Method for preparing cell containing modified DNA, method for preparing organism containing said cell, and method for producing gene product

Abstract

Provided is a means for constructing a modified genomic DNA which has a nucleotide sequence different from an existing genomic DNA sequence, contains a modified gene capable of expressing a polypeptide, a functional RNA, or both, and does not contain exogenous DNA.　This method comprises: performing, at least twice, (a) a step for specifically cleaving genomic DNA of a cell into at least two target sequences by means of a sequence-specific nuclease, and linking the resultants through a DNA double-stranded cleavage repair by the cell; and deleting at least two regions from an existing gene and non-coding regions adjacent thereto to thereby produce a modified gene.

Description

Methods for producing cells containing modified DNA, methods for producing organisms containing the cells, and methods for producing gene products

[0001] The present invention relates to a method for producing cells containing modified DNA that are free of foreign DNA, cells produced by the method, and methods for producing gene products using the cells or organisms containing the cells.

[0002] Genome editing techniques that directly manipulate DNA are known to modify the genome of cells or organisms. For example, the CRISPR / Cas system, zinc finger nucleases (ZFNs), TALENs (transcription activation-like effector nucleases), meganucleases, etc. can be used to recognize and selectively cleave specific sequences in the genome. It is known that the ends of the cleaved genomic DNA are reconnected by the cell's inherent DNA double-strand break (DSB) repair mechanism. When genomic DNA is cleaved at two locations, the region between them is excised and reconnected, allowing for the artificial creation of genomic DNA lacking the excised region. It is known that the DSB repair process can result in deletion, insertion, substitution, etc. of several bases at the cut and ligated sites.

[0003] Mutations introduced by genome editing through deletion manipulation of genomic DNA and DSB repair are essentially indistinguishable from mutations caused by DNA double-strand breaks and repair that occur naturally within cells. Therefore, organisms (including cells) obtained through genome editing that do not introduce exogenous nucleotides are sometimes treated as organisms that do not fall under the category of "genetically modified organisms" (referred to as "non-genetically modified organisms") under the Cartagena Protocol on Biosafety to the Convention on Biological Diversity (Cartagena Protocol). Non-genetically modified organisms are subject to less stringent regulations than genetically modified organisms, potentially offering advantages in terms of distribution, management, and commercial use.

[0004] Patent Document 1 discloses a method for creating a novel gene in an organism, comprising the steps of simultaneously creating DNA breaks at two or more different specific sites in the genome of the organism, the specific sites being genomic sites that can separate different genetic elements or different protein domains, and the DNA breaks creating a new combination of various genetic elements or various protein domains that differ from the original genomic sequence and are linked to each other by non-homologous end joining (NHEJ) or homology repair, thereby creating a new gene.

[0005] Special Publication No. 2022-553598

[0006] Liu G. et al. Molecular Cell, 2022, Vol.82, pp.333-347Gao C. Cell, 2021, Vol.184, pp.1621-1635

[0007] In order to constitutively produce proteins that are not naturally present in cells or that are expressed in low amounts in cells or organisms, a common method is to introduce foreign DNA into cells to create genetically modified organisms. However, genetically modified organisms are subject to legal restrictions under the Cartagena Protocol, and are commercially disadvantaged in that they are subject to strict regulations on their distribution, management, and use.

[0008] Patent Document 1 describes a technical concept of linking promoters or different protein domains of different genes present on existing genomic DNA solely through deletion manipulation of genomic DNA to create a new functional gene. While Patent Document 1 describes cutting genomic DNA at two or more locations (i.e., excising fragments), it does not specify repeating deletion manipulations two or more times, and does not specifically describe creating a sequence dissimilar to any gene on genomic DNA through repeated deletion manipulations, which differs from the present invention. Rather, Patent Document 1 states that gene editing tools such as CRISPR / Cas9 can be used for knockout, but because they mutate existing genes without generating new genes, it is thought that it is difficult to meet certain production needs (see

[0004] of Patent Document 1).

[0009] On the other hand, the present invention aims to provide a means for constructing modified DNA having a nucleotide sequence different from an existing genomic DNA sequence, which contains a modified gene capable of expressing a polypeptide, a functional RNA, or both, and which does not contain any foreign DNA.

[0010] Another objective of the present invention is to provide a new means for constructing genomic DNA that has been modified by deleting a region of genomic DNA that does not contain foreign DNA, and that allows the introduction of foreign DNA and the subsequent removal of the foreign DNA and deletion of the genomic DNA to be confirmed in a simple manner.

[0011] As a result of extensive research, the present inventors have found that a cell containing genomic DNA that does not contain foreign DNA and that contains a modified gene capable of expressing a polypeptide, a functional RNA, or both, can be produced by a method comprising: (a) performing at least twice the steps of specifically cleaving the DNA of a cell at at least two target sequences using a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene to produce a modified gene, and have completed the present invention.

[0012] As a result of further intensive research, the present inventors have found that the removal of foreign DNA and the deletion of genomic DNA can be confirmed in a simple manner by the following method, and that foreign DNA-free genomic DNA modified by deletion manipulation can be constructed, thereby completing the present invention: (i) a step of cleaving the genomic DNA of a cell with a sequence-specific endonuclease, and inserting foreign DNA containing a marker gene into a specific region in the genomic DNA to be deleted (deleted region) or adjacent to the deleted region, or to replace the deleted region, by repair through homologous recombination (HDR) or non-homologous recombination; (ii) a step of cleaving the genomic DNA at at least two sites with a sequence-specific endonuclease to excise the region containing the foreign DNA from the genomic DNA, and repairing DNA double-strand breaks (DSBs) in the cell to obtain foreign DNA-free genomic DNA; and (iii) a step of selecting cells based on the presence or absence of the marker gene, wherein a part of the genomic DNA is excised in step (i), step (ii), or both, The method, wherein step (iii) is performed after step (i), after step (ii), or both.

[0013] The present invention includes, but is not limited to, the following aspects: [1] A method for producing a cell (excluding human cells) containing a modified gene that does not contain foreign DNA, comprising: (a) performing at least twice the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby generating a modified gene that differs in nucleotide sequence from the existing gene; the DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions; and at least one linked fragment that is a region sandwiched between the two deleted regions and remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprise the entire length or a portion of at least one of the linked fragments; and the method is configured to express a polypeptide, a functional RNA, or both from the modified gene. [2] The method according to [1], further comprising: (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism capable of mating with the organism from which the first cell is derived, or into a second cell that can infect the first cell.[3] A cell (excluding human cells) in which genomic DNA has been modified, wherein the genomic DNA comprises, compared to wild-type genomic DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type genomic DNA, at least one linked fragment corresponding to a region in the existing gene sandwiched between the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the cell is configured to express a polypeptide, functional RNA, or both from the modified gene. [4] A cell (excluding human cells) containing a modified gene, obtained by a method comprising the steps of: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both.[5] A cell (excluding human cells) containing a modified gene, comprising: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene having a nucleotide sequence different from that of the existing gene; the DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both of them; and (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell was derived or into a second cell derived from an organism that can mate with the organism from which the first cell was derived.[6] A method for producing modified DNA that does not contain foreign DNA, comprising: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences using a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both. [7] The method according to [6], further comprising: (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell is derived or into a second cell derived from an organism capable of mating with the organism from which the first cell is derived.[8] A modified gene obtained from modified DNA that does not contain foreign DNA, comprising the steps of: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both. [9] Modified DNA comprising, compared to wild-type DNA of a cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type DNA, at least one linked fragment corresponding to a region in the existing gene sandwiched between the deleted regions (deleted regions), and a gene whose nucleotide sequence is modified relative to the existing gene due to the deletion.

[10] A cell produced by any of the methods described above, or an organism (excluding humans) comprising a cell described in any of the methods described above.

[11] A method for producing a gene product, comprising a step of expressing a gene product encoded by the modified gene in a cell produced by any of the methods described above, a cell described in any of the methods described above, or an organism (excluding humans) comprising such a cell.

[12] A marker for genetic testing comprising a modified gene in DNA produced by any of the methods described above, or part or all of the modified gene described in any of the methods described above.

[13] A method for producing cells (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, wherein the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deleted site is performed at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not contained in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and a step of designing an artificially modified gene configured to express a polypeptide, a functional RNA, or both of the modified gene from the modified gene; (Ia3) a step of preparing the artificially modified gene; and (c) a step of introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.

[14] A cell (excluding human cells) containing a modified gene, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deletion site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises at least two deleted regions that are not included in the modified DNA due to the deletion, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and (Ia3) a step of preparing the artificially modified gene: (c) introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.

[15] A method for producing modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, wherein the step of specifically deleting a target sequence from DNA of a first cell and ligating both outer ends of the deletion site is performed at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and (Ia3) a step of preparing the artificially modified gene: (c) introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.

[16] A modified gene obtained from modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificial modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deletion site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not contained in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments has a length of 1 to 150 base pairs; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and a step of designing an artificial modified gene configured to express a polypeptide, a functional RNA, or both of the modified gene from the modified gene; and (Ia3) A modified gene obtained by a method comprising the step of preparing the artificially modified gene.

[17] An organism (excluding humans) comprising a cell prepared by the method described in

[13] above, or the cell described in

[14] , modified DNA obtained by the method described in

[15] , and modified gene described in

[16] .

[18] A method for producing a gene product, comprising the step of expressing a gene product encoded by the modified gene in a cell prepared by the method described in

[13] above, or the cell described in

[14] , modified DNA obtained by the method described in

[15] , or an organism (excluding humans) comprising the modified gene described in

[16] .

[19] A marker for genetic testing, comprising modified DNA obtained by the method described in

[15] above, and part or all of the modified gene described in

[16] .

[20] A method for producing a cell containing modified DNA that is free of foreign DNA, comprising: (i-1a) allowing foreign DNA to coexist with the DNA of the cell, wherein the foreign DNA contains a marker gene and is configured so that the foreign DNA can be inserted into or adjacent to a specific region (deleted region) in the DNA of the cell; (i-2a) cleaving at least one end or the interior of the deleted region with a sequence-specific endonuclease and inserting the foreign DNA into or adjacent to the deleted region; and (iia) cleaving the DNA of the cell at least two sites with the sequence-specific endonuclease to excise the deleted region and the foreign DNA together from the DNA of the cell, and generating modified DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) selecting the cell based on the presence or absence of the marker gene, wherein step (iii) is performed after step (i-2a), after step (iia), or both.

[21] A method for producing a cell containing modified DNA that is free of foreign DNA, comprising: (ib) a step of allowing foreign DNA to coexist with the genomic DNA of the cell, wherein the foreign DNA contains a marker gene, and a step of cleaving both ends of a specific region (deleted region) in the DNA of the cell with a sequence-specific endonuclease and inserting the foreign DNA to replace the deleted region; and (iib) a step of cleaving the genomic DNA at at least two sites with the sequence-specific endonuclease to excise the foreign DNA from the DNA of the cell, and generating modified DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) a step of selecting the cell based on the presence or absence of the marker gene, wherein step (iii) is performed after step (ib), after step (iib), or both.

[22] A method for producing an organism, comprising a step of including a cell containing modified genomic DNA obtained by the production method described in

[20] or

[21] above in an organism.

[0014] The cell production method of the present invention can produce cells that contain modified DNA containing a modified gene capable of expressing a polypeptide, functional RNA, or both, without containing any foreign DNA. Such cells and organisms containing the cells may be subject to less stringent legal restrictions in terms of distribution, storage, use, etc. than genetically modified organisms. Furthermore, the cells or organisms can be used to produce a gene product encoded by the modified gene.

[0015] 1 is a schematic diagram showing an example of a method for producing modified genomic DNA when step (a) is performed once each. FIG. 2 is a schematic diagram showing an example of a method for producing modified genomic DNA when step (a) is performed multiple times at once. FIG. 3 is a diagram showing an example of a method for producing modified genomic DNA in which a modified gene is produced by deleting a portion of the coding region of an existing gene. FIG. 4 is a diagram showing an example of a method for producing modified DNA in which all of the components necessary for protein production are reconstructed, and a method for producing a promoter region shortened from an existing promoter region. FIG. 5 is a schematic diagram showing an example of a method for producing modified genomic DNA, including an embodiment in which foreign DNA containing a marker gene (GFP, ampicillin resistance gene (Amp)) is inserted when the deleted region is excised, and then the foreign DNA is removed. FIG. 6 is a schematic diagram showing an example of a method for producing modified genomic DNA, including an embodiment in which foreign DNA containing a marker gene (RFP, kanamycin resistance gene (Kan)) is inserted, and then the foreign DNA and the deleted region are removed together. FIG. 7 is a diagram showing the sequences of the portions of the sense strand PAM sequence and antisense strand PAM sequence of SpCas9-NG (PAM sequence: NG) on the sense strand. 1 shows specific examples of positions (italicized G or C) at which the type of nucleotide is restricted as a PAM sequence for each cleavage site (↓ or ↑) when SpCas-NG is used to cleave both ends of linked fragments of various lengths. Linked fragments are indicated by underlined sections surrounded by two cleavage sites. 1 shows specific examples of positions (italicized G or C) at which the type of nucleotide is restricted as a PAM sequence for each cleavage site (↓ or ↑) when SpCas-NG is used to cleave both ends of linked fragments of various lengths. Linked fragments are indicated by underlined sections surrounded by two cleavage sites. 1 shows classifications (types 1 to 4) based on the position of the PAM sequence relative to the linked fragments when both ends of the linked fragments are cleaved. Linked fragments are indicated by underlined sections surrounded by two cleavage sites. 1 shows classifications (types 5 and 6) based on the position of the PAM sequence relative to the cleavage site when the cleavage site closest to the 5' or 3' end is cleaved. FIG. 1 is a schematic diagram showing an example of an embodiment in which a first linked fragment is included in a target sequence of CRISPR / Cas9.

[0023] FIG. 1 is a schematic diagram showing an example of an embodiment in which step (a) is performed multiple times simultaneously using a guide RNA in which a first linked fragment is included in a target sequence. In Example 1, this is a schematic diagram showing the positional relationship of the linked fragments in an existing gene and a modified gene, and a diagram showing the mapped search sequence (a fragment consisting of the linked fragments and a PAM sequence). In Example 2, this is a schematic diagram showing the positional relationship of the linked fragments in an existing gene and a modified gene, and a diagram showing the mapped search sequence (a fragment consisting of the linked fragments and a PAM sequence). In Example 3, this is a schematic diagram showing the positional relationship of the linked fragments in an existing gene and a modified gene, and a diagram showing the mapped search sequence (a fragment consisting of the linked fragments and a PAM sequence). In Example 4, this is a schematic diagram showing the positional relationship of the linked fragments in an existing gene and a modified gene. In Example 4, this is a diagram showing the mapped search sequence (a fragment consisting of the linked fragments and a PAM sequence). In Example 5, this is a schematic diagram showing the positional relationship of the linked fragments in an existing gene and a modified gene, and a diagram showing the mapped search sequence (a fragment consisting of the linked fragments and a PAM sequence). (c) A diagram schematically showing one embodiment included in the step. (c) A diagram schematically showing another embodiment included in the step. (c) A diagram schematically showing yet another embodiment included in the step. (c) A diagram schematically showing yet another embodiment included in the step. (c) A diagram schematically showing yet another embodiment included in the step. (c) A diagram schematically showing yet another embodiment included in the step. In Example 7, a diagram showing the positional relationship of the linked fragments in the existing gene and the modified gene, and a diagram showing the mapped search sequence (a fragment consisting of the linked fragments and the PAM sequence). (c) A diagram schematically showing infection (Ic1) in one embodiment included in the step. A diagram showing an example of an embodiment in which foreign DNA is inserted adjacent to the deleted region, and then the foreign DNA and the deleted region are excised. A diagram showing an example of an embodiment in which foreign DNA is inserted into the deleted region, and then the foreign DNA and the deleted region are excised.FIG. 1 shows an example of an embodiment in which foreign DNA is inserted to replace a part of the deleted region by cutting two sites within the deleted region, and then the foreign DNA and the deleted region are excised. FIG. 1 shows an example of an embodiment in which foreign DNA is inserted to replace the deleted region, and then the foreign DNA is excised. FIG. 1 shows a schematic diagram illustrating the positional relationship of the ligated fragments in the existing gene and the modified gene in Example 8. FIG. 13 shows a method for fusing a His6-tag (HHHHHH) downstream of the C-terminus of LacZ in Escherichia coli. FIG. 14 shows a schematic diagram illustrating the step of introducing the modified gene in the first cell into the second cell in Example 8. FIG. 15 shows a schematic diagram illustrating the positional relationship of the ligated fragments in the existing gene and the modified gene in Example 9. FIG. 16 shows a schematic diagram illustrating the step of introducing the modified gene in the first cell into the second cell in Example 9.

[0016] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, the drawings are merely examples, and the present invention is not limited to the embodiments shown in the drawings.

[0017] Unless otherwise specified, nucleotide sequences are described herein from the 5' to the 3' end, and amino acid sequences are described herein from the N-terminus to the C-terminus.

[0018] In this specification, "N" in a nucleotide sequence represents any one of the bases adenine, guanine, cytosine, and uracil in the case of RNA, and any one of the bases adenine, guanine, cytosine, and thymine in the case of DNA.

[0019] As used herein, the term "gene" refers to a polynucleotide that is operably linked to an appropriate control sequence (e.g., a promoter, an enhancer, etc.) and contains a transcribed region that is transcribed from DNA to RNA. A gene in which a polypeptide is expressed by translating the transcribed mRNA may be referred to as a "polypeptide gene" herein. A polypeptide gene contains a ribosome binding sequence and at least one open reading frame (ORF) that is translated. Regions that are not transcribed or translated, such as pseudogenes, are not considered genes. As used herein, gene sequences are represented by the sequence of the sense strand, unless otherwise specified.

[0020] That is, gene products resulting from gene expression may include polypeptides as well as functional RNA. Examples of functional RNA include, but are not limited to, small RNA (such as siRNA, miRNA, and piRNA), tRNA, rRNA, functional tRNA-derived RNA fragments (tRFs; see Alves CS et al., Front. Mol. Biosci., 2021, 8:638911), antisense RNA, ribozymes, and RNA aptamers. A gene may express two or more polypeptides, functional RNAs, or both.

[0021] As used herein, the term "existing gene" refers to a specific gene that is present in the DNA of a cell before modification and whose sequence is to be modified in the method for producing a cell containing modified DNA, modified genomic DNA, or modified extranuclear DNA of the present invention.

[0022] As used herein, the term "modified gene" refers to a gene that results from modifying the nucleotide sequence of an existing gene in a method for producing cells containing modified DNA, modified genomic DNA, or modified nuclear DNA of the present invention.

[0023] In this specification, unless otherwise specified, the "5'-end" or "5'-end" representing a position on a gene or a position relative to a gene refers to the 5'-end or 5'-end of the sense strand of the gene. In this specification, the "3'-end" or "3'-end" representing a position on a gene or a position relative to a gene refers to the 3'-end or 3'-end of the sense strand of the gene, unless otherwise specified.

[0024] As used herein, the term "non-coding region" refers to a region in genomic DNA that does not correspond to a gene. For example, a region encoding a pseudogene is included in the non-coding region.

[0025] As used herein, the term "control sequence" refers to a nucleotide sequence that is necessary for or regulates the expression (transcription or translation) of a gene. Examples of control sequences in prokaryotes include promoters, operator sequences, ribosomal binding sequences, and transcription termination sequences (terminators). Examples of control sequences in eukaryotic cells include promoters, polyadenylation signals, enhancers, transcription termination sequences, internal ribosome entry sites (IRES), and the like.

[0026] As used herein, the term "5'-untranslated region" or "5'-UTR" refers to the region of nucleotides encoding the untranslated region at the 5' end of an mRNA molecule. As used herein, the term "3'-untranslated region" or "3'-UTR" refers to the region of nucleotides encoding the untranslated region at the 3' end of an mRNA molecule.

[0027] As used herein, a "coding region" refers to a region that encodes the amino acid sequence of a polypeptide and is translated into a polypeptide when placed under the control of appropriate control sequences, including a promoter. Unless otherwise specified, the "coding region" of a particular polypeptide refers to a region that encodes the entire amino acid sequence of the polypeptide or a region that encodes a part of the amino acid sequence of the polypeptide.

[0028] As used herein, "exogenous" or "foreign" gene or nucleotide refers to a gene or nucleotide not found in a cell prior to genetic manipulation and that has been or will be introduced into the cell by genetic manipulation. Alternatively, "exogenous" or "foreign" DNA, gene, or nucleotide refers to DNA, gene, or nucleotide that is different from DNA, gene, or nucleotide that may be introduced from a first cell to a second cell by events such as natural hybridization (including cross-breeding such as hybridization) or naturally occurring infection. That is, "exogenous" or "foreign" DNA, gene, or nucleotide does not include DNA, gene, or nucleotide that may be introduced from a first cell to a second cell by events such as natural hybridization (including cross-breeding such as hybridization) or naturally occurring infection. Here, "naturally occurring infection" means that, to the extent that it is naturally possible, DNA or a part thereof carried by a first cell is transferred to a second cell and incorporated into the DNA of the second cell; this is not limited to, for example, the first cell may be an Agrobacterium, and the second cell may be a plant cell that is infected with the Agrobacterium and incorporates DNA (mainly extranuclear DNA) in the Agrobacterium.

[0029] As used herein, the term "genetically modified organism" refers to an organism (including organisms and cells) that contains the above-mentioned "exogenous" or "foreign" DNA, genes, or nucleotides. Therefore, even if, for example, foreign DNA is inserted into genomic DNA or foreign plasmid DNA is introduced into a cell, it does not fall under the category of a genetically modified organism as long as the foreign DNA is completely removed from the final cell and the organism comprising the cell.

[0030] As used herein, the term "non-genetically modified organism" refers to an organism that does not fall under the category of genetically modified organisms.

[0031] As used herein, the term "functional analog" refers to a polypeptide that has an amino acid sequence similar to that of a given polypeptide (e.g., 95% or more, 98% or more, 99% or more, or 99.9% or more amino acid identity) and that exhibits the same qualitative function. Specific examples include polypeptides into which amino acid mutations that do not affect the activity of a given polypeptide have been introduced.

[0032] As used herein, the identity (%) of an amino acid sequence or a nucleotide sequence is the "identity" value in an alignment performed in Protein BLAST or Nucleotide BLAST of NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) with Align two or more sequences selected and default parameters.

[0033] As used herein, the term "cell" refers to a eukaryotic or prokaryotic cell, preferably a eukaryotic cell. As used herein, the term "cell" refers to a cell produced by the production method of the present invention and includes proliferated cells (replicates) obtained by culturing or the like after production.

[0034] As used herein, the term "first cell" refers to a cell in which the modified DNA is directly manipulated. The second cell is a cell of the same species as the organism from which the first cell originates, or a cell derived from a mating-capable organism. Cells of the same species may be derived from the same individual organism as the first cell, or may be derived from a different individual organism of the same species as the first cell. Here, "mating-capable" includes cross-species mating (hybridization). In one embodiment of the method of the present invention, the first cell may be capable of infecting the second cell and introducing extranuclear DNA (e.g., a plasmid) into the second cell.

[0035] Eukaryotes include, for example, animals, plants, fungi, protists, etc., and are preferably animals or plants.

