleavage is preferably performed at a location that does not disrupt the selected marker gene. In the DNA repair process induced by this cleavage, recombination between the exogenous homologous sequence and the endogenous homologous sequence is induced, resulting in the deletion of a series of regions (including the selected marker gene and the target region) located between the exogenous homologous sequence and the endogenous homologous sequence, i.e., between the exogenous homologous sequence and the endogenous homologous sequence. The recombination during this deletion may be homologous recombination or other types of recombination. In this embodiment, there may be only one cleavage site in steps (C1) and (C1'), which may simplify the design of the sequence-specific cleavage molecule compared to the case in which two cleavage sites are introduced at both ends of a series of regions, as in the embodiment of Figure 1C.

[0076] In this embodiment, the donor DNA may further contain a desired nucleic acid sequence. If it contains a desired nucleic acid sequence, the donor DNA preferably contains the left homology arm, the desired nucleic acid sequence, the exogenous homologous sequence, the selection marker gene, and the right homology arm in this order, or the left homology arm, the selection marker gene, the exogenous homologous sequence, the desired nucleic acid sequence, and the right homology arm in this order. By positioning the desired nucleic acid sequence outside the exogenous homologous sequence (i.e., outside the series of regions deleted in steps (C1) and (C1')), the desired nucleic acid sequence is retained on the genome after steps (C1) and (C1'). The desired nucleic acid sequence may include, for example, a nucleic acid sequence that encodes a factor that the cells of this disclosure may express. The desired nucleic acid sequence may include, but is not limited to, transgenes, regulatory sequences, sequences that encode functional molecules, sequences that encode molecules involved in immunomodulation, sequences that encode cell surface molecules, sequences that encode receptors, and so on.

[0077] The exogenous homologous sequence has sufficient length and homology to induce recombination by cleavage with the endogenous homologous sequence. The length of the exogenous homologous sequence is not particularly limited as long as recombination by cleavage is induced, but is 100 bp or more, preferably 200 bp or more, more preferably 250 bp or more, and even more preferably 300 bp or more, for example, about 300 to 5000 bp. The identity between the exogenous homologous sequence and the endogenous homologous sequence is not particularly limited as long as recombination by cleavage is induced, but is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 100%. The sequence identity between the left homology arm and the left region is not particularly limited as long as recombination by cleavage is induced, but is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 100%. The identity between the right homology arm and the right region is not particularly limited as long as recombination is induced by the cleavage, but is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 100%. The cleavage is preferably a double-strand break.

[0078] In a further modification (modification 3), the donor DNA is the same as modification 2 except that it does not contain a selection marker gene (see Figure 5). That is, the present disclosure provides a method for obtaining a genetically modified cell without inserting a selection marker gene into the genome, comprising: (A2) inserting a sequence between the left homology arm and the right homology arm (insertion sequence) into one flanking region of a target region on the genome of a cell in the presence of donor DNA containing a left homology arm, an exogenous homology sequence, and a right homology arm, wherein the exogenous homology sequence is homologous to the sequence of the other flanking region of the target region (endogenous homology sequence), the left homology arm has a sequence homologous to the left region of the insertion site, and the right homology arm has a sequence homologous to the right region of the insertion site, (B2) obtaining a cell having the insertion of the exogenous homology sequence into one flanking region of the target region, and (C2) deleting the sequence between the exogenous homology sequence and the endogenous homology sequence. Furthermore, in Modification 3, a method for producing genetically modified cells is provided, comprising: (A2') inserting an exogenous sequence into a cleavage site on the genome of a cell in the presence of donor DNA containing a desired nucleic acid sequence and an exogenous homologous sequence, wherein the exogenous homologous sequence is a sequence homologous to a sequence (endogenous homologous sequence) that exists on the opposite side of the genome from the desired nucleic acid sequence via an arbitrary genomic region {that is, arranged in the order of desired nucleic acid sequence, exogenous homologous sequence, arbitrary genomic region, and endogenous homologous sequence}; (B2') obtaining a cell having the insertion of the exogenous sequence into the cleavage site; and (C2') deleting a sequence between the exogenous homologous sequence and the endogenous homologous sequence. This provides a method for obtaining genetically modified cells {preferably a method that does not involve inserting a selected marker gene into the genome}.

[0079] In steps (A2) and (A2'), the donor DNA may further contain the desired nucleic acid sequence, preferably consisting of the left homology arm, the desired nucleic acid sequence, the exogenous homologous sequence, and the right homology arm in that order, or the left homology arm, the exogenous homologous sequence, the desired nucleic acid sequence, and the right homology arm in that order. The region sandwiched between the endogenous homologous sequence and the exogenous homologous sequence is removed by recombination induced by cleavage in that region, but the rest, i.e., the sequence outside the endogenous homologous sequence and the exogenous homologous sequence, remains in the genome of the genetically modified cell. This mechanism makes it possible to remove endogenous genes or to remove endogenous genes and then introduce the desired nucleic acid sequence. This mechanism utilizing homologous recombination can increase the efficiency of obtaining modified cells. Step (A1) can be carried out in basically the same way as step (A). Any of the above-mentioned genomic regions can be used as target regions to be deleted by this method.

[0080] Steps (B2) and (B2') do not specifically require cell selection, but cell selection itself is not excluded. For example, if the flanking region of the genome has a landing pad, cell selection may be performed by removing the landing pad (and the negative selection marker gene).

[0081] Steps (C2) and (C2') can preferably be carried out by introducing a sequence-specific cleavage in the region between the endogenous homologous sequence and the exogenous homologous sequence. In the DNA repair process induced by this cleavage, recombination between the exogenous homologous sequence and the endogenous homologous sequence is induced, resulting in the deletion of a series of regions (including the target region) located between the exogenous homologous sequence and the endogenous homologous sequence. In this embodiment, there may be only one cleavage site in steps (C2) and (C2'), which may simplify the design of the sequence-specific cleavage molecule compared to the case where two cleavage sites are introduced at both ends of the series of regions.

[0082] In this embodiment, the donor DNA further comprises the desired nucleic acid sequence. Preferably, the donor DNA comprises the left homology arm, the desired nucleic acid sequence, the exogenous homologous sequence, and the right homology arm in this order, or the left homology arm, the exogenous homologous sequence, the desired nucleic acid sequence, and the right homology arm in this order. By positioning the desired nucleic acid sequence outside the exogenous homologous sequence (i.e., outside the series of regions deleted in steps (C2) and (C2')), the desired nucleic acid sequence is retained on the genome after steps (C2) and (C2'). The desired nucleic acid sequence is not particularly limited, but may include, for example, a transgene, a regulatory sequence, or a sequence encoding a functional molecule (e.g., mRNA, non-coded RNA, etc.).

[0083] The exogenous homologous sequence has sufficient length and homology to induce recombination by cleavage with the endogenous homologous sequence. The length of the exogenous homologous sequence is not particularly limited as long as recombination by cleavage is induced, but is 100 bp or more, preferably 200 bp or more, more preferably 250 bp or more, even more preferably 300 bp or more, for example, about 300 to 5000 bp. The identity (homology) between the exogenous homologous sequence and the endogenous homologous sequence is not particularly limited as long as recombination is induced, but is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 100%. The sequence identity between the left homology arm and the left region is not particularly limited as long as recombination is induced, but is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 100%. The identity between the right homology arm and the right region is not particularly limited as long as recombination is induced, but is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and especially preferably 100%. The breaks are preferably double-strand breaks.

[0084] In some embodiments, the insertion or deletion of nucleic acid sequences into target genomic sites according to this disclosure can be achieved by genome editing methods that do not involve double-strand breaks (DSBs). Examples of such DSB-free methods include twin prime editing (twinPE) and PASTE (Programmable Addition via Site-specific Targeting Elements). twinPE uses a fusion protein of Cas9 nickase and reverse transcriptase (prime editor) and two prime editing guide RNAs (pegRNAs) that target opposing strands of the genome, respectively. Hybridization of complementary DNA flaps synthesized from the reverse transcription templates of each pegRNA replaces the intrinsic sequence between two nick sites with a novel sequence (Anzalone et al., Nature Biotechnology, 40:731-740, 2022). PASTE uses a fusion protein that integrates Cas9 nickase, reverse transcriptase, and serine integrase (e.g., Bxb1 integrase) to achieve prime editing-based programming of integrase-recognized sequences and site-specific integration of donor DNA by integrase (Yarnall et al., Nature Biotechnology, 41:500-512, 2023).