[0036] Examples of animals include mammals such as humans, mice, rats, rabbits, monkeys (chimpanzees, gorillas, orangutans, rhesus monkeys, green monkeys, etc.), sheep, goats, cows, horses, pigs, guinea pigs, dogs, cats, and hamsters; birds such as parakeets and parrots; reptiles such as lizards and snakes; amphibians such as frogs and salamanders; fish such as salmon, tuna, bonito, sea bream, yellowtail, eels, and killifish; and animals of the phylum Arthropoda such as insects and crustaceans.

[0037] Examples of plants include grasses such as wheat, rice, barley, oats, rye, corn, sugarcane, foxtail millet, and barnyard millet; legumes such as soybean, adzuki bean, pea, and kidney bean; solanaceae plants such as tobacco, tomato, eggplant, chili pepper, and potato; cucurbits such as pumpkin, watermelon, cucumber, melon, and oriental melon; roseae plants such as strawberry; seed plants such as Arabidopsis thaliana, buckwheat, cassava, sweet potato, taro, mulberry, pine, cedar, cypress, ginkgo, and eucalyptus; ferns; and bryophytes. Plant cells can be cultured in a medium supplemented with plant hormones to form callus, which can then be further cultured to obtain plants. Callus formation media and media for inducing differentiation into transformed plants are known in the art. The cells may be derived from any of seeds, fruits, cuttings, tubers, tuberous roots, stumps, calluses, protoplasts, and the like.

[0038] Examples of fungi include yeasts such as those of the genus Saccharomyces, Pichia, Schizosaccharomyces, and Candida; filamentous fungi such as those of the genus Rhizopus and Aspergillus; dimorphic fungi such as those of the genus Penicillium; and mushrooms.

[0039] Examples of protists include algae (green algae, red algae, brown algae, cyanobacteria, Euglenophyta, Haptophyta, Cryptophyta, etc.), ciliates, and amoeba.

[0040] Examples of prokaryotes include bacteria (Escherichia coli, Bacillus subtilis, Agrobacterium (such as the genus Rhizobium), thermophilic bacteria (such as the genus Thermus), root nodule bacteria, and cyanobacteria) or archaea.

[0041] The cell is not particularly limited as long as it is genome-editable. In one embodiment, the cell is a cell in tissue isolated from the living body of a human or non-human organism (ex vivo cell; for example, a cell derived from an excised organ, plant leaf, stem, etc.), or an in vitro cell (primary culture cell, passaged cell, cultured cell differentiated from stem cell such as iPS cell, etc.). In one embodiment, the cell is an in vitro cell. In another embodiment, the cell is a cell in the living body of a non-human organism (in vivo). In another embodiment, the cell is a cell in the living body of a human.

[0042] Furthermore, the type of cell is not particularly limited as long as it is genome-editable. When producing a genome-edited organism, germ cells, pluripotent cells (iPS cells, ES cells, etc.), or dedifferentiable cells (e.g., plant cells) are preferred.

[0043] In one embodiment, the cells are cells used in food or in the production of food. Such cells are preferably (i) those contained in food, such as those generally consumed as food as defined in Article 7, Paragraph 2 of the Food Sanitation Act of Japan, or (ii) those generally used in the production of such food. Here, "food" includes not only so-called general foods but also food additives. Specific examples of (ii) include cells used in the production of fermented foods such as soy sauce and sake, as well as cells used for the fermentation production of amino acids, enzymes for food production, other food additives, and the like.

[0044] The method for producing a cell containing modified DNA that does not contain exogenous DNA, which is one embodiment of the present invention, may be a method for modifying intracellular genomic DNA to obtain a modified cell, or a method for modifying extranuclear DNA to obtain a modified cell. The cell may be a modified cell itself (first cell), or a cell into which modified DNA has been transferred (second cell).

[0045] In this specification, step (a) includes an embodiment designated as step (Ia4), but step (Ia4) may be designated as one embodiment of step (a) when distinguishing between the modification of extranuclear DNA. Similarly, step (c) includes step (Ic1), but step (Ic1) may be designated as one embodiment of step (c) when distinguishing between the infection.

[0046] <First Aspect> First, the first aspect of modifying genomic DNA will be described below. [Method for Producing Cells Comprising Modified DNA] One embodiment of the present invention, a method for producing cells containing modified genomic DNA that does not contain foreign DNA (sometimes referred to herein as the "production method of the present invention"), comprises: (a) performing at least twice the steps of specifically cleaving the cellular DNA at at least two target sequences using a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and its adjacent non-coding region to generate a modified gene. In the first aspect, the DNA is genomic DNA.

[0047] According to the production method of the present invention, by linking sequence fragments scattered throughout an existing gene or its adjacent non-coding region, it is possible to construct a modified gene that contains a nucleotide sequence that is significantly different from the sequence contained in the existing gene and is capable of expressing a polypeptide, functional RNA, or both.

[0048] (Step (a)) In one step (a), the genomic DNA is cut at two locations and the region between them is excised. Then, the excised region is deleted in the genomic DNA that has been ligated by DNA double-strand break (DSB) repair in the cell. In some cases, a substitution, insertion, or deletion of one or more base pairs in length is introduced at the site of the cut and ligation during DSB repair. In this specification, the excised region is referred to as the "deleted region." Furthermore, the sites at both ends of the deleted region that are cleaved by the sequence-specific endonuclease are referred to as the "cleavage site."

[0049] By performing step (a) two or more times, two or more deletion regions in the genomic DNA are excised. In this specification, the region sandwiched between the deletion regions and remaining in the modified genomic DNA is referred to as a "ligated fragment." Ultimately, by performing step (a) k times, k deletion regions are lost from the genomic DNA, and k-1 ligated fragments are ligated to construct a modified genomic DNA containing a modified promoter region and / or transcribed region. Figure 1A shows an example of a deletion region and a cleavage site.

[0050] The deletion site on the genomic DNA in the operation differs between one step (a) and another step (a). Thus, in one embodiment, the two target sequences in each step (a) are different from each other.

[0051] Two or more (a) steps may be performed simultaneously or consecutively by a single genetic engineering operation. For example, in Figure 1B, of the three deletion regions, two regions are excised by two simultaneous (a) steps, and the other region is excised by a separate (a) step. In this way, when two or more (a) steps are performed simultaneously, it is not necessary for all (a) steps to be performed simultaneously or consecutively.

[0052] In this specification, one or more steps (a) that are carried out simultaneously or successively by a single genetic engineering operation may be referred to as a "set of steps (a)."

[0053] In the production method of the present invention, the cleavage site does not need to be at a site on the genomic DNA that encodes a site (i.e., a domain boundary) that can separate protein domains of the existing gene. In one embodiment, the existing gene encodes a protein, and in step (a), at least one cleavage and ligation occurs at a position other than a site that encodes a domain boundary of the protein. In a particular embodiment, in step (a), all cleavages occur at positions other than a site that encodes a domain boundary of the protein.

[0054] In one embodiment, the deletion region is present in one existing gene and its adjacent non-coding region in the genomic DNA of the cell. This configuration is preferable because it allows part of the regulatory sequence and transcribed region (e.g., coding region) of the existing gene to be used in constructing the modified gene. In one embodiment, the deletion region includes the non-coding region on the 5' side of the existing gene encoding the polypeptide, and at least one region selected from the group consisting of the regulatory region, coding region, 5' untranslated region, intron, and 3' untranslated region of the existing gene.

[0055] In one embodiment, the deleted region consists of a non-coding region 5' to the existing gene, a promoter region, or a combination thereof. This embodiment is advantageous in that it allows the promoter sequence to be modified and allows the expression of the existing gene to be regulated without affecting other regulatory sequences, splicing factors, and coding regions (see Example 5).

[0056] In one embodiment, the deleted region consists of a transcribed region of an existing gene. When the existing gene encodes a polypeptide, the deleted region consists of, for example, at least one region selected from the group consisting of a coding region, an intron, and a 3'-UTR. This embodiment is preferable because it makes it easy to construct a modified gene while minimizing the introduction of mutations into the regulatory sequence of the existing gene.

[0057] The possible locations of the linked fragments can be understood based on the possible locations of the deleted regions described above. In one embodiment, the linked fragments are present in one existing gene and its adjacent non-coding regions in the genomic DNA of the cell. In a more specific embodiment, the linked fragments include at least one selected from the group consisting of the 5' non-coding region and 3' non-coding region adjacent to the existing gene, and a transcribed region. In one embodiment, the existing gene encodes a polypeptide, and the linked fragments include at least one selected from the group consisting of the 5' non-coding region and 3' non-coding region adjacent to the existing gene, a regulatory region of the existing gene, a coding region, a 5' untranslated region, an intron, and a 3' untranslated region.

[0058] In the production method of the present invention, the length of each linked fragment is not particularly limited, as long as the desired modified gene sequence can be produced by deletion only from the genomic DNA sequence. Longer linked fragments reduce the number of steps (a), thereby saving labor and time. On the other hand, if the linked fragments are too long, it may be difficult to produce a modified gene having a sequence unrelated to existing genes.

[0059] In one embodiment, the length of at least one linked fragment is between 1 and 150 base pairs (bp), for example, 1 bp or more, 2 bp or more, 3 bp or more, 4 bp or more, 5 bp or more, 6 bp or more, 8 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 In one embodiment, the length of at least one linked fragment is 1 to 150 bp, e.g., 1 to 120 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 8 bp, or 1 to 5 bp.

[0060] In one embodiment, the length of at least two, three, four or five linked fragments is between 1 and 150 base pairs (bp), for example, 1 bp or more, 2 bp or more, 3 bp or more, 4 bp or more, 5 bp or more, 6 bp or more, 8 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more. In one embodiment, the length of at least two, three, four, or five linked fragments is 1 to 150 bp, e.g., 1 to 120 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 8 bp, or 1 to 5 bp.

[0061] In one embodiment, the length of all linked fragments is between 1 and 150 base pairs (bp), for example, 1 bp or more, 2 bp or more, 3 bp or more, 4 bp or more, 5 bp or more, 6 bp or more, 8 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 bp or more, 120 bp or more, 130 bp or more, or 140 bp or more, and may be 140 bp or less, 130 bp or less, 120 bp or less, 110 bp or less, 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, 10 bp or less, 8 bp or less, 6 bp or less, or 3 bp or less. In one embodiment, the length of all linked fragments is 1 to 150 bp, and may be, for example, 1 to 120 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 8 bp, or 1 to 5 bp.

[0062] In one embodiment, the total length of the linked fragments of the modified genomic DNA is, for example, 1 to 500 base pairs, preferably 9 to 180 base pairs (bp), for example, 9 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 bp or more, 120 bp or more, 130 bp or more, 140 bp or more, 1 The length may be 50 bp or more, 160 bp or more, or 170 bp or more, and 170 bp or less, 160 bp or less, 150 bp or less, 140 bp or less, 130 bp or less, 120 bp or less, 110 bp or less, 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, 10 bp or less, 8 bp or less, 6 bp or less, or 3 bp or less. In one embodiment, the total length of all linked fragments is 9 to 180 bp, and may be, for example, 9 to 150 bp, 9 to 120 bp, 9 to 100 bp, 9 to 80 bp, 9 to 50 bp, 9 to 30 bp, or 9 to 20 bp. Although it depends on the target sequence of the sequence-specific endonuclease used, when using the CRISPR / Cas9 system, it is possible to construct a modified genomic DNA by only deleting an existing gene, even if the total length of the linked fragments is 150 bp or more (see Example 4).

[0063] In one embodiment, 50% or more, 60% or more, 70% or more, or 80% or more of the linked fragments have a length of 30 bp or less (preferably, 10 to 30 bp or 10 to 20 bp). As shown in Examples 1 to 5, when constructing a sequence different from an existing gene using linked fragments, linked fragments of these lengths tend to be used frequently.

[0064] In one embodiment, the total length of the linked fragments of the modified genomic DNA is 30% or less of the total length of the deleted region, and may be, for example, 20% or less, 10% or less, 5% or less, or 2% or less.

[0065] DSB repair includes non-homologous end joining (NHEJ) repair, in which ends without homologous regions are joined together; microhomology-mediated end joining (MMEJ) repair, in which ends with a complementary sequence of about 5 to 20 bases are joined together; and homology-directed repair (HDR). When no foreign DNA is inserted, repair in step (a) of the present invention is preferably performed by either NHEJ or MMEJ. Among these, ligation by NHEJ repair is more preferred due to its high degree of flexibility in target sequences. As described below, when foreign DNA is inserted together with genomic DNA deletion, MMEJ or HDR can be used for DSB repair.

[0066] During DSB repair, several additional nucleotides may be deleted or inserted at the termini created by the break. Even if such mutations associated with DSB repair exist in the modified genomic DNA, they do not involve the insertion of foreign DNA, and therefore cells or organisms containing the modified genomic DNA are not considered genetically modified organisms.

[0067] In one embodiment, the modified genomic DNA contains mutations associated with DSB repair. The number of mutated bases is defined as the number of substitutions or gaps in sequence alignment with the original genomic DNA. In a more specific embodiment, the modified genomic DNA contains, for example, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3 mutations within 20 base pairs on both sides of at least one cleavage site. However, these mutations may inhibit the intended cleavage or impair the function of the intended gene. Therefore, in the production method of the present invention, it is preferable that mutations associated with DSB repair are not contained in the modified genomic DNA.

[0068] Therefore, it is more preferable that the production method of the present invention includes a step of selecting cells that do not have mutations associated with DSB repair.

[0069] (Existing gene) The existing gene is not particularly limited as long as it is possible to create a modified gene sequence by deletion manipulation, and is preferably a gene that is not essential for cell survival, or a gene that is essential for cell survival but has redundancy as a result of the existence of multiple similar genes.

[0070] In one embodiment, the existing gene is a gene for a highly expressed protein. This embodiment is preferable because the promoter region and ribosome binding sequence can be used as is, and the desired modified gene can be produced with a reduced number of operations in step (a). Examples of such highly expressed protein genes include genes that express proteins in wild-type cells at an amount that accounts for 10% or more, 15% or more, 20% or more, or 30% or more of the total protein amount. Examples of such genes include structural proteins (e.g., collagen) in animal cells and storage proteins (e.g., seed storage proteins) in plant cells.

[0071] (Modified Gene) Modified genomic DNA includes a modified gene configured to express a polypeptide, a functional RNA, or both. The region of the genomic DNA before modification that is included in the modified gene is not particularly limited. In one embodiment, the modified gene comprises or consists of at least one selected from the group consisting of the non-coding regions on the 5'- and 3'-ends of an existing gene, a regulatory region and a transcribed region of the existing gene. In one embodiment, the modified gene comprises or consists of at least one selected from the group consisting of the non-coding regions on the 5'- and 3'-ends of an existing gene, a regulatory region, a coding region, an intron, a 5'-UTR and a 3'-UTR of the existing gene.

[0072] The region of the genomic DNA before modification contained in the promoter region of the modified gene is not particularly limited. In one embodiment, it consists of at least one type selected from the group consisting of the promoter region of an existing gene and the non-coding region adjacent to the 5' side thereof. In such a case, the risk of deleting sequences downstream of the promoter region of the existing gene (e.g., ribosome binding sequence, transcription regulatory sequence) can be reduced, and modification can be achieved by performing step (a) a fewer number of times.

[0073] The region of the genomic DNA before modification contained in the transcribed region of the modified gene is not particularly limited. In one embodiment, the transcribed region of the modified gene consists of the transcribed region of an existing gene. In such a case, it is preferable because it may be possible to use the promoter region of the existing gene as is.

[0074] In one embodiment, the existing gene and the modified gene encode a polypeptide, and the coding region of the modified gene comprises at least one selected from the group consisting of the coding region, intron, 5'-UTR, and 3'-UTR of the existing gene, more preferably at least one selected from the group consisting of the coding region, intron, and 3'-UTR. This is preferable in that it may be possible to utilize the promoter region and ribosome binding sequence of the existing gene as is. In one embodiment, the promoter region, the coding region, or both of the modified gene comprise the full length or a portion of at least one linked fragment. In one embodiment, the coding region of the modified gene comprises the full length or a portion of at least one linked fragment (see FIG. 2 and Examples 1 to 4 for specific examples).

[0075] When the modified gene encodes a polypeptide, a start codon or stop codon of the modified gene may be generated by the ligation in step (a) (see, for example, Example 2-2 in Figure 2 and Examples 1 to 4). The ligation may result in a frameshift, and the frameshift may generate a start codon or stop codon in a region different from that of the existing gene. When a new start codon or stop codon of the modified gene is generated, the start codon or stop codon of the existing gene may be removed by deletion, or the start codon or stop codon of the existing gene may be replaced with another codon during ligation (see, for example, Example 2-2 in Figure 2).

[0076] It is also possible to form a coding region longer than the coding region of an existing gene by step (a) (e.g., Example 2-2 in Figure 2, Example 2). In such an embodiment, if the codon reading frame of the existing gene is maintained, the modified gene encodes a fusion protein of the existing gene.

[0077] It is also possible to generate a coding region that is shorter than an existing gene, as shown in Examples 2-3 of FIG. 2 and Examples 1, 3, and 4.

[0078] In one embodiment, the promoter region of the modified gene comprises the entire length or a portion of at least one of the linked fragments (see Example 5 for an example). In this embodiment, a part or all of the promoter region of an existing gene is deleted to obtain a promoter consisting of a different nucleotide sequence. To construct a modified promoter region, for example, an existing promoter region and its adjacent 5'-terminal non-coding region can be used.

[0079] In one embodiment, the sequence of the promoter region of the modified gene can be a promoter with higher transcription activity than that of the existing gene. This embodiment is preferable because the modified gene obtained by the production method of the present invention has higher transcription activity and is capable of producing a modified gene product. The transcription level of the modified gene can be, for example, 1.1 times or more, 1.2 times or more, 1.5 times or more, 1.8 times or more, 2 times or more, or 3 times or more relative to the transcription level of the existing gene.

[0080] In one embodiment, the promoter of the modified gene has a sequence different from that of the promoter of the existing gene in order to increase the expression level of the modified gene product. Suitable examples of such promoters include: for human cells, the CMV promoter, EF1α promoter, SV40 promoter, RSV promoter, etc.; for plant cells, the RuBisco promoter, ADH promoter, cauliflower mosaic virus (CaMV) 35S promoter, CaMV19S promoter, El2 omega promoter, NOS promoter, etc.; for yeast cells, the Gal1 / 10 promoter, PGK promoter, ADH promoter, PHO5 promoter, etc.; and for Escherichia coli, the T7 promoter, trp promoter, lpp promoter, recA promoter, etc.

[0081] As shown in the following embodiments, if the length of the region formed by the linked fragments in the modified DNA (i.e., the total length of the linked fragments) is a nucleotide sequence encoding amino acids of approximately 60 residues or less, or a nucleotide sequence of 180 base pairs or less, the number of cleavage steps and the number of (a) steps required to obtain modified genomic DNA can be reduced. For example, the number of cleavage sites can be 100 or less, and the number of (a) steps can be 50 or less; the number of cleavage sites can be 50 or less, and the number of (a) steps can be 25 or less; the number of cleavage sites can be 50 or less, and the number of (a) steps can be 20 or less; the number of cleavage sites can be 20 or less, and the number of (a) steps can be 10 or less; or the number of cleavage sites can be 10 or less, and the number of (a) steps can be 5 or less.

[0082] When the existing gene and the modified gene encode a polypeptide, and the coding region of the modified gene includes the entire length or a portion of at least one of the linked fragments, it is preferable that the entire length or a portion of the linked fragment encodes a modified polypeptide region that is not present in the polypeptide region of the existing gene. For example, when a linked fragment of base pairs is present in the coding region of the modified gene, the length of the modified polypeptide region is expressed as an integer length rounded down to 1 / 3 m. In certain embodiments, the length of the modified polypeptide region is 3 to 60 or 3 to 50 amino acids, preferably 3 to 40, 3 to 30, 3 to 20, 3 to 15, or 3 to 10 amino acids.

[0083] In one embodiment, the modified polypeptide region comprises, for example, a functional peptide sequence, an enzyme cleavage sequence, or both. Here, the functional peptide sequence is not particularly limited, but includes, for example, growth factors (epidermal growth factor (EGF), platelet-derived growth factor (PDGF) peptide, etc.), peptide hormones (insulin, glucagon, vasopressin, oxytocin, growth hormone, gastrin, cholecystokinin, secretin, atrial natriuretic peptide (ANP), melanocyte-stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), angiotensin, orexin, endothelin, ghrelin, etc.), peptide pheromones; plant stem cells; Examples of such peptides include signal peptides (CLE41 / 44 (TDIF), EPFL4 / 6, CLE9 / 10, CLE25, CLE46, etc.); peptide sequences having functions such as blood pressure lowering (angiotensin-converting enzyme (ACE) inhibitory peptides, etc.), mineral absorption promotion, cholesterol regulation, immunomodulation, antioxidant, anti-fatigue, anti-stress, β-amyloid toxicity alleviation, bifidobacterium growth promotion, taste, vaccine (for example, peptides containing an epitope sequence of a tumor-associated antigen or a viral antigen); and affinity (6xHis tag, etc.).

[0084] The modified polypeptide region may further comprise a linker sequence.

[0085] In one embodiment, the full-length polypeptide encoded by the modified gene is 50% or more, for example, 60% or more, 70% or more, 80% or more, or 90% or more, of the full-length polypeptide encoded by the existing gene. A typical example of such an embodiment is one in which the modified gene encodes a fusion protein in which a short modified polypeptide region is added to the N-terminus, C-terminus, or internal region of a protein or domain thereof encoded by the existing gene.

[0086] In another embodiment, the full length of the polypeptide encoded by the modified gene is 50% or less, e.g., 40% or less, 30% or less, 20% or less, or 10% or less, of the full length of the polypeptide encoded by the existing gene. A typical example of such an embodiment is one in which the full length of the coding region of the modified gene consists of a nucleotide sequence that encodes a short modified polypeptide that differs from the sequence of the existing gene.

[0087] (Sequence-specific endonuclease, target sequence) To delete a region in genomic DNA in step (a), the genomic DNA is cleaved with a sequence-specific endonuclease specific to the target sequence. As used herein, the term "target sequence" refers to a sequence that the sequence-specific endonuclease needs to identify and cleave.

[0088] The sequence-specific endonuclease is not particularly limited as long as it can cleave a target sequence in a target cell, but it is preferable that it recognizes and cleaves a target sequence that is unique to the genomic DNA of the cell (for example, a specific target sequence of 16 or more bases or 20 or more bases). Examples of such endonucleases include the CRISPR / Cas system, zinc finger nucleases (ZFNs), TALENs (transcription activation-like effector nucleases), meganucleases, etc. The sequence-specific endonuclease may be a wild-type enzyme or a modified mutant.

[0089] The sequence-specific endonuclease used may be of a different type or the same type for each target sequence and each step (a). From the viewpoint of simplifying the operation, it is preferable that all the sequence-specific endonucleases are of the same type.

[0090] If the cell is a eukaryotic cell, the sequence-specific endonuclease preferably contains at least one nuclear localization signal (NLS).