[0085] For example, in the exogenous sequence insertion steps such as (A), (A1), (A1'), (A2), and (A2'), in embodiments using twinPE, first, a serine recombinase recognition sequence (e.g., attB sequence or attP sequence, approximately 38-50 base pairs) is inserted into the target site by twinPE, and then, by the action of serine recombinase (e.g., Bxb1 integrase), donor DNA having the corresponding cognitive recognition sequence is applied to the target site. In embodiments using PASTE, the above-mentioned writing of the recognition sequence and integration of the donor DNA are achieved by a single fusion protein. In either method, the donor DNA does not require homology arms to the target site, and only needs to contain the integrase recognition sequence and the exogenous nucleic acid sequence to be inserted. This simplifies the design of donor nucleic acids compared to homologous recombination repair (HDR)-based methods and enables application to a wide range of cell types, including non-dividing cells.

[0086] Furthermore, in embodiments using twinPE for DNA region deletions such as steps (C), (C1), (C1'), (C2), and (C2'), two pegRNAs are designed to target both ends of the region to be deleted, and the reverse transcription templates of each pegRNA are configured to encode complementary short sequences. The prime editor is induced by each pegRNA to introduce nicks at both ends, and the synthesized complementary flaps hybridize, thereby removing the intrinsic sequence between the two nicks and replacing it with a short sequence (typically tens of base pairs or less) encoded in the reverse transcription template. Alternatively, it is also possible to insert serine recombinase recognition sequences (attB and attP) at both ends of the region to be deleted using twinPE or PASTE, and remove the intervening sequence by the action of serine recombinase.

[0087] According to this disclosure, cells produced by the above method are provided.

[0088] As shown in Figure 2A, a cleavage site was defined in the left-side flanking region of gene 1 (a gene of 10 kb or longer was used as an example). An exogenous sequence containing a marker gene (for example, GFP and a puromycin resistance gene) was inserted between the left-side (HA1L) and right-side (HAR1) homology arms, respectively, which have homologous sequences. The percentage of GFP-expressing cells after puromycin selection was approximately 99.3%. Subsequently, a series of genomic regions including gene 1 were deleted from the marker gene by non-homologous end joining. The deletion efficiency was approximately 5.6%. Thus, according to this disclosure, the genome of cells could be efficiently modified using a simple process.

[0089] Next, as shown in Figure 2B, a cleavage site was defined in the left-side flanking region of gene 1. The left-side region (HA1L) and right-side region (HAR1) of the cleavage site had left and right homology arms with homologous sequences, respectively. An exogenous homologous sequence (HA2R) and an exogenous sequence containing a marker gene (e.g., GFP and a puromycin resistance gene) were inserted between the left and right homology arms. The exogenous homologous sequence was a sequence homologous to (more specifically, identical to) the endogenous homologous sequence (HA2R) present in the right-side flanking region of gene 1, induced by cleavage. The percentage of GFP-expressing cells after puromycin selection was approximately 98.9%. Subsequently, the region between the exogenous homologous sequence (HA2R) and the marker gene was cleaved. This induced recombination between HA2R sequences, resulting in the deletion of a series of genomic regions including gene 1 from the marker gene. The deletion efficiency was approximately 10.4%. Thus, according to this disclosure, the genome of cells can be modified efficiently with a simple process, and the deletion efficiency, particularly in the deletion step, is higher than that of genome editing in the form of Figure 2A. This is thought to be partly due to the fact that deletion in the form of Figure 2B only requires the use of one guide RNA.

[0090] Next, as shown in Figure 3, a cleavage site was defined in the left-side flanking region of the BMP7 gene, and an exogenous sequence containing a marker gene (one allele of GFP and puromycin resistance genes (puroR), and one allele of RFP and blasticidin resistance genes (blasR)) was inserted between the left-side and right-side homology arms, respectively, with homologous sequences in the left-side region (HA1L) and the right-side region (HAR1), respectively (see Figure 3A). After selection of puromycin and blasticidin, the percentage of cells expressing GFP and RFP was approximately 99.7%, while the percentage of cells not expressing either GFP or RFP was 0.08% (see Figure 3B). Subsequently, by cleaving the DNA so as to sandwich the marker gene and a series of regions including BMP7, a series of genomic regions including the BMP7 gene were deleted from the marker gene by non-homologous end joining. The percentage of cells that did not express either GFP or RFP after deletion was 0.32%. These cells that did not express either GFP or RFP were sorted into single cells, cultured in large quantities, and then PCR was used to confirm the deletion of the BMP7 gene and the formation of junctions after deletion. As shown in Figure 3C, the BMP7 gene was confirmed to be deleted in all six clones analyzed, as expected. BMP7 is a large gene extending 98 kb, and it is clear that the invention of this disclosure is useful even for deletions of such large regions.