[0091] Among these, the CRISPR / Cas system is preferred as a sequence-specific endonuclease because it can easily impart specificity to a specific target sequence. The CRISPR / Cas system includes a Cas protein having endonuclease activity and a guide RNA that specifies the target sequence. The Cas pairs with the guide RNA and cleaves nucleotides that have a protospacer adjacent motif (PAM) sequence at a specific position. The Cas protein and guide RNA may be naturally occurring or may be a combination that does not exist in nature.

[0092] Cas9 or Cas12a (Cpf1) are preferred as Cas proteins because they have DNA cleavage activity and pinpoint cleavage activity in target sequences. CRISPR / Cas9 forms blunt ends regardless of whether the PAM sequence is contained on the sense strand or antisense strand. Therefore, Cas9 has the advantage of being less constrained by target sequences than other Cass.

[0093] In nature, CRISPR / Cas9 contains crRNA and tracrRNA as guide RNA components, but the production method of the present invention more preferably uses a system that uses single-stranded guide RNA (sgRNA), in which Cas, tracrRNA, and the target sequence are combined into a single RNA.

[0094] When using the CRISPR / Cas9 system, the Cas9 protein, guide RNA, etc. may be selected based on known literature and be compatible with the target cells. The Cas9 protein is preferably Cas9 from the genus Staphylococcus, and more preferably Cas9 from Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus. These Cas9 proteins may be wild-type or mutant, as long as they have target sequence specificity and DNA double-strand cleavage activity.

[0095] The nucleotide restriction by the PAM sequence complicates the design of guide RNA, so Cas9, which has a small number of positions restricted to specific nucleotides in the PAM sequence, is particularly suitable for use. Examples of such Cas9 include Cas9 derived from Streptococcus pyogenes (SpCas9), which recognizes NGG as the PAM sequence; a mutant of SpCas9 (SpCas-NG) which recognizes NG as the PAM sequence (Nishimasu, H. et al., 2018, Science, Vol. 361, pp. 1259-1262); xCas9-3.7 (Hu, JH et al., 2018, Nature, Vol. 556, pp. 57-63); ScCas9 which recognizes NNG (Chatterjee, P. et al., 2018, Sci. Adv. Vol. 4, eaau0766); and Sc++ (Chatterjee, P. et al. Nat. Biotechnol., 2020, Vol. 38, pp.1154-1158), SpG that recognizes NGN (Walton RT et al., 2020, Science, Vol.368, pp.290-296), or functional analogs thereof.

[0096] In SpCas9 and SpCas-NG, the target sequence consists of 5'-N(17)-(Cas cleavage site)-NNN-PAM sequence-3', where N(17) represents any 17 nucleotides. In this specification, the three nucleotides immediately preceding the PAM sequence are referred to as the spacer sequence.

[0097] When the sequence-specific nuclease is a zinc finger nuclease (ZFN), TALEN (transcription activation-like effector nuclease), or meganuclease, the cleavage in step (a) can be carried out by allowing two nucleases with different target sequences to coexist in a cell.

[0098] When target sequence-specific cleavage is performed using the CRISPR / Cas system, for example, one type of Cas and two types of guide RNAs that pair with the two target sequences to be cleaved can be used in one (a) step. However, the Cas used in each (a) step does not need to be the same. When using multiple Cass, a target sequence containing an appropriate PAM sequence can be selected according to the Cas.

[0099] (Step of selecting cells not containing off-target mutations, step of removing off-target mutations) In addition to the above step (a), the production method of the present invention preferably further comprises step (b) of selecting cells not containing off-target mutations. As used herein, the term "off-target mutation" refers to a mutation that occurs as a result of cleavage and DSB repair at a site that was not originally intended to be cleaved.

[0100] In one embodiment, step (b) is performed after step (a) has been performed one or more times, for example, in a single operation. In one embodiment, step (b) is performed after all deleted regions have been excised from the genomic DNA.

[0101] The presence or absence of off-target mutations is determined, for example, by genomic sequencing of the modified genomic DNA.

[0102] The production method of the present invention may further include a step of removing off-target mutations in addition to the above steps (a) and (b). Specific examples of methods for removing off-target mutations include the step of producing an organism containing a modified genome described below, and backcrossing (backcrossing) an organism containing a modified genome with a non-recombinant organism of the same species that does not contain the off-target mutations (e.g., a wild-type organism having cells before modification).

[0103] (Step of Inserting and Removing Foreign DNA from Genomic DNA) The production method of the present invention may further comprise a step of inserting and removing foreign DNA from genomic DNA. As used herein, removal of foreign DNA means that foreign DNA and its replicas are no longer present in the cell. In one embodiment, removal of foreign DNA is carried out by step (a).

[0104] To insert foreign DNA into genomic DNA, for example, a technique can be used that combines genomic DNA cleavage with sequence-specific endonuclease and DNA repair by recombinational repair. Genomic DNA cleavage can be performed at, for example, one or two locations depending on the purpose, as shown in Figures 3A and 3B described below. The two cleavages can be performed, for example, by the cleavage in step (a). Homologous recombination repair (HRD) is preferred for recombinational repair. Regions homologous to sequences on genomic DNA (referred to as the first and second homologous arms) are added to both ends of the foreign DNA so that recombinational repair can occur at appropriate locations.

[0105] The length of the first homology arm and the second homology arm is not particularly limited as long as it is a length that allows foreign DNA to be inserted, and can be, for example, 5 base pairs (bp) or more, 10 bp or more, 20 bp or more, 50 bp or more, 100 bp or more, 200 bp or more, or 500 bp or more, and can be, for example, 10,000 bp or less, 5,000 bp or less, 2,000 bp or less, or 1,000 bp or less.

[0106] In one embodiment, the foreign DNA is inserted into the deleted region or adjacent to the deleted region, so that the foreign DNA can be removed together with the deleted region in step (a).

[0107] In one embodiment, the foreign DNA is inserted to replace part or all of the deleted region, i.e., when the deleted region is excised by cleavage with a sequence-specific endonuclease in step (a), the foreign DNA is inserted by recombinational repair (see, for example, Figure 3A).

[0108] To enable cleavage of the foreign DNA by step (a), the foreign DNA may be provided with a sequence essential for cleavage of the target sequence, such as a PAM sequence.

[0109] In one embodiment, the first or second homology arm comprises the sequence of at least one linked fragment (referred to as a "first linked fragment"), and all or part of the sequence of the linked fragment or genomic DNA (referred to as a "ligated portion") to be ligated to the first linked fragment in step (a) is connected to at least one of the ends of the first linked fragment. According to this embodiment, insertion of foreign DNA can be regulated depending on the ligation between the first linked fragment and the ligated portion (for a similar example for guide RNA, see the section <Design of guide RNA encompassing linked fragments>).

[0110] The length of the first linked fragment is not particularly limited as long as it is within the range of the length of the homology arm. However, from the viewpoint of reducing the risk of recombination, not limited to the ligation of the first linked fragment, the length of the first linked fragment is preferably, for example, 5% or more of the length of the homology arm, more preferably 10% or more, even more preferably 15% or more, and even more preferably 20% or more. From the same viewpoint, it is also preferable that the end of the first linked fragment is relatively close to the end of the foreign DNA. On the other hand, from the viewpoint of reducing the risk of losing the sequence of the first linked fragment due to cutting by nuclease, it is preferable that the end of the first linked fragment is separated from the end of the foreign DNA. Therefore, it is preferable that the end of the first linked fragment is located, for example, 1 to 30 bp, more preferably 3 to 20 bp, and even more preferably 5 to 15 bp from the end of the foreign DNA.

[0111] In a more specific embodiment, the foreign DNA includes a marker gene for cell selection. In such an embodiment, the deleted region containing or adjacent to the foreign DNA can be labeled with the marker gene.

[0112] Cells into which the foreign DNA has been inserted can be selected based on the expression of marker genes.

[0113] Then, after excising the foreign DNA containing the marker gene, or after excising the region containing the marker gene and the deletion region together, cells that do not express the marker gene can be easily selected to select cells in which the deletion region has been excised. Among the embodiments of the present invention, in embodiments containing a short deletion region (e.g., 50 bp or less, 30 bp or less, 20 bp or less, 10 bp or less, 5 bp or less, 3 bp or less, or 1 bp), it may be difficult to distinguish cells in which the deletion region has been excised using PCR-based methods. However, by labeling the deletion region with a marker gene, cells in which the deletion region has been excised can be selected without undergoing complicated processes such as sequencing.

[0114] As a cell selection marker gene, either a positive selection marker gene or a negative selection marker gene can be used. Positive selection marker genes are genes that allow cells to be selected based on their presence, and examples thereof include fluorescent proteins (GFP, YFP, CFP, etc.), drug resistance genes (neomycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, kanamycin resistance gene, sulfonylurea resistance gene (ALS), glyphosate resistance gene (EPSPS), etc.), and reporter enzyme genes (luciferase, β-galactosidase, β-glucuronidase (GUS), dihydrofolate reductase (DHFR), etc.). Negative selection marker genes are genes that allow cells to be selected based on their absence, and examples thereof include genes encoding toxic proteins and suicide genes (HSV-TK, iCasp9, etc.).

[0115] When a fluorescent protein is used as a marker gene, cells can be easily selected on a large scale using a cell sorter or the like. Furthermore, when a drug resistance gene is used as a marker gene, cells having the resistance gene can be selected by culturing the cells in a medium containing the drug. Therefore, a fluorescent protein or a drug resistance gene is more preferable as the marker gene for cell selection used in the production method of the present invention. In particular, when the marker gene is a fluorescent protein, its absence can be confirmed by the fluorescence intensity of the cells, and both the presence and absence of foreign DNA can be confirmed relatively easily, making it particularly preferable.

[0116] Figure 3A shows an example of an embodiment in which the deletion region (the second region in the figure) is deleted together with the insertion of foreign DNA containing a marker gene. The insertion of foreign DNA can be performed during step (a). If foreign DNA that is not maintained intracellularly is used, cells containing genomic DNA in which the second deletion region has been replaced with the marker gene can be obtained by culturing the cells and selecting cells containing the marker gene. Next, the foreign DNA is removed by cutting the DNA at two sites with a sequence-specific nuclease, and then cells lacking the marker are selected, allowing the selection of cells from which the foreign DNA has been removed.

[0117] Figure 3B shows an example of an embodiment in which foreign DNA containing a marker gene is inserted adjacent to the deleted region (the second region in the figure), and then the foreign DNA and the deleted region are removed together. The insertion is achieved by single-site cleavage with a sequence-specific nuclease and HDR. The foreign DNA and the deleted region are then excised and removed together in step (a). Cell selection can be performed in the same manner as in Figure 3A.

[0118] In one embodiment, two or more marker genes are inserted within or adjacent to the deleted region. In such an embodiment, the marker genes are preferably different. The marker genes are preferably inserted by inserting a single foreign DNA containing multiple marker genes into the genomic DNA (see Figures 3A and 3B). However, the marker genes can also be inserted into the genomic DNA by separately carrying multiple foreign DNAs.

[0119] (Step of introducing sequence-specific endonuclease and / or its function-related factor) In one embodiment, the production method of the present invention can further include a step of introducing foreign DNA containing the target sequence-specific endonuclease or its gene into a cell in order to allow the above-mentioned target sequence-specific endonuclease to function in the cell, and a step of removing the foreign DNA.

[0120] When the target sequence-specific endonuclease is a CRISPR / Cas system, the method may further include the step of introducing an appropriate guide RNA or foreign DNA capable of expressing the guide RNA in cells. These steps may be performed simultaneously or separately.

[0121] When the sequence-specific endonuclease and guide RNA are directly introduced into cells, it is preferable to carry out this before each set of steps (a).

[0122] In one embodiment, the foreign DNA is present in the cell in a form separated from the genomic DNA, making it easier to completely remove it from the cell. Such foreign DNA can be introduced into the cell as a vector, such as a plasmid, cosmid, or artificial chromosome. When the foreign DNA is separated from the genomic DNA, the foreign DNA is naturally lost, and the foreign DNA can sometimes be removed by selecting cells that do not contain the foreign DNA.

[0123] In another embodiment, foreign DNA is inserted into genomic DNA. Such foreign DNA can be introduced into cells as, for example, linear DNA, a viral vector, etc. In this embodiment, the production method of the present invention further comprises a step of removing the foreign DNA. Optionally, it may also comprise a step of selecting cells from which the foreign DNA has been removed.

[0124] To confirm that a sample does not contain foreign DNA, for example, primers capable of specifically amplifying foreign DNA can be designed and used to detect the foreign DNA using various PCR methods. The absence of foreign DNA in genomic DNA can also be confirmed by, for example, genome sequencing.

[0125] The gene encoding the target sequence-specific endonuclease of the above-mentioned foreign DNA may be adjusted to have a codon usage frequency similar to that of the cell (so-called codon optimization) in order to improve expression in the cell.

[0126] In addition to the gene of interest, for example, a promoter, an enhancer, an insulator, an intron, a terminator, a poly A addition signal, a selection marker gene, etc. can be ligated to the vector.

[0127] The target gene to be inserted into a vector may be one or more types per vector.

[0128] In this specification, the introduction of substances such as nucleotides and proteins into cells is not particularly limited as long as it is a means capable of delivering RNA and proteins to living cells, and can be carried out by, for example, the liposome method (lipofection, etc.), particle gun (gene gun) method, electroporation method, polyethylene glycol (PEG) method, plasma method (see, for example, WO2018016217), whisker method, laser injection method, etc. When the cells are plant cells, the particle gun method is preferred.

[0129] (Design process of ligated fragments and target sequences) By determining which parts of a genomic DNA region containing an existing gene are to be cleaved by a sequence-specific endonuclease, it is possible to determine the target sequences recognized by each endonuclease. The determination of the cleavage site is not particularly limited, and can be carried out, for example, by the following steps (i) to (v). The design of the target sequence, including the steps described below, may be carried out manually or based on a computer program or a trained machine learning or artificial intelligence (AI) algorithm.

[0130] (i) First, prepare the DNA sequence to be included in the modified gene. When creating a coding region, multiple sequences with different codon combinations can be used as candidates. Furthermore, since frameshifts can occur due to deletions in step (a), a sequence may be generated with a partially frameshifted reading frame. Therefore, if the linked fragments cannot be mapped in order in step (iv) below for the DNA sequence of a coding region, steps (ii) to (iv) can be retried for substitution with synonymous codons or frameshift sequences.

[0131] (ii) The sequence of (i) is fragmented into two or more linked fragments. The boundary between the linked fragments is the cutting point. Here, the smaller the linked fragments, the easier it is to find the target existing gene, but there is a disadvantage in that the number of times step (a) is increased. On the other hand, the larger the linked fragments, the more limited the number of target existing genes, but the more likely it is that the increase in step (a) can be suppressed.

[0132] (iii) If there is a sequence essential for cleavage of the target sequence of the sequence-specific nuclease to be used (e.g., a PAM sequence in the CRISPR / Cas system), the sequence is added to the resulting ligated fragment to design a search sequence.

[0133] (iv) The search sequence designed in (iii) above is mapped onto genomic DNA. For a given existing gene, it is examined whether the search sequence can be mapped so that the linked fragments are arranged in the same order as the linked fragments of the modified gene on the existing gene and its adjacent non-coding regions. For example, multiple linked fragments may be mapped at once, and one in which the linked fragments are arranged in the same order as the modified gene may be selected. However, sequential mapping of the search sequences one by one is preferable because it reduces the amount of calculation required for the search. That is, if a second linked fragment exists adjacent to a first linked fragment, the first search sequence corresponding to the first linked fragment is first mapped, and then it is examined whether the second search sequence corresponding to the second linked fragment can be mapped near the mapped first search sequence. The vicinity may be set, for example, within 1000 base pairs (bp), 500 bp, 300 bp, 200 bp, 100 bp, or 50 bp of the search sequence.

[0134] (v) If all linked fragments can be finally mapped to the existing gene in the same order as the modified gene, the cleavage site on the genomic DNA has been identified. The target sequence can be determined so that cleavage occurs at the identified cleavage site. Of those that can be mapped, the one with the fewest number of linked fragments is preferred in terms of minimizing the number of steps (a). In one embodiment, the target sequence is determined based on the genomic DNA sequence before modification. In another embodiment, the target sequence is determined based on the genomic DNA sequence during modification, i.e., the genomic DNA from which one or more deletion regions have been excised. Note that one of these embodiments can be selected independently for each target sequence corresponding to each cleavage site.

[0135] A more specific embodiment of (iii) when CRISPR / Cas9 is used can be carried out, for example, by the following procedure.

[0136] (iii)-1: A sequence (search sequence) is generated by adding a PAM sequence to each ligated fragment. In the case of Cas9, since a blunt end is formed, the PAM sequence may be present on either the sense strand or the antisense strand. Therefore, in terms of the positional relationship with the cleavage site on the sense strand, either (1) 5'-(cleavage site)-NNN-(PAM sequence)-3' or (2) 5'-(complementary sequence of the PAM sequence)-NNN-(cleavage site)-3' is allowed (Figure 4A).

[0137] When SpCas-NG was used, search sequences generated by arranging PAM sequences so as to cleave both ends of ligated fragments of various lengths are shown in Figures 4B and 4C.

[0138] (iii)-2: When one of the two cleavage sites is cleaved and ligated, the PAM sequence for the other cleavage site may be lost. For example, when the PAM sequence is eccentrically located on the right side of the ligated fragments, as in Type 1 of Figure 4D, cleavage and ligation may occur on the right side of the ligated fragments, and cleavage on the left side may become impossible. Conversely, when the PAM sequence is eccentrically located on the left side of the ligated fragments, as in Type 2, cleavage and ligation may occur on the left side, and cleavage on the right side may become impossible. Therefore, in these types of PAM sequence arrangements, it is preferable to limit the cleavage order, or if the cleavage order is not limited, to apply Type 1 only to the 5'-end ligated fragments and Type 2 only to the 3'-end ligated fragments. On the other hand, when two PAM sequences are not contained in the ligated fragments (Type 3 of Figure 4D) or when two PAM sequences are present in the ligated fragments (Type 4 of Figure 4D), even if the first cleavage occurs, cleavage on the other side is possible, and this is more preferable in that there are no restrictions on the cleavage order. Of the possible cleavage sites on the genomic DNA, the cleavage site closest to the 5' or 3' end may be designed to include either the sequence (1) or (2) in Figure 4A (see types 5 and 6 in Figure 4E).

[0139] Based on the cleavage site and PAM sequence identified in steps (i) to (iv) above, a guide RNA for the CRISPR / Cas system can be designed. When Cas9 is used, a guide RNA can be designed that includes the PAM sequence and a 20-nucleotide sequence 5' upstream of it (17 nucleotides + 3 nucleotide spacer sequence) in the target sequence.

[0140] Optionally, the method may further include a step of predicting off-target sequences based on the target sequences obtained, and a step of selecting target sequences that are less likely to be off-targets. Off-target prediction can be performed using, for example, a known prediction service or software.

[0141] <Design of guide RNA encompassing linked fragments> When a linked fragment (referred to as a first linked fragment) is 16 base pairs or less, it is preferable to design and use a guide RNA such that the sequence of at least one linked fragment is encompassed in the target sequence, and the linked fragment, genomic DNA, or foreign DNA (referred to as the "linked portion of the first linked fragment") that will be ligated to the first linked fragment in step (a) is located at either end of the first linked fragment. The first linked fragment may be encompassed within the 17 nucleotides on the 5' side of the cleavage site, or within the 6 nucleotides consisting of the spacer sequence and PAM sequence on the 3' side. The ligated portion may be located on the 5' side, the 3' side, or both the 5' and 3' sides of the first linked fragment. Multiple linked fragments (e.g., two, three, four, or five) may be encompassed.

[0142] A guide RNA containing the above-mentioned linked fragments can be used not only in step (a) of the present invention, but also in inserting foreign DNA into genomic DNA using a sequence-specific endonuclease.

[0143] Figure 5A (I) and (II) show examples of regions where the ligated portion is present on the 5' and 3' sides of the first ligated fragment, consisting of the target sequence of the guide RNA and the PAM sequence. The portion on the 5' side that is not the ligated region (e.g., the left end portion of Figure 5A (II)) may be derived from the (adjacent) ligated fragment, a genomic DNA region, a deleted region, or foreign DNA inserted into the genomic DNA. In (II), the 3' side of the first ligated fragment further includes the sequence of another ligated fragment.

[0144] The guide RNA containing the above-mentioned linked fragment can cleave the intended cleavage site only after the first linked fragment and its ligated portion are ligated. This reduces the possibility of unintended ligation occurring when multiple (a) steps are performed simultaneously or consecutively using three or more guide RNAs. Furthermore, by using this guide RNA, step (a) proceeds only if the included linked fragment does not contain unintended ligation (e.g., ligation with another linked fragment or inverted ligation) or mutations associated with DSB repair at the ligated portion. Therefore, the use of such a guide RNA can reduce the risk of unintended mutations remaining.

[0145] FIG. 5B shows a case where cleavage and ligation are performed in the coexistence of three types of guide RNA (guide RNAs 1 to 3), including a guide RNA (guide RNA 3) that encompasses the linked fragment. In the first cleavage, guide RNAs 1 and 2 are cleaved and ligated, but cleavage by guide RNA 3 does not occur. This ensures that fragments 1 and 3 are ligated in the correct combination after the first cleavage. Subsequently, in the second cleavage, the genomic DNA in which fragments 1 and 3 are ligated in the correct orientation is cleaved between fragments 3 and 4 by guide RNA 3. On the other hand, when three types of guide RNA that do not encompass the linked fragment and are cleavable against the pre-ligation sequence are used, cleavage is performed by guide RNAs 1 to 3, resulting in fragments 1 to 4. Since ligation between fragments 1 to 4 can occur in various combinations, the efficiency of producing genomic DNA in which the linked fragments are correctly ligated decreases.

[0146] (Other Steps) The production method of the present invention preferably includes a step of determining the sequence of the obtained modified genomic DNA. The production method of the present invention preferably further includes a step of selecting cells having the modified genomic DNA of interest based on the determined sequence. By including these steps, cells having the modified genomic DNA of interest can be isolated and concentrated.

[0147] The production method of the present invention may further include a step of growing cells containing the modified genomic DNA. The cell growth can be performed using known methods used in growing the original cells (e.g., in vitro cell culture using a medium that can be used to culture the cells). The step of growing cells containing the modified genomic DNA can be performed before, during, or after the production of the modified genomic DNA, but is preferably performed after the production of the cells.

[0148] In the production method of the present invention, the cells may be subjected to an appropriate dedifferentiation step, differentiation step, etc. depending on the intended use. These steps can be carried out before, during, or after the production of the modified genomic DNA.

[0149] [Cells containing modified genomic DNA] One embodiment of the present invention is a cell whose genomic DNA has been modified, characterized in that the genomic DNA of the cell contains, compared to the wild-type genomic DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type genomic DNA, at least one linked fragment corresponding to a region in the existing gene sandwiched between the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and is configured to express a polypeptide, functional RNA, or both from the modified gene.

[0150] In a more specific aspect, a cell having modified genomic DNA, which is one embodiment of the present invention, is characterized in that: the cell has modified genomic DNA, wherein the genomic DNA comprises, compared to wild-type genomic DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type genomic DNA, at least one linked fragment corresponding to a region in the existing gene sandwiched between the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and at least one of the linked fragments is 1 to 180 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprise the full length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both.