[0091] Next, as shown in Figure 4, a cleavage site was defined in the left-side flanking region of the B2M gene. The left-side region (HA1L) and right-side region (HAR1) of the cleavage site had homologous sequences in the left-side and right-side homology arms, respectively. An exogenous homologous sequence (HA2R) and a marker gene (one allele of GFP and puromycin resistance genes, and one allele of RFP and blasticidin resistance genes) were inserted between the left-side and right-side homology arms (see Figure 4A). The exogenous homologous sequence was a sequence homologous to (more specifically, identical to) the endogenous homologous sequence (HA2R) present in the right-side flanking region of the B2M gene. After selection for puromycin and blasticidin, the percentage of cells expressing GFP and RFP was approximately 98.9%, while the percentage of cells not expressing either GFP or RFP was 0.5% (see Figure 4B). Subsequently, the region between the two HA2Rs, more specifically between the exogenous homologous sequence (HA2R) and the marker gene, was cleaved. This induced recombination between the HA2Rs, resulting in the deletion of a series of genomic regions, including the B2M gene, from the marker gene. The percentage of cells that did not express either GFP or RFP after deletion was 26.9% (see Figure 4B). These cells that did not express either GFP or RFP were sorted into single cells, and after expansion culture, B2M gene deletion and subsequent junction formation were confirmed by PCR. Of the 12 clones analyzed, B2M gene deletion was confirmed in 10 clones as expected (see Figure 4C).

[0092] As shown in Figure 5, the results in Figure 4 suggest that cells undergoing genome modification according to this disclosure can be obtained without the use of selection markers. For example, when used for deletion of a target region (Target in Figure 5), an exogenous homologous sequence of 100 bp or more (HA on the left in Figure 5) is introduced into the genome by some genome editing method, not limited to homologous recombination. In recent years, with the invention of genome modification technologies such as Twin Prime Editing and the PASTE method, it has become possible to efficiently insert target sequences into the genome, and the insertion of endogenous homologous sequences of 100 bp or more can be performed by these methods without necessarily relying on homologous recombination. After insertion of the exogenous homologous sequence (HA on the left) into the genome, cleavage is induced, causing the HA-HA space to be removed, and the deletion of the target region in Figure 5 is induced. Deleting the target region has been shown to yield cells with a high efficiency (26.9% in Figure 5), as illustrated in Figure 4 (corresponding to the lower right figure in Figure 5). Therefore, it is considered that selection markers may not necessarily be used when obtaining cells after deletion of the target region.

[0093] Next, as shown in Figure 6, a cleavage site was defined in the left-side flanking region of the CIITA gene. The left-side region (HA1L) and right-side region (HAR1) of the cleavage site had homologous sequences in the left-side and right-side homology arms, respectively. An exogenous homologous sequence (HA2R) and a marker gene (one allele of GFP and puromycin resistance genes, and one allele of RFP and blasticidin resistance genes) were inserted between the left-side and right-side homology arms (see Figure 6A). The exogenous homologous sequence was a sequence homologous to (more specifically, identical to) the endogenous homologous sequence (HA2R) present in the right-side flanking region of the CIITA gene. At this time, six types of exogenous homologous sequences (HA2R) were tested, starting from the rightmost sequence: approximately 4300 bp, approximately 2500 bp, approximately 1000 bp, approximately 500 bp, approximately 300 bp, and approximately 100 bp. After selecting puromycin and blasticidin, the region between the exogenous homologous sequence (HA2R) and the marker gene was cleaved. Subsequently, cells that did not express either GFP or RFP were sorted into single cells, and after expansion culture, deletion of the CIITA gene and junction formation after deletion were confirmed by PCR. As a result, clones with the expected CIITA gene deletion were obtained with an efficiency of over 80% in all five conditions: endogenous homologous sequence (HA2R) of approximately 4300 bp, 2500 bp, 1000 bp, 500 bp, and 300 bp (see Figure 6B). Even with an endogenous homologous sequence (HA2R) of 100 bp, cells with the CIITA gene deletion could be obtained in 1 out of 14 clones, and the efficiency of obtaining deletion cells increased at 300 bp.