[0151] Specific embodiments of the above-mentioned cells, existing genes, modified genes, deleted regions, and linked fragments conform to the respective embodiments described in the above-mentioned production methods of the present invention. Specific embodiments of the positional relationship between the existing gene or modified gene and the linked fragments or deleted regions also conform to the respective embodiments described in the above-mentioned production methods of the present invention.

[0152] The method for producing the cell of the present invention in which the genomic DNA has been modified is not particularly limited, and the cell can be produced, for example, by the production method of the present invention described above.

[0153] [Organism] An organism according to one embodiment of the present invention comprises cells produced by the production method of the present invention or cells containing the modified DNA of the present invention. These cells may be present throughout the organism or in parts thereof. If present in parts, they may be present locally or scattered. In one embodiment, these cells are present throughout the organism.

[0154] In one embodiment, the organism of the present invention is a non-genetically modified organism.

[0155] The method for producing an organism according to the present invention can be embodied, for example, in the following manner: (I) Cells containing modified DNA are produced from cells in a living organism by the production method of the present invention. (II) An organism is produced by applying a known method for producing transgenic organisms or cloned organisms to cells produced by the production method of the present invention (including cells replicated after production) or cells containing modified DNA of the present invention.

[0156] [Method for producing a gene product] The method for producing a gene product of the present invention comprises the step of expressing a gene product encoded by a modified gene contained in the modified genomic DNA using cells obtained by the production method of the present invention, cells containing the modified genomic DNA of the present invention, or an organism of the present invention.

[0157] In one embodiment, the gene product is a polypeptide.

[0158] Expression of the gene product may be appropriately induced using a regulatory sequence such as a promoter of the modified gene.

[0159] The method for producing a gene product of the present invention may further include a step of growing the cells or growing or propagating the organisms used. Growing the cells or growing or propagating the organisms can be carried out, for example, using a medium, feed, fertilizer, or the like that is commonly used for cells or organisms, based on a commonly used method.

[0160] The method for producing a gene product of the present invention may further include a step of isolating the gene product. Examples of the step of isolating the gene product include disruption of cells or organisms, centrifugation, filtration, solubilization, concentration, separation, and purification. The method for producing a gene product of the present invention may further include steps of sterilization, drying, packaging, and the like. Known techniques can be used as appropriate for these steps.

[0161] <Second Aspect> to <Fifth Aspect> The second to fifth aspects of the present invention will be described below. Here, the second aspect includes a step of modifying genomic DNA in a first cell and then introducing the modified DNA into a second cell. The third aspect includes a step of creating an artificial gene and introducing the gene into a second cell. The fourth aspect includes modifying extranuclear DNA in a first cell and introducing it into a second cell. The fifth aspect is similar to the first aspect, but modifies extranuclear DNA in a cell and retains the modified DNA in the cell.

[0162] [Method for producing cells containing modified DNA] <Second aspect> A second method for producing cells containing modified genomic DNA that does not contain foreign DNA, which is one embodiment of the present invention (sometimes referred to herein as the "production method of the second aspect"), comprises: (a) performing at least twice the steps of specifically cleaving the genomic DNA of a first cell at at least two target sequences with a sequence-specific nuclease and religating the sequences by cellular DNA double-strand break repair, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to produce a modified gene that differs in nucleotide sequence from the existing gene; the genomic DNA of the cell in step (a) comprises: at least two deleted regions that are not contained in the modified genomic DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and that remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; (c) introducing the modified gene obtained by step (a) into a second cell of the same species as the organism from which the first cell was derived or into a second cell derived from an organism that can mate with the organism from which the first cell was derived.

[0163] <Third Aspect> A third method for producing a cell containing modified DNA that does not contain foreign DNA, which is one embodiment of the present invention (sometimes referred to in this specification as the "production method of the third aspect"), is a method for producing a cell (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deleted site at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene; the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not contained in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; (Ia3) a step of preparing the artificially modified gene; and (c) a step of introducing the artificially modified gene obtained by step (Ia3) into a second cell of the same species as the organism from which the first cell is derived, or a second cell derived from an organism that can mate with the organism from which the first cell is derived, or a second cell that can infect the first cell.

[0164] <Fourth Aspect> A fourth method for producing a cell containing modified DNA that does not contain foreign DNA, which is one embodiment of the present invention (sometimes referred to herein as the "production method of the fourth aspect"), is a method for producing a cell (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia4) performing at least twice the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and religating the sequences by cellular DNA double-strand break repair, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to produce a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not contained in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; (Ic1) introducing the modified gene obtained by step (Ia4) into a second cell, wherein the modified gene is configured to express a polypeptide, a functional RNA, or both.

[0165] <Fifth Aspect> A fifth method for producing a cell containing modified DNA that does not contain foreign DNA, which is one embodiment of the present invention (sometimes referred to herein as the "production method of the fifth aspect"), is a method for producing a cell (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia4) performing at least twice the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and religating the sequences by cellular DNA double-strand break repair, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to produce a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not contained in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; The modified gene is configured to express a polypeptide, a functional RNA, or both.

[0166] <Second Aspect> to <Fifth Aspect> (Step (a)) In one step (a), the genomic DNA is cut at two sites and the region between them is excised. Then, the excised region is deleted in the genomic DNA that has been ligated by DNA double-strand break (DSB) repair in the cell. In some cases, a substitution, insertion, or deletion of one or more base pairs in length is introduced at the site of cleavage and ligation during DSB repair. In this specification, the excised region is referred to as the "deleted region." Furthermore, the sites at both ends of the deleted region that are cleaved by the sequence-specific endonuclease are referred to as the "cleavage site."

[0167] By performing step (a) two or more times, two or more deletion regions in the genomic DNA are excised. In this specification, the region sandwiched between the deletion regions and remaining in the modified genomic DNA is referred to as a "ligated fragment." Ultimately, by performing step (a) k times, k deletion regions are lost from the genomic DNA, and k-1 ligated fragments are ligated to construct a modified genomic DNA containing a modified promoter region and / or transcribed region. Figure 1A shows an example of a deletion region and a cleavage site.

[0168] In one step (a) and another step (a), the deletion site on the genomic DNA in the operation is different. Thus, in one embodiment, the two target sequences in each step (a) are different from each other.

[0169] Two or more (a) steps may be performed simultaneously or consecutively by a single genetic engineering operation. For example, in Figure 1B, of the three deletion regions, two regions are excised by two simultaneous (a) steps, and the other region is excised by a separate (a) step. In this way, when two or more (a) steps are performed simultaneously, it is not necessary for all (a) steps to be performed simultaneously or consecutively.

[0170] In this specification, one or more steps (a) that are carried out simultaneously or successively by a single genetic engineering operation may be referred to as a "set of steps (a)."

[0171] In the production method of the present invention, the cleavage site does not need to be at a site on the genomic DNA that encodes a site (i.e., a domain boundary) that can separate protein domains of the existing gene. In one embodiment, the existing gene encodes a protein, and in step (a), at least one cleavage and ligation occurs at a position other than a site that encodes a domain boundary of the protein. In a particular embodiment, in step (a), all cleavages occur at positions other than a site that encodes a domain boundary of the protein.

[0172] In one embodiment, the deletion region is present in one existing gene and its adjacent non-coding region in the genomic DNA of the cell. This configuration is preferable because it allows part of the regulatory sequence and transcribed region (e.g., coding region) of the existing gene to be used in constructing the modified gene. In one embodiment, the deletion region includes the non-coding region on the 5' side of the existing gene encoding the polypeptide, and at least one region selected from the group consisting of the regulatory region, coding region, 5' untranslated region, intron, and 3' untranslated region of the existing gene.

[0173] In one embodiment, the deleted region consists of a non-coding region 5' to the existing gene, a promoter region, or a combination thereof. This embodiment is advantageous in that it allows the promoter sequence to be modified and allows the expression of the existing gene to be regulated without affecting other regulatory sequences, splicing factors, and coding regions (see Example 5).

[0174] In one embodiment, the deleted region consists of a transcribed region of an existing gene. When the existing gene encodes a polypeptide, the deleted region consists of, for example, at least one region selected from the group consisting of a coding region, an intron, and a 3'-UTR. This embodiment is preferable because it makes it easy to construct a modified gene while minimizing the introduction of mutations into the regulatory sequence of the existing gene.

[0175] The possible locations of the linked fragments can be understood based on the possible locations of the deleted regions described above. In one embodiment, the linked fragments are present in one existing gene and its adjacent non-coding regions in the genomic DNA of the cell. In a more specific embodiment, the linked fragments include at least one selected from the group consisting of the 5' non-coding region and 3' non-coding region adjacent to the existing gene, and a transcribed region. In one embodiment, the existing gene encodes a polypeptide, and the linked fragments include at least one selected from the group consisting of the 5' non-coding region and 3' non-coding region adjacent to the existing gene, a regulatory region of the existing gene, a coding region, a 5' untranslated region, an intron, and a 3' untranslated region.

[0176] In the production method of the present invention, the length of each linked fragment is not particularly limited, as long as the desired modified gene sequence can be produced by deletion only from the genomic DNA sequence. Longer linked fragments reduce the number of steps (a), thereby saving labor and time. On the other hand, if the linked fragments are too long, it may be difficult to produce a modified gene having a sequence unrelated to existing genes.

[0177] In one embodiment, the length of at least one linked fragment is between 1 and 150 base pairs (bp), for example, 1 bp or more, 2 bp or more, 3 bp or more, 4 bp or more, 5 bp or more, 6 bp or more, 8 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 In one embodiment, the length of at least one linked fragment is 1 to 150 bp, e.g., 1 to 120 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 8 bp, or 1 to 5 bp.

[0178] In one embodiment, the length of at least two, three, four or five linked fragments is between 1 and 150 base pairs (bp), for example, 1 bp or more, 2 bp or more, 3 bp or more, 4 bp or more, 5 bp or more, 6 bp or more, 8 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more. In one embodiment, the length of at least two, three, four, or five linked fragments is 1 to 150 bp, e.g., 1 to 120 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 8 bp, or 1 to 5 bp.

[0179] In one embodiment, the length of all linked fragments is between 1 and 150 base pairs (bp), for example, 1 bp or more, 2 bp or more, 3 bp or more, 4 bp or more, 5 bp or more, 6 bp or more, 8 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 bp or more, 120 bp or more, 130 bp or more, or 140 bp or more, and may be 140 bp or less, 130 bp or less, 120 bp or less, 110 bp or less, 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, 10 bp or less, 8 bp or less, 6 bp or less, or 3 bp or less. In one embodiment, the length of all linked fragments is 1 to 150 bp, and may be, for example, 1 to 120 bp, 1 to 100 bp, 1 to 80 bp, 1 to 50 bp, 1 to 30 bp, 1 to 20 bp, 1 to 10 bp, 1 to 8 bp, or 1 to 5 bp.

[0180] In one embodiment, the total length of the linked fragments of the modified genomic DNA is, for example, 1 to 500 base pairs, preferably 9 to 180 base pairs (bp), for example, 9 bp or more, 10 bp or more, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 bp or more, 120 bp or more, 130 bp or more, 140 bp or more, 1 The length may be 50 bp or more, 160 bp or more, or 170 bp or more, and 170 bp or less, 160 bp or less, 150 bp or less, 140 bp or less, 130 bp or less, 120 bp or less, 110 bp or less, 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, 10 bp or less, 8 bp or less, 6 bp or less, or 3 bp or less. In one embodiment, the total length of all linked fragments is 9 to 180 bp, and may be, for example, 9 to 150 bp, 9 to 120 bp, 9 to 100 bp, 9 to 80 bp, 9 to 50 bp, 9 to 30 bp, or 9 to 20 bp. Although it depends on the target sequence of the sequence-specific endonuclease used, when using the CRISPR / Cas9 system, it is possible to construct a modified genomic DNA by only deleting an existing gene, even if the total length of the linked fragments is 150 bp or more (see Example 4).

[0181] In one embodiment, 50% or more, 60% or more, 70% or more, or 80% or more of the linked fragments have a length of 30 bp or less (preferably, 10 to 30 bp or 10 to 20 bp). As shown in Examples 1 to 5, when constructing a sequence different from an existing gene using linked fragments, linked fragments of these lengths tend to be used frequently.

[0182] In one embodiment, the total length of the linked fragments of the modified genomic DNA is 30% or less of the total length of the deleted region, and may be, for example, 20% or less, 10% or less, 5% or less, or 2% or less.

[0183] DSB repair includes non-homologous end joining (NHEJ) repair, in which ends without homologous regions are joined together; microhomology-mediated end joining (MMEJ) repair, in which ends with a complementary sequence of about 5 to 20 bases are joined together; and homology-directed repair (HDR). When no foreign DNA is inserted, repair in step (a) of the present invention is preferably performed by either NHEJ or MMEJ. Among these, ligation by NHEJ repair is more preferred due to its high degree of flexibility in target sequences. As described below, when foreign DNA is inserted together with genomic DNA deletion, MMEJ or HDR can be used for DSB repair.

[0184] During DSB repair, several additional nucleotides may be deleted or inserted at the termini created by the break. Even if such mutations associated with DSB repair exist in the modified genomic DNA, they do not involve the insertion of foreign DNA, and therefore cells or organisms containing the modified genomic DNA are not considered genetically modified organisms.

[0185] In one embodiment, the modified genomic DNA contains mutations associated with DSB repair. The number of mutated bases is defined as the number of substitutions or gaps in sequence alignment with the original genomic DNA. In a more specific embodiment, the modified genomic DNA contains, for example, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3 mutations within 20 base pairs on both sides of at least one cleavage site. However, these mutations may inhibit the intended cleavage or impair the function of the intended gene. Therefore, in the production method of the present invention, it is preferable that mutations associated with DSB repair are not contained in the modified genomic DNA.

[0186] Therefore, it is more preferable that the production method of the present invention includes a step of selecting cells that do not have mutations associated with DSB repair.

[0187] (Step (Ia2)) Step (Ia2) can be carried out in silico using information on a sequence in the genomic DNA present in the first cell or an extranuclear DNA sequence (e.g., a sequence in a plasmid) in accordance with procedures and conditions similar to those of step (a) above. In other words, an artificial gene corresponding to the modified gene can be designed in silico without any intracellular manipulation. The extranuclear DNA is not limited to, but is preferably DNA (e.g., a plasmid) present outside the nucleus of a cell that can infect other organisms, such as Agrobacterium (e.g., Rhizobium tumefaciens, Rhizobium rhizogenes, etc.).

[0188] (Step (Ia3)) In step (Ia3), the artificial gene designed in step (Ia2) is prepared. Typically, it can be synthesized by a chemical DNA synthesis method.

[0189] (Step (Ia4)) Step (Ia4) is one aspect included in step (a), and refers to modification of extranuclear DNA. In this specification, step (Ia4) may also be referred to as step (a), but may also be written as (Ia4) when specifically referring to modification of extranuclear DNA. This can be carried out using a DNA sequence present in the first cell, following procedures and conditions similar to those of step (a) above. Here, the DNA sequence present in the first cell may be DNA present outside the nucleus. Although not limited thereto, it is preferable that the DNA sequence be DNA (e.g., a plasmid) present outside the nucleus of a cell that can infect other organisms, such as Agrobacterium (e.g., Rhizobium tumefaciens, Rhizobium rhizogenes, etc.).

[0190] (Step (c)) The step of introducing the DNA fragment or artificial gene obtained in step (a) or (Ia3) above into a second cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, can be carried out using a conventionally known method. Note that after step (a), the selected DNA fragment can also be subjected to step (c) via step (b) described below. When the artificial gene is prepared based on information from extranuclear DNA (e.g., a plasmid) in a cell that can infect other biological species, the step of introducing the artificial gene into the second cell to be infected can also be carried out using a conventionally known method.

[0191] For the gene introduction in step (c), known methods for introducing DNA into cells can be used as appropriate depending on the type of cell. Examples of such methods include electroporation, microinjection, particle gun technology, calcium phosphate technology, polyethyleneimine (PEI) technology, liposome technology (lipofection), DEAE-dextran technology, cationic lipid-mediated transfection, viruses (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium ionization, lithium acetate technology, spheroplast technology, and heat shock technology (calcium chloride technology, rubidium chloride technology). In this process, manipulation is performed so that only the modified gene portion is incorporated into the DNA of the second cell. A homologous recombination technique may also be used, although this is not limited thereto. Homologous recombination of a DNA fragment into the DNA of a cell can be performed using a common homologous recombination method. Specifically, for example, a DNA sequence for a homologous recombination region is added to both ends of the DNA fragment before introduction. During homologous recombination, only the coding region of the modified gene may be introduced, but the coding region of the modified gene may also be introduced together with adjacent regions.

[0192] A vector can also be designed for homologous recombination. The DNA fragment excised in step (a) or the artificial gene prepared in step (Ia3) can be amplified by a gene amplification method in a second cell of the same species as the organism from which the first cell is derived, or in a second cell derived from an organism capable of mating with the organism from which the first cell is derived, and can be ligated to an appropriate vector and subjected to homologous recombination. Insertion of the DNA fragment into the vector can be performed by conventional methods, for example, by a ligase reaction using a restriction enzyme site. Alternatively, if the artificial gene prepared in step (Ia3) is an artificial gene obtained from information about extranuclear DNA, the extranuclear DNA in a cell capable of infecting other organisms, such as Agrobacterium (e.g., Rhizobium tumefaciens, Rhizobium rhizogenes, etc.), can be introduced into the second cell by a method such as homologous recombination.

[0193] (Step (Ic1)) The step of introducing the DNA fragment obtained in (Ia4) above into a second cell can be carried out using a conventionally known method. After step (Ia4), the selected DNA fragment can also be subjected to step (Ic1) via step (b) described below. Step (Ic1) particularly represents an infection-like aspect of step (c).

[0194] For example, the extranuclear DNA in the first cell can carry the desired DNA through step (Ia4), and the cell carrying the desired DNA is then infected with the second cell. In this case, it is particularly preferred that the first cell is derived from a bacterium capable of infecting other cells, such as Agrobacterium (e.g., Rhizobium tumefaciens, Rhizobium rhizogenes, etc., which are Rhizobium species).

[0195] (Existing gene) The existing gene is not particularly limited as long as it is possible to create a modified gene sequence by deletion manipulation, and is preferably a gene that is not essential for cell survival, or a gene that is essential for cell survival but has redundancy as a result of the existence of multiple similar genes.

[0196] In one embodiment, the existing gene is a gene for a highly expressed protein. This embodiment is preferable because the promoter region and ribosome binding sequence can be used as is, and the desired modified gene can be produced with a reduced number of operations in step (a), step (Ia2), or step (Ia4). Examples of such highly expressed protein genes include genes that express proteins in wild-type cells at an amount that accounts for 10% or more, 15% or more, 20% or more, or 30% or more of the total protein amount. Examples of such genes include structural proteins (e.g., collagen) in animal cells and storage proteins (e.g., seed storage proteins) in plant cells.

[0197] (Modified Gene) Modified DNA includes a modified gene configured to express a polypeptide, a functional RNA, or both. The region of the DNA before modification that is included in the modified gene is not particularly limited. In one embodiment, the modified gene comprises or consists of at least one selected from the group consisting of the non-coding regions on the 5'- and 3'-ends of an existing gene, a regulatory region, and a transcribed region of the existing gene. In one embodiment, the modified gene comprises or consists of at least one selected from the group consisting of the non-coding regions on the 5'- and 3'-ends of an existing gene, a regulatory region, a coding region, an intron, a 5'-UTR, and a 3'-UTR of the existing gene.

[0198] The region of DNA before modification contained in the promoter region of the modified gene is not particularly limited. In one embodiment, it consists of at least one type selected from the group consisting of the promoter region of an existing gene and the non-coding region adjacent to the 5' side thereof. In such a case, the risk of deleting sequences downstream of the promoter region of the existing gene (e.g., ribosome binding sequence, transcription regulatory sequence) can be reduced, and modification can be achieved by performing step (a) or step (Ia2) a small number of times. Step (Ia2) or step (Ia4) is similar to the case of manipulating extranuclear DNA, for example.

[0199] The region of the DNA before modification contained in the transcribed region of the modified gene is not particularly limited. In one embodiment, the transcribed region of the modified gene consists of the transcribed region of an existing gene. In such a case, it is preferable because it may be possible to use the promoter region of the existing gene as is.

[0200] In one embodiment, the existing gene and the modified gene encode a polypeptide, and the coding region of the modified gene comprises at least one selected from the group consisting of the coding region, intron, 5'-UTR, and 3'-UTR of the existing gene, more preferably at least one selected from the group consisting of the coding region, intron, and 3'-UTR. In such a case, it is preferable because it may be possible to use the promoter region and ribosome binding sequence of the existing gene as is. In one embodiment, the promoter region, the coding region, or both of the modified gene comprise the full length or a portion of at least one linked fragment. In one embodiment, the coding region of the modified gene comprises the full length or a portion of at least one linked fragment (see FIG. 2A and Examples 1 to 4 for specific examples).

[0201] When the modified gene encodes a polypeptide, the ligation in step (a) or step (Ia2) may generate a start codon or stop codon for the modified gene. The ligation may result in a frameshift, which may generate a start codon or stop codon in a region different from that of the existing gene. When a new start codon or stop codon for the modified gene is generated, the start codon or stop codon of the existing gene may be deleted, or the start codon or stop codon of the existing gene may be replaced with another codon during ligation. Steps (Ia2) and (Ia4) are similar, for example, when manipulating extracellular DNA. Steps (a) and (Ia2) can also form a coding region longer than the coding region of the existing gene. In this embodiment, if the codon reading frame of the existing gene is maintained, the modified gene encodes a fusion protein of the existing gene. Steps (Ia2) and (Ia4) are similar, for example, when manipulating extracellular DNA.

[0202] It is also possible to generate a coding region that is shorter than an existing gene, as shown in Examples 2-3 of FIG. 2A and Examples 1, 3, and 4.

[0203] In addition, as shown in Example 2-4 of Figure 2B, it is possible to reconstruct and generate a set of genes necessary for protein production, and as shown in Example 2-5 of Figure 2B, it is also possible to adjust the expression level of a gene by shortening only the promoter region from an existing promoter region.

[0204] In one embodiment, the promoter region of the modified gene comprises the entire length or a portion of at least one of the linked fragments (see Example 5 for an example). In this embodiment, a part or all of the promoter region of an existing gene is deleted to obtain a promoter consisting of a different nucleotide sequence. To construct a modified promoter region, for example, an existing promoter region and its adjacent 5'-terminal non-coding region can be used.

[0205] In one embodiment, the sequence of the promoter region of the modified gene can be a promoter with higher transcription activity than that of the existing gene. This embodiment is preferable because the modified gene obtained by the production method of the present invention has higher transcription activity and is capable of producing a modified gene product. The transcription level of the modified gene can be, for example, 1.1 times or more, 1.2 times or more, 1.5 times or more, 1.8 times or more, 2 times or more, or 3 times or more relative to the transcription level of the existing gene.

[0206] In one embodiment, the promoter of the modified gene has a sequence different from that of the promoter of the existing gene in order to increase the expression level of the modified gene product. Suitable examples of such promoters include: for human cells, the CMV promoter, EF1α promoter, SV40 promoter, RSV promoter, etc.; for plant cells, the RuBisco promoter, ADH promoter, cauliflower mosaic virus (CaMV) 35S promoter, CaMV19S promoter, El2 omega promoter, NOS promoter, etc.; for yeast cells, the Gal1 / 10 promoter, PGK promoter, ADH promoter, PHO5 promoter, etc.; and for Escherichia coli, the T7 promoter, trp promoter, lpp promoter, recA promoter, etc.

[0207] As will be shown in the embodiments below, if the length of the region formed by the linked fragments in the modified DNA (i.e., the total length of the linked fragments) is a nucleotide sequence encoding amino acids of approximately 60 residues or less, or a nucleotide sequence of 180 base pairs or less, the number of cleavage steps and the number of steps (a), (Ia2), or (Ia4) required to obtain modified genomic DNA can be reduced. For example, the number of cutting locations can be 100 or less, and the number of steps (a), (Ia2), or (Ia4) can be 50 or less; the number of cutting locations can be 50 or less, and the number of steps (a), (Ia2), or (Ia4) can be 25 or less; the number of cuttings can be 50 or less, and the number of steps (a), (Ia2), or (Ia4) can be 20 or less; the number of cuttings can be 20 or less, and the number of steps (a), (Ia2), or (Ia4) can be 10 or less; or the number of cuttings can be 10 or less, and the number of steps (a), (Ia2), or (Ia4) can be 5 or less.

[0208] When the existing gene and the modified gene encode a polypeptide, and the coding region of the modified gene includes the entire length or a portion of at least one of the linked fragments, it is preferable that the entire length or a portion of the linked fragment encodes a modified polypeptide region that is not present in the polypeptide region of the existing gene. For example, when a linked fragment of m base pairs is present in the coding region of the modified gene, the length of the modified polypeptide region is expressed as an integer length of 1 / 3m, rounded down to the nearest whole number. In certain embodiments, the length of the modified polypeptide region is 3 to 60 or 3 to 50 amino acids, preferably 3 to 40, 3 to 30, 3 to 20, 3 to 15, or 3 to 10 amino acids.

[0209] In one embodiment, the modified polypeptide region comprises, for example, a functional peptide sequence, an enzyme cleavage sequence, or both. Here, the functional peptide sequence is not particularly limited, but includes, for example, growth factors (epidermal growth factor (EGF), platelet-derived growth factor (PDGF) peptide, etc.), peptide hormones (insulin, glucagon, vasopressin, oxytocin, growth hormone, gastrin, cholecystokinin, secretin, atrial natriuretic peptide (ANP), melanocyte-stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), angiotensin, orexin, endothelin, ghrelin, etc.), peptide pheromones; plant stem cells; Examples of such peptides include signal peptides (CLE41 / 44 (TDIF), EPFL4 / 6, CLE9 / 10, CLE25, CLE46, etc.); peptide sequences having functions such as blood pressure lowering (angiotensin-converting enzyme (ACE) inhibitory peptides, etc.), mineral absorption promotion, cholesterol regulation, immunomodulation, antioxidant, anti-fatigue, anti-stress, β-amyloid toxicity alleviation, bifidobacterium growth promotion, taste, vaccine (for example, peptides containing an epitope sequence of a tumor-associated antigen or a viral antigen); and affinity (6xHis tag, etc.).

[0210] The modified polypeptide region may further comprise a linker sequence.

[0211] In one embodiment, the full-length polypeptide encoded by the modified gene is 50% or more, for example, 60% or more, 70% or more, 80% or more, or 90% or more, of the full-length polypeptide encoded by the existing gene. A typical example of such an embodiment is one in which the modified gene encodes a fusion protein in which a short modified polypeptide region is added to the N-terminus, C-terminus, or internal region of a protein or domain thereof encoded by the existing gene.

[0212] In another embodiment, the full length of the polypeptide encoded by the modified gene is 50% or less, e.g., 40% or less, 30% or less, 20% or less, or 10% or less, of the full length of the polypeptide encoded by the existing gene. A typical example of such an embodiment is one in which the full length of the coding region of the modified gene consists of a nucleotide sequence that encodes a short modified polypeptide that differs from the sequence of the existing gene.

[0213] (Sequence-specific endonuclease, target sequence) To delete a region on DNA in step (a) or step (Ia4), genomic DNA is cleaved with a sequence-specific endonuclease specific to a target sequence. As used herein, the term "target sequence" refers to a sequence that the sequence-specific endonuclease needs to identify and cleave.

[0214] The sequence-specific endonuclease is not particularly limited as long as it can cleave a target sequence in a target cell, but it is preferable that it recognizes and cleaves a target sequence that is unique to the genomic DNA of the cell (for example, a specific target sequence of 16 or more bases or 20 or more bases). Examples of such endonucleases include the CRISPR / Cas system, zinc finger nucleases (ZFNs), TALENs (transcription activation-like effector nucleases), meganucleases, etc. The sequence-specific endonuclease may be a wild-type enzyme or a modified mutant.

[0215] The sequence-specific endonuclease used may be of a different type or of the same type for each target sequence, step (a), or step (Ia4). From the viewpoint of simplifying the operation, it is preferable that all the sequence-specific endonucleases are of the same type.

[0216] If the cell is a eukaryotic cell, the sequence-specific endonuclease preferably contains at least one nuclear localization signal (NLS).

[0217] Among these, the CRISPR / Cas system is preferred as a sequence-specific endonuclease because it can easily impart specificity to a specific target sequence. The CRISPR / Cas system includes a Cas protein having endonuclease activity and a guide RNA that specifies the target sequence. The Cas pairs with the guide RNA and cleaves nucleotides that have a protospacer adjacent motif (PAM) sequence at a specific position. The Cas protein and guide RNA may be naturally occurring or may be a combination that does not exist in nature.

[0218] Cas9 or Cas12a (Cpf1) are preferred as Cas proteins because they have DNA cleavage activity and pinpoint cleavage activity in target sequences. CRISPR / Cas9 forms blunt ends regardless of whether the PAM sequence is contained on the sense strand or antisense strand. Therefore, Cas9 has the advantage of being less constrained by target sequences than other Cass.

[0219] In nature, CRISPR / Cas9 contains crRNA and tracrRNA as guide RNA components, but the production method of the present invention more preferably uses a system that uses single-stranded guide RNA (sgRNA), in which Cas, tracrRNA, and the target sequence are combined into a single RNA.

[0220] When using the CRISPR / Cas9 system, the Cas9 protein, guide RNA, etc. may be selected based on known literature and be compatible with the target cells. The Cas9 protein is preferably Cas9 from the genus Staphylococcus, and more preferably Cas9 from Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus. These Cas9 proteins may be wild-type or mutant, as long as they have target sequence specificity and DNA double-strand cleavage activity.

[0221] The nucleotide restriction by the PAM sequence complicates the design of guide RNA, so Cas9, which has a small number of positions restricted to specific nucleotides in the PAM sequence, is particularly suitable for use. Examples of such Cas9 include Cas9 derived from Streptococcus pyogenes (SpCas9), which recognizes NGG as the PAM sequence; a mutant of SpCas9 (SpCas-NG) which recognizes NG as the PAM sequence (Nishimasu, H. et al., 2018, Science, Vol. 361, pp. 1259-1262); xCas9-3.7 (Hu, JH et al., 2018, Nature, Vol. 556, pp. 57-63); ScCas9 which recognizes NNG (Chatterjee, P. et al., 2018, Sci. Adv. Vol. 4, eaau0766); and Sc++ (Chatterjee, P. et al. Nat. Biotechnol., 2020, Vol. 38, pp.1154-1158), SpG that recognizes NGN (Walton RT et al., 2020, Science, Vol.368, pp.290-296), or functional analogs thereof.

[0222] In SpCas9 and SpCas-NG, the target sequence consists of 5'-N(17)-(Cas cleavage site)-NNN-PAM sequence-3', where N(17) represents any 17 nucleotides. In this specification, the three nucleotides immediately preceding the PAM sequence are referred to as the spacer sequence.

[0223] When the sequence-specific nuclease is a zinc finger nuclease (ZFN), TALEN (transcription activation-like effector nuclease), or meganuclease, the cleavage in step (a) or (Ia4) can be carried out by allowing two nucleases with different target sequences to coexist in a cell.

[0224] When target sequence-specific cleavage is performed using the CRISPR / Cas system, for example, one type of Cas and two types of guide RNAs paired with the two target sequences to be cleaved can be used in a single step (a) or (Ia4) in a cell. However, the Cas used in each step (a) or (Ia4) does not need to be the same. When using multiple Cass, a target sequence containing an appropriate PAM sequence can be selected depending on the Cas.

[0225] (Step of selecting cells not containing off-target mutations, step of removing off-target mutations) In addition to the above step (a) or (Ia4), the production method of the present invention preferably further comprises step (b) of selecting cells not containing off-target mutations. As used herein, the term "off-target mutation" refers to a mutation that occurs as a result of cleavage and DSB repair at a site that is not originally intended to be cleaved.

[0226] In one embodiment, step (b) is performed after one or more steps (a) or (Ia4), for example, in a single operation. In one embodiment, step (b) is performed after all deleted regions have been excised from the DNA.

[0227] The presence or absence of off-target mutations is determined, for example, by sequencing the modified DNA.

[0228] The production method of the present invention may further include a step of removing off-target mutations in addition to the above step (b). Specific examples of methods for removing off-target mutations include the step of producing an organism containing modified DNA described below, and backcrossing (backcrossing) an organism containing modified DNA with a non-recombinant organism of the same species that does not contain the off-target mutations (e.g., a wild-type organism having cells before modification).

[0229] (Step of Inserting and Removing Foreign DNA into DNA) The production method of the present invention may further comprise a step of inserting and removing foreign DNA into DNA. As used herein, removal of foreign DNA means that foreign DNA and its replicas are no longer present in the cell. In one embodiment, removal of foreign DNA is carried out by step (a) or step (Ia4).

[0230] To insert foreign DNA into genomic DNA (or extranuclear DNA), for example, a technique can be used that combines genomic DNA (or extranuclear DNA) cleavage with sequence-specific endonuclease and DNA repair by recombinational repair. DNA cleavage can be performed at, for example, one or two locations depending on the purpose, as shown in Figures 3A and 3B described below. The two cleavages can be performed, for example, by the cleavage in step (a) or step (Ia4). Homologous recombination repair (HRD) is preferred for recombinational repair. Regions homologous to sequences on the DNA (referred to as the first and second homology arms) are added to both ends of the foreign DNA so that recombinational repair can occur at appropriate positions.

[0231] The length of the first homology arm and the second homology arm is not particularly limited as long as it is a length that allows foreign DNA to be inserted, and can be, for example, 5 base pairs (bp) or more, 10 bp or more, 20 bp or more, 50 bp or more, 100 bp or more, 200 bp or more, or 500 bp or more, and can be, for example, 10,000 bp or less, 5,000 bp or less, 2,000 bp or less, or 1,000 bp or less.

[0232] In one embodiment, the foreign DNA is inserted into the deleted region or adjacent to the deleted region, so that the entire deleted region can be removed in step (a) or step (Ia4).

[0233] In one embodiment, the foreign DNA is inserted to replace part or all of the deleted region, i.e., when the deleted region is excised by cleavage with a sequence-specific endonuclease in step (a) or (Ia4), the foreign DNA is inserted by recombinational repair (see, for example, Figure 3A).

[0234] To enable cleavage of the foreign DNA by step (a) or (Ia4), the foreign DNA may be provided with a sequence essential for cleavage of the target sequence, such as a PAM sequence.

[0235] In one embodiment, the first or second homology arm comprises the sequence of at least one linked fragment (referred to as a "first linked fragment"), and all or part of the sequence of the linked fragment or genomic DNA (referred to as a "ligated portion") ligated to the first linked fragment in step (a) or (Ia4) is connected to at least one of the ends of the first linked fragment. According to this embodiment, insertion of foreign DNA can be regulated depending on the ligation between the first linked fragment and the ligated portion (for a similar example for guide RNA, see the section <Design of guide RNA encompassing linked fragments>).

[0236] The length of the first linked fragment is not particularly limited as long as it is within the range of the length of the homology arm. However, from the viewpoint of reducing the risk of recombination, not limited to the ligation of the first linked fragment, the length of the first linked fragment is preferably, for example, 5% or more of the length of the homology arm, more preferably 10% or more, even more preferably 15% or more, and even more preferably 20% or more. From the same viewpoint, it is also preferable that the end of the first linked fragment is relatively close to the end of the foreign DNA. On the other hand, from the viewpoint of reducing the risk of losing the sequence of the first linked fragment due to cutting by nuclease, it is preferable that the end of the first linked fragment is separated from the end of the foreign DNA. Therefore, it is preferable that the end of the first linked fragment is located, for example, 1 to 30 bp, more preferably 3 to 20 bp, and even more preferably 5 to 15 bp from the end of the foreign DNA.

[0237] In a more specific embodiment, the foreign DNA includes a marker gene for cell selection. In such an embodiment, the deleted region containing or adjacent to the foreign DNA can be labeled with the marker gene.

[0238] Cells into which the foreign DNA has been inserted can be selected based on the expression of marker genes.

[0239] The manner of use and type of the cell selection marker gene are the same as those described in the first embodiment.

[0240] (Step of introducing sequence-specific endonuclease and / or its function-related factor) In one embodiment, the production method of the present invention can further include a step of introducing foreign DNA containing the target sequence-specific endonuclease or its gene into a cell in order to allow the above-mentioned target sequence-specific endonuclease to function in the cell, and a step of removing the foreign DNA.

[0241] When the target sequence-specific endonuclease is a CRISPR / Cas system, the method may further include the step of introducing an appropriate guide RNA or foreign DNA capable of expressing the guide RNA in cells. These steps may be performed simultaneously or separately.

[0242] When the sequence-specific endonuclease and guide RNA are directly introduced into cells, it is preferable to carry out this before each set of step (a) or step (Ia4).

[0243] In one embodiment, the foreign DNA is present in the cell in a form separated from the genomic DNA, making it easier to completely remove it from the cell. Such foreign DNA can be introduced into the cell as a vector, such as a plasmid, cosmid, or artificial chromosome. When the foreign DNA is separated from the genomic DNA, the foreign DNA is naturally lost, and the foreign DNA can sometimes be removed by selecting cells that do not contain the foreign DNA.

[0244] In another embodiment, foreign DNA is inserted into the DNA. Such foreign DNA can be introduced into cells as, for example, linear DNA, a viral vector, etc. In this embodiment, the production method of the present invention further comprises a step of removing the foreign DNA. Optionally, it may also comprise a step of selecting cells from which the foreign DNA has been removed.

[0245] To confirm that the DNA does not contain foreign DNA, for example, primers that can specifically amplify foreign DNA can be designed and used to detect the foreign DNA using various PCR methods. The absence of foreign DNA in the DNA can also be confirmed by, for example, sequencing.

[0246] The gene encoding the target sequence-specific endonuclease of the above-mentioned foreign DNA may be adjusted to have a codon usage frequency similar to that of the cell (so-called codon optimization) in order to improve expression in the cell.

[0247] In addition to the gene of interest, for example, a promoter, an enhancer, an insulator, an intron, a terminator, a poly A addition signal, a selection marker gene, etc. can be ligated to the vector.

[0248] The target gene to be inserted into a vector may be one or more types per vector.

[0249] In this specification, the introduction of substances such as nucleotides and proteins into cells is not particularly limited as long as it is a means capable of delivering RNA and proteins to living cells, and can be carried out by, for example, the liposome method (lipofection, etc.), particle gun (gene gun) method, electroporation method, polyethylene glycol (PEG) method, plasma method (see, for example, WO2018016217), whisker method, laser injection method, etc. When the cells are plant cells, the particle gun method is preferred.

[0250] (Design process of ligated fragments and target sequences) By determining which parts of a DNA region containing an existing gene are to be cleaved by a sequence-specific endonuclease, it is possible to determine the target sequences recognized by each endonuclease. The determination of the cleavage site is not particularly limited, and can be carried out, for example, by the following steps (i) to (v). The design of the target sequence, including the steps described below, may be carried out manually or based on a computer program or a trained machine learning or artificial intelligence (AI) algorithm.

[0251] (i) First, prepare the DNA sequence to be included in the modified gene. When creating a coding region, multiple sequences with different codon combinations can be used as candidates. Furthermore, because frameshifts can occur due to deletions in step (a) or (Ia4), a sequence may be generated with a partially frameshifted reading frame. Therefore, if the linked fragments cannot be mapped in order in step (iv) below for the DNA sequence of a coding region, steps (ii) to (iv) can be retried for substitution with synonymous codons or frameshift sequences.

[0252] (ii) The sequence of (i) is fragmented into two or more linked fragments. The boundary between the linked fragments is the cleavage site. Here, the smaller the linked fragments, the easier it is to find the target existing gene, but this is disadvantageous in that the number of steps (a) or (Ia4) increases. On the other hand, the larger the linked fragments, the more limited the number of target existing genes, but the more likely it is that the number of steps (a) or (Ia4) can be suppressed.

[0253] (iii) If there is a sequence essential for cleavage of the target sequence of the sequence-specific nuclease to be used (e.g., a PAM sequence in the CRISPR / Cas system), the sequence is added to the resulting ligated fragment to design a search sequence.

[0254] (iv) The search sequence designed in (iii) above is mapped onto DNA. For a given existing gene, it is examined whether the search sequence can be mapped so that the concatenated fragments are arranged in the same order as the concatenated fragments of the modified gene on the existing gene and its adjacent non-coding regions. For example, multiple concatenated fragments may be mapped at once, and one in which the concatenated fragments are arranged in the same order as the modified gene may be selected. However, sequential mapping of the search sequences one by one is preferable because it reduces the amount of calculation required for the search. That is, if a second concatenated fragment exists adjacent to a first concatenated fragment, the first search sequence corresponding to the first concatenated fragment is first mapped, and then it is examined whether the second search sequence corresponding to the second concatenated fragment can be mapped near the mapped first search sequence. The vicinity may be set, for example, within 1,000 base pairs (bp), 500 bp, 300 bp, 200 bp, 100 bp, or 50 bp of the search sequence.

[0255] (v) If all linked fragments can be ultimately mapped to the existing gene in the same order as the modified gene, the cleavage site on the DNA has been identified. The target sequence can be determined so that cleavage occurs at the identified cleavage site. Of those that can be mapped, the one with the fewest number of linked fragments is preferred in terms of minimizing the number of times step (a) or step (Ia4) is performed. In one embodiment, the target sequence is determined based on the DNA sequence before modification. In another embodiment, the target sequence is determined based on the DNA sequence during modification, i.e., the DNA from which one or more deletion regions have been excised. Note that one of these embodiments can be selected independently for each target sequence corresponding to each cleavage site.

[0256] A more specific embodiment of (iii) when CRISPR / Cas9 is used can be carried out, for example, by the following procedure.

[0257] (iii)-1: A sequence (search sequence) is generated by adding a PAM sequence to each ligated fragment. In the case of Cas9, since a blunt end is formed, the PAM sequence may be present on either the sense strand or the antisense strand. Therefore, in terms of the positional relationship with the cleavage site on the sense strand, either (1) 5'-(cleavage site)-NNN-(PAM sequence)-3' or (2) 5'-(complementary sequence of the PAM sequence)-NNN-(cleavage site)-3' is allowed (Figure 4A).

[0258] When SpCas-NG was used, search sequences generated by arranging PAM sequences so as to cleave both ends of ligated fragments of various lengths are shown in Figures 4B and 4C.

[0259] (iii)-2: When one of the two cleavage sites is cleaved and ligated, the PAM sequence for the other cleavage site may be lost. For example, when the PAM sequence is eccentrically located on the right side of the ligated fragments, as in Type 1 of Figure 4D, cleavage and ligation may occur on the right side of the ligated fragments, and cleavage on the left side may become impossible. Conversely, when the PAM sequence is eccentrically located on the left side of the ligated fragments, as in Type 2, cleavage and ligation may occur on the left side, and cleavage on the right side may become impossible. Therefore, in these types of PAM sequence arrangements, it is preferable to limit the cleavage order, or if the cleavage order is not limited, to apply Type 1 only to the 5'-end ligated fragments and Type 2 only to the 3'-end ligated fragments. On the other hand, when two PAM sequences are not contained in the ligated fragments (Type 3 of Figure 4D) or when two PAM sequences are present in the ligated fragments (Type 4 of Figure 4D), even if the first cleavage occurs, cleavage on the other side is possible, and this is more preferable in that there are no restrictions on the cleavage order. Of the possible cleavage sites on the DNA, the cleavage site closest to the 5' or 3' end can be designed to include either the sequence (1) or (2) in Figure 4A (see types 5 and 6 in Figure 4E).

[0260] Based on the cleavage site and PAM sequence identified in steps (i) to (iv) above, a guide RNA for the CRISPR / Cas system can be designed. When Cas9 is used, a guide RNA can be designed that includes the PAM sequence and a 20-nucleotide sequence 5' upstream of it (17 nucleotides + 3 nucleotide spacer sequence) in the target sequence.

[0261] Optionally, the method may further include a step of predicting off-target sequences based on the target sequences obtained, and a step of selecting target sequences that are less likely to be off-targets. Off-target prediction can be performed using, for example, a known prediction service or software.

[0262] <Design of Guide RNA Comprising Linked Fragments> When a linked fragment (referred to as a first linked fragment) is 16 base pairs or less, it is preferable to design and use a guide RNA such that the target sequence contains the sequence of at least one linked fragment, and the linked fragment, genomic DNA, extranuclear DNA, or foreign DNA (referred to as the "linked portion of the first linked fragment") to be linked to the first linked fragment in step (a) or (Ia4) is located at either end of the first linked fragment. The first linked fragment may be contained within the 17 nucleotides on the 5' side of the cleavage site, or within the 6 nucleotides consisting of the spacer sequence and PAM sequence on the 3' side. The linked portion may be located on the 5' side, the 3' side, or both the 5' and 3' sides of the first linked fragment. Multiple linked fragments (e.g., two, three, four, or five) may be contained.

[0263] A guide RNA comprising the above-mentioned linked fragments can be used in step (a) or (Ia4) of the present invention, as well as for inserting foreign DNA into genomic DNA or extracellular DNA using a sequence-specific endonuclease.

[0264] Figure 5A (I) and (II) show examples of regions where the ligated portion is present on the 5' and 3' sides of the first ligated fragment, consisting of the target sequence of the guide RNA and the PAM sequence. The portion on the 5' side that is not the ligated region (e.g., the left end portion of Figure 5A (II)) may be derived from an (adjacent) ligated fragment, a genomic DNA region, a deleted region, or foreign DNA inserted into genomic DNA or extranuclear DNA. In (II), the 3' side of the first ligated fragment further includes the sequence of another ligated fragment.

[0265] The guide RNA containing the above-mentioned linked fragment can cleave the intended cleavage site only after the first linked fragment and its ligated portion are ligated. This reduces the possibility of unintended ligation occurring when multiple steps (a) or (Ia4) are performed simultaneously or consecutively using three or more guide RNAs. Furthermore, by using this guide RNA, step (a) or (Ia4) proceeds only if the included linked fragment does not undergo unintended ligation (e.g., ligation with another linked fragment or inverted ligation) or mutations associated with DSB repair at the ligated portion. Therefore, the use of such a guide RNA can reduce the risk of unintended mutations remaining.