Claims

1. A method for producing cells having a genome with a deletion of a target region from cells having a genome with a target region, comprising: (A) inserting an exogenous sequence containing a selection marker gene into (i) the left flanking region or (ii) the right flanking region of the target region on the genome of the cell; (B) selecting cells having an insertion of a selection marker gene in the flanking region of the target region; and (C) deleting a series of regions (contiguous region) on the genome containing the selection marker gene and the target region, thereby obtaining cells having a genome with a deletion of a target region without leaving an insertion of the selection marker gene on the genome.

2. The method according to claim 1, wherein the foreign sequence includes a left homologous sequence homologous to the left flanking sequence of the left flanking region in the case of (i) above, and includes a right homologous sequence homologous to the right flanking sequence of the right flanking region in the case of (ii) above, and step (C) is achieved by inducing a cleavage at a site other than the selection marker gene between the right or left flanking sequence and the homologous sequence.

3. The method according to claim 1 or 2, wherein step (A) includes (i) introducing a sequence-specific cleavage into a left flanking region or (ii) a right flanking region, and inducing homologous recombination by applying donor DNA having a left homology arm and a right homology arm that are homologously recombinable with the left and right regions of the cleavage, respectively, and containing a selection marker gene between the left and right homology arms, to the flanking region.

4. The method according to claim 3, wherein, when the donor DNA is (i) acted upon the left flanking region to induce homologous recombination, the donor DNA contains a unique sequence that is targeted by a sequence-specific cleavage molecule to the left of the selection marker gene.

5. The method according to any one of claims 1 to 4, wherein the selection marker gene comprises (i) a positive selection marker gene and a negative selection marker gene; or (ii) a marker gene that can be used for both positive and negative selection.

6. The method according to any one of claims 1 to 5, wherein step (C) includes introducing sequence-specific cuts at both ends of the region to be deleted to induce deletion of the selection marker gene and the target region, and selecting cells in which the expression of the negative selection marker has been lost.

7. A method for producing genetically modified cells, comprising: (A) inserting an exogenous sequence into one flanking region of a target region on the genome of a cell in the presence of donor DNA containing an exogenous homologous sequence and an exogenous sequence containing a selection marker gene, wherein the exogenous homologous sequence is homologous to the sequence of the other flanking region of the target region (endogenous homologous sequence); (B) selecting cells having the insertion of the exogenous homologous sequence and the selection marker gene into one flanking region of the target region; and (C) deleting the sequence between the exogenous homologous sequence and the endogenous homologous sequence, thereby obtaining genetically modified cells without leaving an insertion of the selection marker gene on the genome.

8. The method according to claim 7, wherein the donor DNA contains a desired nucleic acid sequence, an exogenous homologous sequence, and a selection marker gene in that order, or contains a selection marker gene, an exogenous homologous sequence, and a desired nucleic acid sequence in that order, and the desired nucleic acid sequence remains on the genome after the region containing the selection marker gene is deleted by inducing recombination between the exogenous homologous sequence and the endogenous homologous sequence.

9. A method for producing genetically modified cells, comprising: (A) inserting an exogenous sequence into a cleavage site on the cell's genome in the presence of donor DNA containing an exogenous sequence containing a desired nucleic acid sequence and an exogenous homologous sequence, wherein the exogenous homologous sequence is homologous to a sequence (endogenous homologous sequence) that exists on the opposite side of the genome from the desired nucleic acid sequence via an arbitrary region; (B) selecting cells having the insertion of the exogenous sequence into the cleavage site; and (C) deleting a sequence between the exogenous homologous sequence and the endogenous homologous sequence, thereby obtaining genetically modified cells.

10. The method according to claim 9, wherein the donor DNA contains a desired nucleic acid sequence and an exogenous homologous sequence in that order, and the desired nucleic acid sequence remains on the genome by inducing recombination between the exogenous homologous sequence and the endogenous homologous sequence.

11. A method for producing genetically modified cells, comprising: (A) inserting an exogenous sequence into a cleavage site on the genome of a cell in the presence of donor DNA containing an exogenous sequence containing an exogenous homologous sequence, wherein the exogenous homologous sequence is homologous to a sequence present via a target region (endogenous homologous sequence); (B) selecting cells having the insertion of the exogenous sequence into the target region; and (C) deleting a sequence between the exogenous homologous sequence and the endogenous homologous sequence, thereby obtaining cells with a deleted target region.

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