[0266] FIG. 5B shows a case where cleavage and ligation are performed in the coexistence of three types of guide RNA (guide RNAs 1 to 3), including a guide RNA (guide RNA 3) that encompasses the linked fragment. In the first cleavage, guide RNAs 1 and 2 are cleaved and ligated, but cleavage by guide RNA 3 does not occur. This ensures that fragments 1 and 3 are ligated in the correct combination after the first cleavage. Subsequently, in the second cleavage, the DNA in which fragments 1 and 3 are ligated in the correct orientation is cleaved between fragments 3 and 4 by guide RNA 3. On the other hand, when three types of guide RNA that do not encompass the linked fragment and are cleavable against the pre-ligation sequence are used, cleavage is performed by guide RNAs 1 to 3, resulting in fragments 1 to 4. Since ligation between fragments 1 to 4 can occur in various combinations, the efficiency of producing DNA in which the linked fragments are correctly ligated decreases.

[0267] (Other Steps) The production method of the present invention preferably includes a step of determining the sequence of the obtained modified DNA. The production method of the present invention preferably further includes a step of selecting cells having the modified DNA of interest based on the determined sequence. By including these steps, cells having the modified DNA of interest can be isolated and concentrated.

[0268] The production method of the present invention may further include a step of growing cells containing the modified DNA. The cell growth can be performed using known methods used for growing the original cells (e.g., in vitro cell culture using a medium that can be used to culture the cells). The step of growing the cells containing the modified DNA can be performed before, during, or after the production of the modified DNA, but is preferably performed after the production of the cells.

[0269] In the production method of the present invention, the cells may be subjected to an appropriate dedifferentiation step, differentiation step, etc. depending on the intended use. These steps can be carried out before, during, or after the production of the modified DNA.

[0270] [Cells containing modified genomic DNA] One embodiment of the present invention is a cell whose DNA has been modified, characterized in that the DNA of the cell contains, compared to wild-type DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type DNA, at least one linked fragment corresponding to a region in the existing gene flanked by the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and is configured to express a polypeptide, functional RNA, or both from the modified gene.

[0271] In a more specific aspect, a cell with modified DNA, which is one embodiment of the present invention, is a cell with modified DNA, characterized in that: the DNA comprises, compared to wild-type DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type DNA, at least one linked fragment corresponding to a region in the existing gene sandwiched between the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and at least one of the linked fragments is 1 to 180 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprise the full length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both.

[0272] Specific embodiments of the above-mentioned cells, existing genes, modified genes, deleted regions, and linked fragments conform to the respective embodiments described in the above-mentioned production methods of the present invention. Specific embodiments of the positional relationship between the existing gene or modified gene and the linked fragments or deleted regions also conform to the respective embodiments described in the above-mentioned production methods of the present invention.

[0273] The method for producing the cell of the present invention in which the genomic DNA has been modified is not particularly limited, and the cell can be produced, for example, by the production method of the present invention described above. That is, a method for producing a cell (excluding human cells) containing modified genomic DNA that does not contain foreign DNA, comprising: (a) performing the steps of specifically cleaving the genomic DNA of a first cell at at least two target sequences with a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the genomic DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified genomic DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both; (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell is derived or into a second cell derived from an organism that can mate with the organism from which the first cell is derived.

[0274] Alternatively, there is provided a method for producing cells (excluding human cells) containing modified genomic DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deleted site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and (Ia3) a step of preparing the artificially modified gene: (c) introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived or into a second cell derived from an organism that can breed with the organism from which the first cell is derived.

[0275] [Cells containing modified DNA] One embodiment of the present invention is a cell whose DNA has been modified, characterized in that the DNA (particularly extranuclear DNA) in the cell contains, compared to the wild-type DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type DNA, at least one linked fragment corresponding to a region in the existing gene flanked by the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and the modified gene is configured to express a polypeptide, functional RNA, or both.

[0276] In a more specific embodiment, the method for producing the DNA-modified cells according to the present invention is not particularly limited, but for example, the cells can be produced by the production method according to the third embodiment of the present invention. That is, a method for producing cells (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia4) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both of them; (Ic1) A cell obtained by a method comprising the step of introducing the modified gene obtained by step (Ia4) into a second cell, wherein the first cell is infected with the second cell.

[0277] Alternatively, there is provided a method for producing cells (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising the steps of: (Ia4) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and the non-coding regions adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, functional RNA, or both.

[0278] [Organism] An organism according to one embodiment of the present invention comprises cells produced by the production method of the present invention or cells containing the modified DNA of the present invention. These cells may be present throughout the organism or in parts thereof. If present in parts, they may be present locally or scattered. In one embodiment, these cells are present throughout the organism.

[0279] In one embodiment, the organism of the present invention is a non-genetically modified organism.

[0280] The method for producing an organism according to the present invention can be embodied, for example, in the following manner: (I) Cells containing modified DNA are produced from cells in a living organism by the production method of the present invention. (II) An organism is produced by applying a known method for producing transgenic organisms or cloned organisms to cells produced by the production method of the present invention (including cells replicated after production) or cells containing modified DNA of the present invention.

[0281] [Method for producing a gene product] The method for producing a gene product of the present invention comprises the step of expressing a gene product encoded by a modified gene contained in the modified genomic DNA using cells obtained by the production method of the present invention, cells containing the modified genomic DNA of the present invention, or an organism of the present invention.

[0282] In one embodiment, the gene product is a polypeptide.

[0283] Expression of the gene product may be appropriately induced using a regulatory sequence such as a promoter of the modified gene.

[0284] The method for producing a gene product of the present invention may further include a step of growing the cells or growing or propagating the organisms used. Growing the cells or growing or propagating the organisms can be carried out, for example, using a medium, feed, fertilizer, or the like that is commonly used for cells or organisms, based on a commonly used method.

[0285] The method for producing a gene product of the present invention may further include a step of isolating the gene product. Examples of the step of isolating the gene product include disruption of cells or organisms, centrifugation, filtration, solubilization, concentration, separation, and purification. The method for producing a gene product of the present invention may further include steps of sterilization, drying, packaging, and the like. Known techniques can be used as appropriate for these steps.

[0286] [Marker for Genetic Testing] One aspect of the present invention relates to a marker for genetic testing that contains part or all of the modified gene in the DNA prepared by the above method. Such a marker is provided in the form of a probe or primer, and can be used to detect the presence of a modified gene that is different from the wild type.

[0287] <Sixth Aspect of the Invention> [Method for Producing Cells Comprising Modified Genomic DNA] A method for producing cells comprising modified DNA by deletion, which is one embodiment of the present invention (sometimes referred to herein as the "production method of the sixth aspect of the present invention"), comprises: (i) a first step of cleaving the genomic DNA of a cell with a sequence-specific endonuclease, and inserting, by homologous recombination (HDR) or non-homologous recombination repair, foreign DNA comprising a marker gene (herein simply referred to as "foreign DNA" or "first foreign DNA") into a specific region in the genomic DNA to be deleted (deleted region), or adjacent to the deleted region, or replacing the deleted region; (ii) a second step of cleaving the genomic DNA at at least two sites with a sequence-specific endonuclease to excise the region comprising the foreign DNA from the genomic DNA, and obtaining genomic DNA free of foreign DNA by DNA double-strand break (DSB) repair; and (iii) a third step of selecting cells based on the presence or absence of the marker gene. The method is characterized in that a portion of the genomic DNA is excised with a sequence-specific endonuclease in the first step, the second step, or both, and the third step is performed after the first step, the second step, or both. In a preferred, but not limited to, embodiment, step (iii) is performed once after step (i) and once after step (ii), and cells are selected after step (i) based on the presence of the marker gene, and cells are selected after step (ii) based on the absence of the marker gene.

[0288] (Step 1) One embodiment of step 1 is shown below, but the present invention is not limited to this. For example, the first foreign DNA to which a homology arm is added may have no homology arm. Furthermore, the repair step does not necessarily have to be performed by homologous recombination.

[0289] In the first step, genomic DNA is site-specifically cleaved using a sequence-specific endonuclease. When a first foreign DNA, which has added homology arms (first and second homology arms) capable of homologous recombination with the ends (first and second ends) of the genomic DNA generated by the cleavage, is present near the genomic DNA, homologous recombination occurs between the first end and the first homology arm, and between the second end and the second homology arm, by the cell's inherent repair mechanism. As a result, the first foreign DNA can be inserted into the cleaved site of the genomic DNA.

[0290] By using an appropriate sequence-specific endonuclease, the cleavage site can be controlled, and as a result, the insertion site of the foreign DNA can be controlled. In the present invention, the first foreign DNA can be inserted into a specific region of the genomic DNA to be deleted by modification (deletion region), adjacent to the deletion region, or to replace the deletion region.

[0291] In one embodiment, the foreign DNA is inserted within or adjacent to the deletion region (referred to as embodiment i-2a). In this embodiment, in the second step, the marker gene and the deletion region of the foreign DNA can be easily excised together, and cells having genomic DNA that does not contain the deletion region can be easily selected by selecting cells that do not contain the marker gene. Figure 10A shows an example of an embodiment in which foreign DNA containing a marker gene (GFP, ampicillin resistance gene) is inserted adjacent to the deletion region. Figure 10B shows an example of an embodiment in which foreign DNA containing a marker gene (RFP, kanamycin resistance gene) is inserted within the deletion region. Furthermore, as shown in Figure 10C, cleavage does not have to be performed at a single site; cleavage may be performed at two or more sites selected from both ends and within the deletion region. This allows genomic DNA in which at least a portion of the deletion region is deleted during the insertion of foreign DNA in the first step. In this aspect, the first homology arm or the second homology arm is not particularly limited as long as it is configured to enable insertion of foreign DNA into or adjacent to a specific region (deleted region) on the genomic DNA by homologous recombination repair. In this aspect, at least one of the first homology arm or the second homology arm includes part or all of the deleted region, and the homology arm may also include a region adjacent to the deleted region.

[0292] In another embodiment, a sequence-specific endonuclease is used to cleave both ends of the deleted region, excising the entire deleted region, and then foreign DNA is inserted to replace the deleted region (referred to as embodiment i-2b). An example is shown in Figure 2. In such a case, the first and second homology arms are sequences capable of homologous recombination with sequences (referred to as "outside sequences") located outside both ends of the deleted region (towards the genomic DNA remaining after the deletion manipulation).

[0293] The first step may include allowing the foreign DNA used for homologous recombination to coexist with the genomic DNA of the cell. Examples of means for allowing the foreign DNA to coexist with the genomic DNA of the cell include introducing the foreign DNA itself into the cell, and introducing nucleotides that serve as precursors of the foreign DNA (e.g., circular DNA or linear DNA containing the sequence of the foreign DNA) into the cell.

[0294] (Foreign DNA, Homology Arm) The length of the first or second homology arm is not particularly limited as long as it is a length that allows homologous recombination with the above-mentioned first end or second end sequence on the genomic DNA, and can be, independently, for example, 5 base pairs (bp) or more, 10 bp or more, 20 bp or more, 50 bp or more, 100 bp or more, 200 bp or more, or 500 bp or more, and can be, for example, 10,000 bp or less, 5,000 bp or less, 2,000 bp or less, or 1,000 bp or less.

[0295] In one embodiment, the first and second homology arms are sequences homologous to the sequences of the first end or second end on the genomic DNA, and do not contain sequence substitutions, insertions, or gaps on the genomic DNA. This embodiment is preferable because it prevents the introduction of substitutions, insertions, or gaps derived from the homology arms during insertion of foreign DNA by homologous recombination, thereby reducing the risk that the resulting cell containing the modified genomic DNA is a genetically modified organism.

[0296] In another embodiment, the first or second homology arm may contain a substitution, insertion, or gap in the sequence of the first or second end, as long as homologous recombination is possible. In such an embodiment, a mutation derived from the homology arm is introduced into the genomic DNA. Therefore, to make the finally obtained cell a non-genetically modified organism, a separate step of removing the region containing the mutation from the genomic DNA is required.

[0297] The foreign DNA includes a marker gene for cell selection. This allows the presence or absence of foreign DNA in the genomic DNA of a cell to be determined by the presence or absence of the marker gene product. As the marker gene, either a positive selection marker gene or a negative selection marker gene can be used. A positive selection marker gene is a gene that allows cells to be selected based on its presence, and examples include fluorescent proteins (GFP, YFP, CFP, etc.), drug resistance genes (neomycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, kanamycin resistance gene, sulfonylurea resistance gene (ALS), glyphosate resistance gene (EPSPS), etc.), and reporter enzyme genes (luciferase, β-galactosidase, β-glucuronidase (GUS), dihydrofolate reductase (DHFR), etc.). Negative selection marker genes are genes that allow cells to be selected based on their absence, and include, for example, genes encoding toxic proteins, suicide genes (HSV-TK, iCasp9, etc.), and the like.

[0298] When a fluorescent protein is used as a marker gene, cells can be easily selected on a large scale using a cell sorter or the like. Furthermore, when a drug resistance gene is used as a marker gene, cells having the resistance gene can be selected by culturing the cells in a medium containing the drug. Therefore, a fluorescent protein or a drug resistance gene is more preferable as the marker gene for cell selection used in the production method of the present invention. In particular, when the marker gene is a fluorescent protein, its absence can be confirmed by the fluorescence intensity of the cells, and both the presence and absence of foreign DNA can be confirmed relatively easily, making it particularly preferable.

[0299] In one embodiment, one exogenous DNA contains one marker gene. In another embodiment, as illustrated in Figures 10A to 10C, one exogenous DNA contains two or more marker genes. This embodiment is preferable because the accuracy of selecting the target cells can be further improved by combining selection using multiple marker genes.

[0300] The foreign DNA may contain a sequence essential for cleavage of the target sequence, such as a PAM sequence in a CRISPR / Cas system, to enable cleavage of the foreign DNA in a second step.

[0301] (Deletion region) The location, function, and sequence of the deletion region on the genomic DNA are not particularly limited as long as both ends of the deletion region can be cleaved by the presence of a target sequence for the sequence-specific endonuclease, and can be appropriately selected depending on the purpose of modifying the genomic DNA.

[0302] The length of the deletion region is not particularly limited and may be, for example, 1 to 100,000 base pairs (bp), 1 to 10,000 bp, 1 to 1,000 bp, or 1 to 100 bp. The shorter the deletion region, the more difficult it is to determine whether the deletion region has been correctly removed using a simple technique such as polymerase chain reaction (PCR). On the other hand, the production method of the present invention combines labeling of the deletion region with a marker gene and cleavage with a sequence-specific endonuclease, making it possible to easily determine whether the target region has been correctly removed. From the perspective of more significantly achieving the effects of the present invention, the length of the deletion region is preferably such that differences in base length are difficult to distinguish using PCR, for example, 1 to 60 bp, more preferably 1 to 50 bp, even more preferably 1 to 40 bp, and even more preferably 1 to 30 bp.

[0303] (Step 2) In step 2, the genomic DNA is cleaved at least two times using a sequence-specific endonuclease to excise the region containing the foreign DNA from the genomic DNA, and then DNA double-strand break (DSB) repair is performed to obtain genomic DNA that does not contain foreign DNA. DSB repair is performed by either non-homologous end joining (NHEJ) repair, in which ends without homologous regions are joined, or microhomology-mediated end joining (MMEJ) repair, in which ends having complementary sequences of about 5 to 20 bases are joined. However, due to the high degree of freedom in target sequences, it is more preferable to use ligation by NHEJ repair.

[0304] During DSB repair, several additional nucleotides may be deleted or inserted at the termini created by the break. Even if such mutations associated with DSB repair exist in the modified genomic DNA, they do not involve the insertion of foreign DNA, and therefore cells or organisms containing the modified genomic DNA are not considered genetically modified organisms.

[0305] In one embodiment, the modified genomic DNA contains mutations associated with DSB repair. The number of mutated bases is defined as the number of substitutions or gaps in a sequence alignment with the original genomic DNA. In a more specific embodiment, the modified genomic DNA contains, for example, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3 mutations within 20 base pairs on either side of at least one break.

[0306] Depending on the embodiment of the first step, in the second step, the deleted region in the genomic DNA is also excised and removed from the genomic DNA along with the foreign DNA. For example, when the first step is the above-mentioned embodiment (i-2a), it is preferable to excise the deleted region and the foreign DNA together in a form that does not include other regions (referred to as embodiment iia). In such an embodiment, removal of the deleted region from the genomic DNA can be easily confirmed by the absence of a marker gene in the foreign DNA.

[0307] Furthermore, when the deleted region has already been removed in the first step, as in the above-mentioned embodiment (1-2b), the foreign DNA can be removed by cleaving both ends of the foreign DNA (referred to as embodiment iib).

[0308] The first and second steps can be performed on two or more deletion regions. In this case, in the first step, foreign DNA containing the same marker gene or different markers can be inserted into two or more deletion regions.

[0309] (Step 3) Step 3 is a step of selecting cells based on the presence or absence of a marker gene. By performing step 3 after step 1 and selecting cells in which the marker gene is present, cells in which foreign DNA has been inserted into their genomic DNA can be selected. Alternatively, by performing step 3 after step 2 and selecting cells in which the marker gene is absent, cells in which foreign DNA containing the marker gene has been removed by step 2 can be selected. Step 3 can be performed after step 1, step 2, or both. However, from the viewpoint of easily distinguishing between the insertion and removal of foreign DNA and the removal of the deleted region, it is preferable to perform step 3 after both steps 1 and 2. As illustrated in Figures 10A-C and 11, the insertion of foreign DNA in step 1 or the removal of foreign DNA in step 2 is associated with the removal of the deleted region. Therefore, by performing step 3 after steps 1 and 2, cells in which foreign DNA and the deleted region have been removed can be easily selected.

[0310] When steps 1 and 2 are performed on two or more deletion regions, in one embodiment, step 3 is performed after steps 1 and 2 have been performed on each of the deletion regions. In another embodiment, step 3 is performed after steps 1 and 2 have been performed on two or more deletion regions, preferably all of the deletion regions. In particular, step 3 is preferably performed after steps 1 and 2 have been performed on all of the deletion regions. When selection is performed after inserting or removing foreign DNA collectively in this manner, the foreign DNA inserted into each deletion region may be labeled with the same marker gene or with different marker genes.

[0311] (Sequence-specific endonuclease, target sequence) The sequence-specific endonuclease and target sequence are the same as those in the first aspect of the present invention.

[0312] (Step of Introducing Sequence-Specific Endonuclease and / or Factor Associated with Its Function) The step of introducing a sequence-specific endonuclease and / or factor associated with its function conforms to the content of the first aspect of the present invention.

[0313] (Step of selecting cells that do not contain off-target mutations, and step of removing off-target mutations) The production method of the present invention preferably further comprises a step of selecting cells that do not contain off-target mutations after the above steps 1 to 3. As used herein, the term "off-target mutation" refers to a mutation that occurs as a result of cleavage and DSB repair at a site that was not originally intended to be cleaved.

[0314] The presence or absence of off-target mutations is determined, for example, by genomic sequencing of the modified genomic DNA.

[0315] The production method of the present invention may further include a step of removing off-target mutations in addition to the above steps 1 to 3. Specific examples of methods for removing off-target mutations include the step of producing an organism containing a modified genome described below, and mating (backcrossing) an organism containing a modified genome with a non-recombinant organism of the same species that does not contain the off-target mutations (e.g., a wild-type organism having cells before modification).

[0316] (Other Steps) The production method of the present invention preferably includes a step of determining the sequence of the obtained modified genomic DNA. The production method of the present invention preferably further includes a step of selecting cells having the modified genomic DNA of interest based on the determined sequence. By including these steps, cells having the modified genomic DNA of interest can be isolated and concentrated.

[0317] The production method of the present invention may further include a step of growing cells containing the modified genomic DNA. The cell growth can be performed using known methods used in growing the original cells (e.g., in vitro cell culture using a medium that can be used to culture the cells). The step of growing cells containing the modified genomic DNA can be performed before, during, or after the production of cells containing the modified genomic DNA, but is preferably performed after the production of the cells.

[0318] In the production method of the present invention, the cells may be subjected to an appropriate dedifferentiation step, differentiation step, etc. depending on the intended use. These steps can be carried out before, during, or after the production of the modified genomic DNA.

[0319] [Method for producing an organism] One embodiment of the method for producing an organism of the present invention includes a step of incorporating cells containing modified genomic DNA obtained by the above-mentioned [Method for producing cells containing modified genomic DNA] into an organism.

[0320] Examples of processes for incorporating cells containing modified genomic DNA into an organism include the following, and each can be produced using a known method used to produce organisms: (I) introducing cells containing modified genomic DNA into an organism or its embryo; (II) inducing germ cells from cells containing modified genomic DNA to generate an organism; (III) applying a method for producing cells containing modified genomic DNA directly to cells of the organism itself; (IV) propagating an organism obtained by any of (I) to (III).

[0321] (Aspects of the Invention) The present invention includes the following aspects: [1] A method for producing a cell containing modified genomic DNA that does not contain foreign DNA, comprising: (a) performing at least twice the steps of specifically cleaving the genomic DNA of a cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby generating a modified gene that differs in nucleotide sequence from the existing gene; the genomic DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified genomic DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the method is configured to express a polypeptide, a functional RNA, or both from the modified gene. [2] The method of [1], wherein the step (a) is carried out at least twice simultaneously or consecutively with different combinations of the target sequences. [3] The method of [1] or [2], further comprising the step of (b) selecting the cells containing no off-target mutations. [4] The method of any one of [1] to [3], wherein at least one of the target sequences comprises the sequence of at least one linked fragment (first linked fragment), and at least one of the ends of the first linked fragment is connected to the whole or part of the sequence of the linked fragment or genomic DNA linked to the first linked fragment in the step (a). [5] The method of any one of [1] to [4], which does not comprise the step of inserting foreign DNA into the genomic DNA of the cells.[6] The method of any one of [1] to [4], further comprising the step of inserting foreign DNA, to both ends of which a first homology arm and a second homology arm capable of homologous recombination with the genomic DNA, into at least one of the deleted regions, inserting the foreign DNA so that the foreign DNA is adjacent to at least one of the deleted regions, or inserting the foreign DNA so that the foreign DNA replaces part or all of at least one of the deleted regions, by recombinational repair of the cell, wherein in step (a), the foreign DNA is removed from the genomic DNA by the deletion, and further comprising the step of selecting the cells free of foreign DNA. [7] The method of [6], wherein the foreign DNA comprises a cell selection marker gene. [8] The method of [6] or [7], wherein the first or second homology arm encompasses the sequence of at least one linked fragment (first linked fragment), and wherein at least one of the ends of the first linked fragment is connected to the whole or part of the sequence of the linked fragment or genomic DNA linked to the first linked fragment in step (a). [9] The method according to any one of [1] to [8], wherein the total length of the linked fragments is 9 to 180 base pairs.

[10] The method according to any one of [1] to [8], wherein the total length of the linked fragments is 30% or less of the total length of the deleted regions.

[11] The method according to any one of [1] to

[10] , wherein the existing gene and the modified gene encode a polypeptide, and in a wild-type cell of the cell, the polypeptide encoded by the existing gene accounts for 10% or more of the mass of the total protein.

[12] The method according to any one of [1] to

[11] , wherein the existing gene and the modified gene encode a polypeptide, and the coding region of the modified gene comprises the full length or a portion of at least one of the linked fragments, and the full length or a portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids that is not encoded by the existing gene.

[13] The method according to

[12] , wherein the modified polypeptide region comprises a functional peptide sequence, an enzyme cleavage sequence, or both.

[14] The method according to any one of [1] to

[13] , wherein the existing gene and the modified gene encode polypeptides, and the full-length polypeptide encoded by the modified gene is 20% or less of the full-length polypeptide encoded by the existing gene.

[15] The method according to any one of [1] to

[13] , wherein the existing gene and the modified gene encode polypeptides, and the full-length polypeptide encoded by the modified gene is 50% or more of the full-length polypeptide encoded by the existing gene.

[16] The method according to any one of [1] to

[15] , wherein the cell is used in food or is used in the production of food.

[17] The method according to any one of [1] to

[16] , wherein the sequence-specific nuclease is performed by a CRISPR / Cas system.

[18] The method according to

[17] , wherein the CRISPR / Cas system comprises SpCas9-NG or a functional analogue thereof, or ScCas9 or a functional analogue thereof.

[19] A cell whose genomic DNA has been modified, wherein the genomic DNA contains, compared to wild-type genomic DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type genomic DNA, at least one linked fragment corresponding to a region in the existing gene sandwiched between the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the cell is configured to express a polypeptide, a functional RNA, or both from the modified gene.

[20] The cell according to

[19] , wherein the total length of the linked fragments is 9 to 180 base pairs.

[21] The cell according to

[19] or

[20] , wherein the total length of the linked fragments is 30% or less of the total length of the deleted regions.

[22] The cell according to any one of

[19] to

[21] , wherein the existing gene and the modified gene encode polypeptides, and in a wild-type cell of the cell, the amount of protein encoded by the existing gene accounts for 10% or more of the mass of the total protein.

[23] The cell according to any one of

[19] to

[22] , wherein the existing gene and the modified gene encode polypeptides, and the coding region of the modified gene comprises the full length or a portion of at least one of the linked fragments, and the full length or a portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids that is not encoded by the existing gene.

[24] The cell according to

[23] , wherein the modified polypeptide region comprises a functional peptide, an enzyme cleavage sequence, or both.

[25] The cell according to any one of

[19] to

[24] , wherein the existing gene and the modified gene encode polypeptides, and the full length of the polypeptide encoded by the modified gene is 20% or less of the full length of the polypeptide encoded by the existing gene.

[26] The cell according to any one of

[19] to

[24] , wherein the existing gene and the modified gene encode a polypeptide, and the full-length polypeptide encoded by the modified gene is 50% or more of the full-length polypeptide encoded by the existing gene.

[27] The cell according to any one of

[19] to

[26] , wherein the cell is used in food or for producing food.

[28] An organism comprising a cell produced by the method according to any one of [1] to

[18] , or a cell according to any one of

[19] to

[27] .

[29] A method for producing a gene product, comprising a step of expressing a gene product encoded by the modified gene in a cell produced by the method according to any one of [1] to

[18] , the cell according to any one of

[19] to

[27] , or an organism comprising such a cell.[2-1] A method for producing cells (excluding human cells) containing modified genomic DNA that does not contain foreign DNA, comprising: (a) performing at least twice the steps of specifically cleaving the genomic DNA of a first cell at at least two target sequences with a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the genomic DNA of the cell in step (a) comprises: at least two deleted regions that are not contained in the modified genomic DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both of them; (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell was derived or into a second cell derived from an organism that can mate with the organism from which the first cell was derived.[2-2] A method for producing cells (excluding human cells) containing modified genomic DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deleted site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not contained in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and a step of designing an artificially modified gene configured to express a polypeptide, a functional RNA, or both of the modified gene from the modified gene; A method comprising: (Ia3) a step of preparing the artificially modified gene; and (c) a step of introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or a second cell derived from an organism capable of mating with the organism from which the first cell is derived, or a second cell to be infected with the first cell. [2-3] The method described in [2-1], wherein step (a) is performed at least twice simultaneously or consecutively with different combinations of the target sequences. [2-4] The method described in [2-1] or [2-2], further comprising (b) a step of selecting the cells that do not contain off-target mutations. [2-5] The method described in [2-1], wherein at least one of the target sequences includes the sequence of at least one linked fragment (first linked fragment), and at least one of the ends of the first linked fragment is connected to the sequence of all or a part of the linked fragment or genomic DNA linked to the first linked fragment in step (a). [2-6] The method described in [2-1] or [2-2], which does not comprise a step of inserting foreign DNA into the DNA of the cell.[2-7] The method of [2-1] further comprising the step of inserting foreign DNA, to both ends of which are added a first homology arm and a second homology arm capable of homologous recombination with the genomic DNA, into at least one of the deleted regions, inserting the foreign DNA so that the foreign DNA is adjacent to at least one of the deleted regions, or inserting the foreign DNA so that the foreign DNA replaces part or all of at least one of the deleted regions, by recombinational repair of the cell, wherein in the step (a), the foreign DNA is removed from the genomic DNA by the deletion, and further comprising the step of selecting the cells free of foreign DNA. [2-8] The method of [2-7], wherein the foreign DNA comprises a cell selection marker gene. [2-9] The method of [2-7], wherein the first or second homology arm encompasses the sequence of at least one linked fragment (first linked fragment), and wherein at least one of the ends of the first linked fragment is connected to the whole or part of the sequence of the linked fragment or genomic DNA linked to the first linked fragment in the step (a). [2-10] The method according to any one of [2-1] to [2-9], wherein the total length of the linked fragments is 9 to 180 base pairs. [2-11] The method according to any one of [2-1] to [2-10], wherein the total length of the linked fragments is 30% or less of the total length of the deleted regions. [2-12] The method according to any one of [2-1] to [2-11], wherein the existing gene and the modified gene encode a polypeptide, and in a wild-type cell of the cell, the polypeptide encoded by the existing gene accounts for 10% or more of the total protein mass. [2-13] The method according to any one of [2-1] to [2-12], wherein the existing gene and the modified gene encode a polypeptide, and the coding region of the modified gene comprises the full length or a portion of at least one of the linked fragments, and the full length or a portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids that is not encoded by the existing gene. [2-14] The method according to [2-13], wherein the modified polypeptide region comprises a functional peptide sequence, an enzyme cleavage sequence, or both.[2-15] The method according to any one of [2-1] to [2-14], wherein the existing gene and the modified gene encode polypeptides, and the full-length polypeptide encoded by the modified gene is 20% or less of the full-length polypeptide encoded by the existing gene. [2-16] The method according to any one of [2-1] to [2-15], wherein the existing gene and the modified gene encode polypeptides, and the full-length polypeptide encoded by the modified gene is 50% or more of the full-length polypeptide encoded by the existing gene. [2-17] The method according to any one of [2-1] to [2-16], wherein the cell is used for food or for producing food. [2-18] The method according to [2-1], wherein the sequence-specific nuclease is operated by a CRISPR / Cas system. [2-19] The method according to [2-18], wherein the CRISPR / Cas system comprises SpCas9-NG or a functional analogue thereof, or ScCas9 or a functional analogue thereof.[2-20] A cell (excluding human cells) containing a modified gene, comprising: (a) performing the steps of specifically cleaving the genomic DNA of a first cell at at least two target sequences with a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene having a nucleotide sequence different from that of the existing gene; the genomic DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified genomic DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both of them; (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell was derived or into a second cell derived from an organism that can mate with the organism from which the first cell was derived.[2-21] A cell (excluding human cells) containing a modified gene, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deletion site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs in nucleotide sequence from the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not contained in the modified genomic DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and (Ia3) a step of preparing the artificially modified gene: (c) introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can breed with the organism from which the first cell is derived. [2-22] The cell according to [2-20] or [2-21], wherein the total length of the linked fragments is 9 to 180 base pairs. [2-23] The cell according to [2-20] or [2-21], wherein the total length of the linked fragments is 30% or less of the total length of the deleted regions. [2-24] The cell according to [2-20] or [2-21], wherein the existing gene and the modified gene encode polypeptides, and wherein the amount of protein encoded by the existing gene accounts for 10% or more of the total protein mass in a wild-type cell of the cell.[2-25] The cell according to [2-20] or [2-21], wherein the existing gene and the modified gene encode a polypeptide, and the coding region of the modified gene comprises the full length or a portion of at least one of the linked fragments, and the full length or a portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids not encoded by the existing gene. [2-26] The cell according to [2-25], wherein the modified polypeptide region comprises a functional peptide, an enzyme cleavage sequence, or both. [2-27] The cell according to [2-20] or [2-21], wherein the existing gene and the modified gene encode a polypeptide, and the full length of the polypeptide encoded by the modified gene is 20% or less of the full length of the polypeptide encoded by the existing gene. [2-28] The cell according to [2-20] or [2-21], wherein the existing gene and the modified gene encode a polypeptide, and the full length of the polypeptide encoded by the modified gene is 50% or more of the full length of the polypeptide encoded by the existing gene. [2-29] The cell according to [2-20] or [2-21], wherein the cell is used in a food product or in the production of a food product.[2-30] A method for producing modified genomic DNA that does not contain foreign DNA, comprising: (a) performing the steps of specifically cleaving the genomic DNA of a first cell at at least two target sequences with a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the genomic DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified genomic DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both; (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell was derived or into a second cell derived from an organism that can mate with the organism from which the first cell was derived.[2-31] A method for producing modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from DNA of a first cell and ligating both outer ends of the deletion site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; a promoter region, a transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and (Ia3) a step of preparing the artificially modified gene: (c) introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.[2-32] A modified gene obtained from modified genomic DNA that does not contain foreign DNA, comprising the steps of: (a) performing the steps of specifically cleaving the genomic DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and non-coding regions adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the genomic DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified genomic DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified genomic DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both.[2-33] A modified gene obtained from modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificial modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deletion site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not contained in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and a step of designing an artificial modified gene configured to express a polypeptide, a functional RNA, or both of them from the modified gene; and (Ia3) A modified gene obtained by a method comprising the step of preparing the artificially modified gene. [2-34] A cell (excluding human cells) into which one or more modified genes according to [2-32] or [2-33] have been incorporated, wherein the cell belongs to a species that can be crossed with the organism species from which the modified gene was derived. [2-35] Modified DNA comprising, compared to the wild-type DNA of a cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding region of the wild-type DNA, at least one linked fragment corresponding to a region sandwiched between the deleted regions (deleted regions) in the existing gene, and a gene whose nucleotide sequence is modified relative to the existing gene due to the deletions. [2-36] An organism (excluding humans) comprising a cell produced by the method described in any one of [2-1] to [2-19], or a cell described in any one of [2-20] to [2-29], a cell described in [2-34], a modified DNA obtained by the method described in [2-30] or [2-31], a modified gene described in [2-32] or [2-33], or a modified DNA described in [2-35].[2-37] A method for producing a gene product, comprising a step of expressing a gene product encoded by a modified gene in an organism (excluding humans) containing a cell prepared by the method of any one of [2-1] to [2-19], a cell described in any one of [2-20] to [2-29], a cell described in [2-34], a modified DNA obtained by the method of [2-30] or [2-31], a modified gene described in [2-32] or [2-33], or a modified DNA described in [2-35]. [2-38] A marker for genetic testing, comprising a modified gene in DNA prepared by the method of any one of [2-20] or [2-21], or part or all of the modified gene described in any one of [2-32] or [2-33]. [2-39] A method for producing a cell (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia4) performing at least twice the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and reconnecting the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both of them; (Ic1) a step of introducing the modified gene obtained by step (Ia4) into a second cell. [2-40] The method according to [2-39] above, wherein at least two rounds of step (Ia4) are carried out simultaneously or consecutively using different combinations of the target sequences.[2-41] The method according to [2-39] or [2-40] above, further comprising the step of (b) selecting the cells that do not contain off-target mutations. [2-42] The method according to [2-39] above, wherein at least one of the target sequences comprises the sequence of at least one linked fragment (first linked fragment), and the sequence of all or part of the linked fragment or genomic DNA that is linked to the first linked fragment in step (a) is connected to at least one of the ends of the first linked fragment. [2-43] The method according to

[39] above, which does not comprise the step of inserting foreign DNA into the DNA of the cells. [2-44] The method of

[39] , further comprising the step of inserting foreign DNA, to both ends of which a first homology arm and a second homology arm capable of homologous recombination with the DNA, into at least one of the deleted regions, inserting the foreign DNA so that the foreign DNA is adjacent to at least one of the deleted regions, or inserting the foreign DNA so that the foreign DNA replaces part or all of at least one of the deleted regions, by recombinational repair of the cell, wherein in step (Ia4), the foreign DNA is removed from the DNA by the deletion, and further comprising the step of selecting the cells free of foreign DNA. [2-45] The method of

[44] , wherein the foreign DNA comprises a cell selection marker gene. [2-46] The method of [2-44], wherein the first or second homology arm encompasses the sequence of at least one linked fragment (first linked fragment), and wherein at least one of the ends of the first linked fragment is connected to the whole or part of the sequence of the linked fragment or genomic DNA linked to the first linked fragment in step (Ia4). [2-47] The method according to [2-39], wherein the total length of the linked fragments is 9 to 180 base pairs. [2-48] The method according to [2-39], wherein the total length of the linked fragments is 30% or less of the total length of the deleted region. [2-49] The method according to [2-39], wherein the existing gene and the modified gene encode polypeptides, and in a wild-type cell of the cell, the polypeptide encoded by the existing gene accounts for 10% or more of the mass of all proteins.[2-50] The method of [2-39], wherein the existing gene and the modified gene encode a polypeptide, the coding region of the modified gene comprises the entire length or a portion of at least one of the linked fragments, and the entire length or a portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids that is not encoded by the existing gene. [2-51] The method of [2-39], wherein the modified polypeptide region comprises a functional peptide sequence, an enzyme cleavage sequence, or both. [2-52] The method of [2-39], wherein the existing gene and the modified gene encode a polypeptide, and the full-length polypeptide encoded by the modified gene is 20% or less of the full-length polypeptide encoded by the existing gene. [2-53] The method of [2-39], wherein the existing gene and the modified gene encode a polypeptide, and the full-length polypeptide encoded by the modified gene is 50% or more of the full-length polypeptide encoded by the existing gene. [2-54] The method of [2-39], wherein the cell is used in a food product or for producing a food product. [2-55] The method according to [2-39], wherein the sequence-specific nuclease is performed by a CRISPR / Cas system. [2-56] The method according to [2-55], wherein the CRISPR / Cas system comprises SpCas9-NG or a functional analog thereof, or ScCas9 or a functional analog thereof.[2-57] A cell (excluding human cells) containing a modified gene, comprising: (Ia4) performing at least twice the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene having a nucleotide sequence different from that of the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both of them; (Ic1) A cell obtained by a method comprising a step of introducing the modified gene obtained by step (Ia4) into a second cell, wherein the step comprises infecting the second cell with the first cell. [2-58] The cell according to [2-57], wherein the total length of the linked fragments is 9 to 180 base pairs. [2-59] The cell according to [2-57], wherein the total length of the linked fragments is 30% or less of the total length of the deleted region. [2-60] The cell according to [2-57], wherein the existing gene and the modified gene encode a polypeptide, and wherein the amount of protein encoded by the existing gene accounts for 10% or more of the total protein mass in a wild-type cell of the cell. [2-61] The cell according to [2-57], wherein the existing gene and the modified gene encode a polypeptide, and wherein the coding region of the modified gene comprises the full length or a portion of at least one of the linked fragments, and wherein the full length or a portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids that is not encoded by the existing gene.[2-62] The cell according to [2-61], wherein the modified polypeptide region comprises a functional peptide, an enzyme cleavage sequence, or both. [2-63] The cell according to [2-57], wherein the existing gene and the modified gene encode polypeptides, and the full-length polypeptide encoded by the modified gene is 20% or less of the full-length polypeptide encoded by the existing gene. [2-64] The cell according to [2-57], wherein the existing gene and the modified gene encode polypeptides, and the full-length polypeptide encoded by the modified gene is 50% or more of the full-length polypeptide encoded by the existing gene. [2-65] The cell according to [2-57], wherein the cell is used in food or used in the production of food. [2-66] A method for producing modified DNA that does not contain foreign DNA, comprising the steps of: (Ia4) performing at least twice the steps of specifically cleaving the DNA of a first cell at at least two target sequences using a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both.[2-67] A modified gene obtained from modified DNA that does not contain foreign DNA, comprising the steps of: (Ia4) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both. [2-68] A cell (excluding human cells) into which one or more modified genes according to [2-67] have been incorporated. [2-69] A cell (excluding human cells) into which one or more modified genes according to any of the above have been incorporated, wherein the cell is obtained by infecting an organism from which the modified genes are derived. [2-70] Modified DNA comprising, compared to the wild-type DNA of a cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions in the wild-type DNA, and at least one linked fragment corresponding to a region in the existing gene sandwiched between the deleted regions (deleted regions), and a gene whose nucleotide sequence is modified relative to the existing gene due to the deletion. [2-71] A cell produced by the method according to any one of the above, or a cell according to any one of the above, a modified DNA obtained by the method according to any one of the above, or an organism (excluding humans) comprising the modified gene according to any one of the above.[2-72] A method for producing a gene product, comprising a step of expressing a gene product encoded by the modified gene in a cell prepared by the method described in any one of the above, in a modified DNA obtained by the method described in any one of the above, or in an organism (excluding humans) containing the modified gene described in any one of the above. [2-73] A marker for genetic testing, comprising a modified gene in DNA prepared by the method described in any one of the above, or part or all of the modified gene described in any one of the above. [2-74] A method for producing cells (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia4) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences using a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and configuring the modified gene to express a polypeptide, functional RNA, or both. [2-75] The method according to [2-74], wherein the modified DNA is extranuclear DNA.[2-76] A cell containing modified DNA, obtained by a method comprising the steps of: (Ia4) performing at least twice the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, to generate a modified gene having a nucleotide sequence different from that of the existing gene; the DNA in the cell in step (Ia4) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and configuring the modified gene to express a polypeptide, a functional RNA, or both. [2-77] The cell according to [2-76], wherein the modified DNA is extranuclear DNA. [2-78] An organism (excluding humans) comprising a cell produced by the method according to [2-74] or [2-75], or the cell according to [2-76] or [2-77]. [2-79] A method for producing a gene product, comprising a step of expressing a gene product encoded by the modified gene in an organism (excluding humans) comprising a cell produced by the method according to [2-74] or [2-75], or the cell according to [2-76] or [2-77]. The present invention includes, but is not limited to, the following aspects.[3-1] A method for producing a cell containing modified genomic DNA that does not contain foreign DNA, comprising: (i-1a) a step of allowing foreign DNA to coexist with the genomic DNA of the cell, wherein the foreign DNA contains a marker gene and is configured so that the foreign DNA can be inserted into or adjacent to a specific region (deleted region) on the genomic DNA; (i-2a) a step of cleaving at least one end or the interior of the deleted region with a sequence-specific endonuclease and inserting the foreign DNA into or adjacent to the deleted region by homologous recombination repair in the cell; and (iia) a step of cleaving the genomic DNA at least two sites with the sequence-specific endonuclease, excising the deleted region and the foreign DNA together from the genomic DNA, and generating modified genomic DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) a step of selecting the cell based on the presence or absence of the marker gene, [3-2] The method according to [3-1], wherein in (i-1a), the foreign DNA is added with a first homology arm and a second homology arm, the first homology arm and the second homology arm being sequences homologous to the genomic DNA of the cell, and the first homology arm and the second homology arm are configured so that the foreign DNA can be inserted into or adjacent to a specific region (deleted region) in the genomic DNA by homologous recombination (HDR) repair. [3-3] The method according to [3-1] or [3-2], wherein a part of the deleted region is deleted upon insertion of the foreign DNA.[3-4] A method for producing a cell containing modified genomic DNA that is free of foreign DNA, comprising: (ib) a step of allowing foreign DNA to coexist with the genomic DNA of the cell, wherein the foreign DNA contains a marker gene, and a step of cleaving both ends of a specific region (deleted region) on the genomic DNA with a sequence-specific endonuclease and inserting the foreign DNA to replace the deleted region; and (iib) a step of cleaving the genomic DNA at at least two sites with the sequence-specific endonuclease to excise the foreign DNA from the genomic DNA and generating modified genomic DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) a step of selecting the cell based on the presence or absence of the marker gene, wherein step (iii) is performed after step (ib), after step (iib), or both. [3-5] The method according to [3-4], wherein in (ib), the foreign DNA has a first homology arm and a second homology arm attached thereto, the first homology arm being homologous to an outer sequence at one end of a specific region (deleted region) on the genomic DNA, and the second homology arm being homologous to an outer sequence at the other end, and the first homology arm and the second homology arm being configured to insert the foreign DNA so as to replace the specific region (deleted region) on the genomic DNA by homologous recombination (HDR) repair. [3-6] The method according to any one of [3-1] to [3-5], wherein the length of the deleted region is 1 to 60 bp. [3-7] A method for producing an organism, comprising the step of incorporating cells containing modified genomic DNA obtained by the production method according to any one of [3-1] to [3-6] into an organism.[3-8] A method for producing a cell containing modified genomic DNA that does not contain foreign DNA, comprising: (i-1a) allowing foreign DNA to coexist with the genomic DNA of the cell, the foreign DNA having a first homology arm and a second homology arm added, the first homology arm being a sequence homologous to the genomic DNA of the cell, wherein the foreign DNA comprises a marker gene, and the first homology arm and the second homology arm are configured so that the foreign DNA can be inserted into or adjacent to a specific region (deleted region) on the genomic DNA by homologous recombination (HDR) repair; (i-2a) cleaving at least one end or the inside of the deleted region with a sequence-specific endonuclease, and inserting the foreign DNA into or adjacent to the deleted region by homologous recombination repair in the cell; and (ii) cleaving the genomic DNA at least two sites with a sequence-specific endonuclease to excise the deleted region and the foreign DNA together from the genomic DNA, and generating modified genomic DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) selecting the cell based on the presence or absence of the marker gene, wherein step (iii) is performed once after step (i-2a) and once after step (iia), and wherein after step (i-2a), cells are selected based on the presence of the marker gene, and after step (iia), cells are selected based on the absence of the marker gene. [3-8] The method according to [3-7], in which a part of the deleted region is deleted upon insertion of the foreign DNA.[3-9] A method for producing a cell containing modified genomic DNA that does not contain foreign DNA, comprising: (i-1b) allowing foreign DNA to coexist with the genomic DNA of the cell, the foreign DNA having a first homology arm and a second homology arm added, the first homology arm being a sequence homologous to the genomic DNA of the cell, wherein the foreign DNA comprises a marker gene, and the first homology arm is a sequence homologous to an outer sequence at one end of a specific region (deleted region) on the genomic DNA, and the second homology arm is a sequence homologous to an outer sequence at the other end; (i-2b) cleaving both ends of the deleted region with a sequence-specific endonuclease, and inserting the foreign DNA to replace the deleted region by homologous recombination (HDR) repair in the cell; and (iib) cleaving the genomic DNA at at least two sites with the sequence-specific endonuclease, excising the foreign DNA from the genomic DNA, and generating modified genomic DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) a step of selecting the cells based on the presence or absence of the marker gene, wherein step (iii) is performed once after step (i-2b) and once after step (iib), and wherein after step (i-2b), cells are selected based on the presence of the marker gene, and after step (iib), cells are selected based on the absence of the marker gene.

[0322] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0323] The following examples show specific examples of combinations of cleavage sites and PAM sequences that can be used to construct specific modified genes from specific genome sequences using the production method of the present invention. These were all manually specified based on the genomic DNA sequence. By designing a target sequence based on the information on the specified cleavage site and PAM sequence and using a CRISPR / Cas9 system appropriate for each cell, those skilled in the art can prepare cells containing modified DNA.

[0324] In each figure in the examples, the upper wedge of the sequence represents the cleavage site by the sense strand PAM sequence, and the lower wedge of the sequence represents the cleavage site by the antisense strand PAM sequence. The sequences enclosed in boxes and written in italics represent the PAM sequence and spacer sequence. The underlined portion represents the ligated fragment or the genomic DNA sequence maintained in the modified genomic DNA. The deleted region is indicated as the region between the two cleavage sites and includes the dashed omission. The numbers in the table indicate the corresponding positions in the genome sequence listed in NCBI (https: / / www.ncbi.nlm.nih.gov / ) using the reference numbers described in each example.

[0325] Example 1: Modification of the Coding Region of an Existing Gene

[0049] The coding region of the β-conglycinin α subunit 2 (CG-2) gene on chromosome 20 of the soybean (Glycine max) genome (NCBI Reference Number: NC_038256.2) was modified solely by deletion using SpCas9 (PAM sequence: NGG) to create a gene encoding a polypeptide in which a new 4-residue amino acid sequence was added to the N-terminal 57 residues of CG-2. To identify the cleavage site, a search sequence mapped based on the above design procedure is shown in Figure 6A. A modified gene can be constructed by five iterations of Step (a) or Step (Ia2). The stop codon of the existing gene is eliminated by deletion, and a new stop codon is generated by ligation. When the polypeptide encoded by the modified gene is produced in soybean cells, it is cleaved at the C-terminus of the asparagine residue by vacuolar protease (VPE) in ripening seeds, producing an umami-enhancing peptide consisting of Ala-Glu-Asp (AED) (Maehashi K. et al., Biosci. Biotechnol. Biochem., 1999, Vol. 63, No. 3, pp. 555-559). The sequence surrounding the modified site shown in Figure 6A, the modified polypeptide, and the identified cleavage site and PAM sequence are shown in Table 1.

[0326]

[0327] Example 2: Modification of the Coding Region of an Existing Gene (Example 2) The 3'-UTR region of lipoxygenase-3 (LOX-3) on chromosome 15 of the soybean (Glycine max) genome (NCBI Reference Number: NC_038251.2) was modified solely by deletion using SpCas9-NG (PAM sequence: NG) to create a gene encoding a polypeptide with a linker and His tag attached to the C-terminus of LOX3. To identify the cleavage site, a search sequence mapped based on the above design procedure is shown in Figure 6B. A modified gene can be constructed through five iterations of step (a) or step (Ia2). A new stop codon is generated by ligation. The sequence surrounding the modified site shown in Figure 6B, the modified polypeptide, and the identified cleavage site and PAM sequence are shown in Table 2.

[0328]

[0329] Example 3: Modification of the Coding Region of an Existing Gene Example 3 The region from the coding region of the β-conglycinin α-subunit (CG-2) to the 3'-end non-coding region of soybean (Glycine max) genome chromosome 20 (NCBI Reference Number: NC_038256.2) was modified solely by deletion using SpCas9-NG (PAM sequence: NG) to create a gene encoding a new polypeptide. The new polypeptide comprises four tandem tripeptide sequences with angiotensin-converting enzyme (ACE) inhibitory activity in the N-terminal fragment of CG-2. To identify the cleavage site, a search sequence mapped based on the above design procedure is shown in Figure 6C. A modified gene can be constructed by seven iterations of Step (a) or Step (Ia2). The stop codon and terminator sequence of the existing gene are deleted, and a new stop codon is generated by ligation instead. From this polypeptide, two molecules of Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP) peptides are obtained by thermolysin. The sequences around the modified site shown in Figure 6C, the modified polypeptide, and the identified cleavage site and PAM sequence are shown in Table 3.

[0330]

[0331] Example 4: Modification of the Coding Region of an Existing Gene Example 4 The coding region of the β-conglycinin α-subunit (CG-2) of soybean (Glycine max) genome chromosome 20 (NCBI Reference Number: NC_038256.2) through the non-coding region at the 3' end was modified solely by deletion using SpCas9-NG (PAM sequence: NG) to create a gene encoding a protein in which human epidermal growth factor (EGF) is fused to the N-terminal fragment of CG-2. To identify the cleavage site, the locations of the ligated fragments mapped based on the above design procedure are shown in Figure 6D, and the search sequence is shown in Figure 6E. A modified gene can be constructed by 33 iterations of Step (a) or Step (Ia2). The stop codon and terminator sequence of the existing gene are deleted, and a new stop codon is generated by ligation instead. The sequences around the modified portions shown in Figure 6D, the sequences around the modified portions, the modified polypeptides, and the identified cleavage sites and PAM sequences are shown in Tables 4-1 and 4-2.

[0332]

[0333]

[0334] Example 5: Example of modification of the promoter region of an existing gene The lipoxygenase-3 (LOX-3) gene on chromosome 15 of the soybean (Glycine max) genome (NCBI Reference Number: NC_038251.2) was modified solely by deletion using SpCas9-NG (PAM sequence: NG) from the 5' non-coding region to the promoter region to create a gene in which the promoter was changed to a minimal CaMV 35S promoter. To identify the cleavage site, a search sequence mapped based on the above design procedure is shown in Figure 6F. A modified gene can be constructed by nine iterations of step (a) or step (Ia2). The sequence surrounding the modification site shown in Figure 6F, the modified polypeptide, and the identified cleavage site and PAM sequence are shown in Table 5.

[0335]

[0336] As described above, even when the existing genes are limited to the CG-2 gene and the LOX-3 gene, modified genes with various sequences can be produced. Therefore, it is easy to understand that various modified genes can be constructed from the genomic DNA sequence by deletion manipulation alone.

[0337] The CG-2 and LOX-3 genes are highly expressed in soybean seeds. Therefore, it is expected that the modified polypeptides can be produced in large quantities from the modified genes in Examples 1 to 4, which have the same promoters as these genes, and from the modified genes in Example 5, which have an even stronger promoter.

[0338] Example 6: Example of modification of promoter and coding regions of an existing gene The coding region of the β-conglycinin α subunit 2 (CG-2) gene on chromosome 20 of the soybean (Glycine max) genome (NCBI Reference Number: NC_038256.2) was modified solely by deletion using SpCas9 (PAM sequence: NGG) to create a gene encoding a polypeptide in which a new 4-residue amino acid sequence was added to the N-terminal 57 residues of CG-2. The search sequence mapped based on the above design procedure to identify the cleavage site is shown in Figure 6A, as in Example 1. A modified gene can be constructed by five iterations of Step (a), Step (Ia2), or Step (Ia4). The stop codon of the existing gene is eliminated by deletion, and a new stop codon is generated by ligation.

[0339] The resulting modified gene is introduced into soybean cells of a different individual from the individual from which the cells used to create the modified gene were derived. Introduction can be achieved by homologous recombination, as shown in Figures 7A, 7B, 7C, or 7D, or non-homologous recombination, as shown in Figures 7E or 7F. When the polypeptide encoded by the modified gene is produced in soybean cells, it is cleaved at the C-terminus of the asparagine residue by vacuolar protease (VPE) in ripening seeds, producing an umami-enhancing peptide consisting of Ala-Glu-Asp (AED) (Maehashi K. et al., Biosci. Biotechnol. Biochem., 1999, Vol. 63, No. 3, pp. 555-559). The sequence surrounding the modified portion, the modified polypeptide, the identified cleavage site, and the PAM sequence are shown in Table 1.

[0340] Example 7: Modification of the Coding Region of an Existing Gene The T-DNA region of the Agropin synthase (ags) gene in the Ti plasmid (NCBI Reference Number: AF242881.1) of Agrobacterium tumefaciens was modified solely through deletion manipulation using SpCas9 (PAM sequence: NGG) from the coding region to the 3'-end non-coding region to create a gene encoding a new polypeptide. Here, a gene encoding a polypeptide in which the delicious peptide (KGDEESLA) was added to the N-terminal fragment of ags was created. To identify the cleavage site, a search sequence mapped based on the above design procedure is shown in Figure 8. A modified gene can be constructed through four steps (Ia4). The stop codon and terminator sequence of the existing gene are deleted, and instead a new stop codon is generated by ligation. The sequences around the modified portion shown in Figure 8, the modified polypeptide, and the identified cleavage site and PAM sequence are shown in Table 6.

[0341] The resulting Agrobacterium containing the modified gene is introduced into plant cells by infection as shown in Figure 9.

[0342] Example 8: Example of modification of the coding region of an existing gene The 3'-UTR region of β-galactosidase (LacZ) from Escherichia coli (NCBI Reference Number: U00096.3) was modified solely by deletion using ScCas9 (PAM sequence: NNG) to create a gene encoding a polypeptide with a His tag attached to the C-terminus of LacZ. To identify the cleavage site, a search sequence mapped based on the design procedure described above is shown in Figure X1. A modified gene can be constructed through three (a) steps. A new stop codon is generated by ligation. The sequence surrounding the modified site, the modified polypeptide, and the identified cleavage site and PAM sequence shown in Figure 12 are shown in Table 7.

[0343] (Example of synthesis of pScCas9 (ampicillin resistance) plasmid) The sequence of tracrRNA derived from Streptococcus pyogenes was obtained from the genome of Streptococcus pyogenes (CP151447.1) and synthesized using an artificial gene. Using this artificial gene as a template, amplification was performed using Tra1 / Tra2 primers. On the other hand, the Cas9 (ScCas9) gene sequence derived from Streptococcus canis was obtained from the genome of Streptococcus canis (WP_003043819.1), and the gene sequence was optimized for E. coli and synthesized using an artificial gene. Using this artificial gene as a template, amplification was performed using SC1 / SC2 / SC3 primers. The two amplified fragments were amplified with the Tra1 / SC4 primers to obtain the tracrRNA-ScCas9 expression cassette. pUC19 was amplified with the UC1 / UC2 primers, and the tracrRNA-ScCas9 expression cassette was ligated by seamless cloning using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) to obtain the pScCas9 (ampicillin-resistant) plasmid.

[0344] (Example of synthesis of pLigKu (chloramphenicol resistance) plasmid) The LigD and Ku genes derived from M. tuberculosis were obtained from the genome of M. tuberculosis H37Rv (NC_000962.3), and the gene sequence was optimized for E. coli. The LigD and Ku genes were linked with a spacer sequence (SP-LigKu), and an artificial gene was synthesized by adding a 5LigKu sequence to the 5' end and a 3LigKu sequence to the 3' end. Furthermore, pUC19c, in which the ampicillin resistance gene had been replaced with a chloramphenicol resistance gene, was cleaved with SmaI, and the artificial gene was inserted in the forward direction relative to the direction of the Lac promoter, followed by cloning to obtain pLigDKu.

[0345] (Example of synthesis of pL2a, pL4a, pL6a (kanamycin resistance) plasmid) The pL2a plasmid was constructed by incorporating two gRNAs, each with an independent promoter. The sgRNA was connected directly below the J23119 promoter, and the two sgRNAs were linked with a spacer sequence (SP-PL). Furthermore, a 5PL sequence was added to the 5' side and a 3PL sequence was added to the 3' side, and the plasmid was inserted between Psci and NdeI of the plasmid pUC57-Kan using an artificial gene (Genscript) to obtain pL2a. pL4a and pL6a were constructed using the same procedure. The respective target sequences and PAM sequences are shown in Table 10, and the target sequences in the plasmids are shown in Table 11.

[0346] (Example of pScCas9 transformation) 100 ng of the vector pScCas9 plasmid was added to 10 μL of E. coli BL21 (DE3) heat shock competent cells (Invitrogen catalog number C600003), placed on ice for 20 minutes, heated at 42°C for 45 seconds, and finally placed on ice for 5 minutes. 100 μL of SOC medium was added, and the cells were osmotically cultured at 200 rpm and 30°C for 2 hours. The cells were then spread onto an LB plate containing 100 μg / mL of ampicillin antibiotic and incubated in an incubator at 30°C for 18 hours to obtain single colonies. The resulting colonies were osmotically cultured in LB medium containing 100 μg / mL of ampicillin antibiotic at 200 rpm and 30°C until logarithmic growth phase. After the culture, the cells were collected by centrifugation, washed twice with 10% glycerol, and suspended in 10% glycerol to obtain electrocompetent E. coli cells carrying the pScCas9 plasmid.

[0347] (Example of pLigKu transformation) 100 ng of the pLigKu vector plasmid was added to 80 μL of E. coli electrocompetent cells carrying the pScCas9 plasmid, and electroporation was performed under the conditions of the MicroPlusr (Bio-Rad) program Ec1. 500 μL of SOC medium was added, and the cells were osmotically cultured at 200 rpm and 30 ° C for 6 hours. The cells were then spread onto an LB plate containing 100 μg / mL of ampicillin and 20 μg / mL of chloramphenicol antibiotics, and 500 μL of 1 M glucose was added. The cells were then incubated in an incubator at 30 ° C for 18 hours to obtain single colonies. The resulting colonies were osmotically cultured at 200 rpm and 30 ° C until the logarithmic growth phase in LB medium containing 20 mM glucose and 100 μg / mL of ampicillin and 20 μg / mL of chloramphenicol antibiotics. After the culture, the medium was replaced with LB medium containing 0.1 mM IPTG and osmotic culture was performed at 200 rpm and 30° C. for 1 hour. After the culture, the cells were collected by centrifugation, washed twice with 10% glycerol, and suspended in 10% glycerol to obtain electrocompetent E. coli cells carrying the pScCas9 and pLigKu plasmids.

[0348] (Example of transformation of pL2a, pL4a, or pL6a) 100 ng of vector pL2s plasmid was added to 80 μL of electrocompetent E. coli cells carrying pScCas9 and pLigKu plasmids, and electroporation was performed using a MicroPlusr (Bio-Rad) (conditions: program Ec1). 500 μL of SOC medium was added, and the cells were osmotic cultured at 200 rpm and 30°C for 6 hours. The cells were then spread onto an LB plate containing 100 μg / mL ampicillin, 20 μg / mL chloramphenicol, and 50 μg / mL kanamycin antibiotics, and 500 μL of 1 M glucose. The plate was then incubated in an incubator at 30°C for 18 hours to obtain multiple colonies. Multiple colonies were obtained for pL4a and pL6a using the same procedure.

[0349] (Example of confirming genome editing) Figure 13 shows a method for fusing a His6-tag (HHHHHH) downstream of the C-terminus of E. coli LacZ. The resulting colonies were subjected to direct PCR and electrophoresis on 2% agarose to confirm the presence or absence of chromosomal cleavage / repair. Table 12 shows the primers used for the colonies obtained with pL2a, pL4a, and pL6a. When pL2a was amplified with DP1 / DP2 primers, the unprocessed chromosomal fragment was 1,539 bp, and when cleavage / repair occurred as planned, it was 1,000 bp. When pL4a was amplified with DP1 / DP3 primers, the unprocessed chromosomal fragment was 2,822 bp, and when cleavage / repair occurred as planned, it was 1,557 bp. Furthermore, pL6a was amplified with DP1 / DP4 primers, resulting in an unprocessed chromosomal fragment of 3,465 bp and a 2,840 bp fragment if the expected cleavage and repair occurred. Colonies that yielded bands of the expected size were further sequenced using the same primers to confirm the sequence of the PCR fragment. From these results, the three-site cleavage and repair model could be implemented.

[0350] The resulting modified gene is introduced from one E. coli into another E. coli. The introduction can be carried out in the manner shown in Figure 14. The sequences of the ligated fragment, junction sequence 1, junction sequence 2, and deleted region shown in Figure 14 are as follows:

[0351] Example 9: Example of modification of the coding region of an existing gene The coding region of the Maltose binding protein (MalE) gene of Escherichia coli (NCBI reference number: NZ_JRYM01000001.1) was modified solely by deletion using ScCas9 (PAM sequence: NNG) to create a gene encoding a polypeptide in which a new human GLP-1 was added to the C-terminus of the signal sequence 26 residues from the N-terminus of MalE. To identify the cleavage site, a search sequence mapped based on the above design procedure is shown in Figure 15. A modified gene can be constructed through 18 iterations of step (a). A new stop codon is generated by ligation. When the polypeptide encoded by the modified gene is transported into the periplasm, the signal sequence 26 residues from the N-terminus is cleaved, producing human GLP-1. The sequences around the modified portions shown in Figure 15, the modified polypeptides, and the identified cleavage sites and PAM sequences are shown in Table 14.

[0352] The resulting modified gene is introduced from one E. coli into another E. coli. The introduction can be carried out in the manner shown in Figure 16. Examples of junction sequence 1 and junction sequence 2 shown in Figure 16 are as follows. The sequences of the ligated fragment, junction sequence 1, junction sequence 2, and deleted region are as follows:

Claims

1. A method for producing a cell (excluding human cells) containing a modified gene that does not contain foreign DNA, comprising: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining the sequences by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and non-coding regions adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, functional RNA, or both.

2. The method according to claim 1, wherein at least two of the steps (a) are carried out simultaneously or consecutively using different combinations of the target sequences.

3. The method of claim 1, wherein the DNA of the first cell is genomic DNA.

4. The method of claim 1, wherein the DNA of the first cell is extranuclear DNA.

5. The method of claim 1, further comprising (b) selecting the cells that do not contain off-target mutations.

6. The method of claim 1, further comprising the step of: (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.

7. The method of claim 1, wherein at least one of the target sequences comprises the sequence of at least one linked fragment (first linked fragment), and at least one of the ends of the first linked fragment is connected to the sequence of all or part of the linked fragment or DNA that is linked to the first linked fragment in step (a).

8. The method of claim 1, which does not include the step of inserting foreign DNA into the DNA of said cell.

9. The method of claim 1, further comprising the step of inserting foreign DNA, to both ends of which a first homology arm and a second homology arm capable of homologous recombination with the DNA, into at least one of the deleted regions, adjacent to at least one of the deleted regions, or replacing part or all of at least one of the deleted regions, by recombinational repair of the cell, wherein in step (a), the foreign DNA is removed from the DNA by the deletion, and further comprising the step of selecting the cells in which the foreign DNA is not present.

10. The method of claim 9, wherein the foreign DNA comprises a cell selectable marker gene.

11. The method according to claim 9, wherein the first or second homology arm comprises the sequence of at least one linked fragment (first linked fragment), and the sequence of all or part of the linked fragment or DNA linked to the first linked fragment in step (a) is connected to at least one of the ends of the first linked fragment.

12. The method of claim 1, wherein the total length of the linked fragments is between 9 and 180 base pairs.

13. The method of claim 1, wherein the total length of the linked fragments is 30% or less of the total length of the deleted region.

14. The method of claim 1, wherein the existing gene and the modified gene encode polypeptides, and in a wild-type cell of the cell, the polypeptides encoded by the existing genes account for 10% or more by mass of total proteins.

15. The method of claim 1, wherein the existing gene and the modified gene encode a polypeptide, the coding region of the modified gene comprises a full length or a portion of at least one of the linked fragments, and the full length or portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids that is not encoded by the existing gene.

16. The method of claim 15, wherein the modified polypeptide region comprises a functional peptide sequence, an enzymatic cleavage sequence, or both.

17. The method of claim 1, wherein the existing gene and the modified gene encode polypeptides, and the total length of the polypeptide encoded by the modified gene is 20% or less of the total length of the polypeptide encoded by the existing gene.

18. The method of claim 1, wherein the existing gene and the modified gene encode polypeptides, and the full length of the polypeptide encoded by the modified gene is at least 50% of the full length of the polypeptide encoded by the existing gene.

19. The method of claim 1, wherein the cells are cells used in or for the manufacture of food products.

20. The method of claim 1, wherein the sequence-specific nuclease is performed by a CRISPR / Cas system.

21. The method of claim 20, wherein the CRISPR / Cas system comprises SpCas9-NG or a functional analogue thereof, or ScCas9 or a functional analogue thereof.

22. A cell (excluding a human cell) in which genomic DNA has been modified: the genomic DNA comprises, relative to wild-type genomic DNA of the cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type genomic DNA, at least one linked fragment corresponding to a region in the existing gene between the deleted regions (deleted regions), and a modified gene whose nucleotide sequence has been modified relative to the existing gene by the deletion; and at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the cell is configured to express a polypeptide, functional RNA, or both from the modified gene.

23. A cell (excluding human cells) containing a modified gene, obtained by a method comprising the steps of: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and non-coding regions adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; wherein the DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, functional RNA, or both.

24. A cell (excluding human cells) comprising a modified gene, comprising: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining by DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and non-coding regions adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA of the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions; and at least one linked fragment that is a region between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both; and (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell is derived or into a second cell derived from an organism that can mate with the organism from which the first cell is derived.

25. The cell of claim 22, wherein the total length of the linked fragments is 9 to 180 base pairs.

26. The cell of claim 22, wherein the total length of the linked fragments is 30% or less of the total length of the deleted region.

27. The cell of claim 22, wherein the existing gene and the modified gene encode polypeptides, and in a wild-type cell of the cell, the amount of protein encoded by the existing gene accounts for 10% or more of the mass of total protein.

28. The cell of claim 22, wherein the existing gene and the modified gene encode a polypeptide, the coding region of the modified gene comprises a full length or a portion of at least one of the linked fragments, and the full length or portion of the at least one linked fragment encodes a modified polypeptide region of 3 to 60 amino acids that is not encoded by the existing gene.

29. The cell of claim 28, wherein the modified polypeptide region comprises a functional peptide, an enzymatic cleavage sequence, or both.

30. The cell of claim 22, wherein the existing gene and the modified gene encode polypeptides, and the total length of the polypeptide encoded by the modified gene is 20% or less of the total length of the polypeptide encoded by the existing gene.

31. The cell of claim 22, wherein the existing gene and the modified gene encode polypeptides, and the full length of the polypeptide encoded by the modified gene is at least 50% of the full length of the polypeptide encoded by the existing gene.

32. The cell of claim 22, wherein the cell is a cell used in a food product or used in the manufacture of a food product.

33. A method for producing modified DNA that does not contain foreign DNA, comprising the steps of: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining through DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and non-coding regions adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; the DNA in the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both.

34. The method of claim 33, further comprising the step of: (c) introducing the modified gene obtained by step (a) into a cell of the same species as the organism from which the first cell is derived or into a second cell derived from an organism that can mate with the organism from which the first cell is derived.

35. A modified gene obtained from modified DNA that does not contain foreign DNA, comprising the steps of: (a) performing the steps of specifically cleaving the DNA of a first cell at at least two target sequences with a sequence-specific nuclease and rejoining through DNA double-strand break repair by the cell at least twice, thereby deleting at least two regions from an existing gene and non-coding regions adjacent to the existing gene, to generate a modified gene that differs in nucleotide sequence from the existing gene; wherein the DNA in the cell in step (a) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions, and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the full length or a portion of at least one of the linked fragments; and the modified gene is configured to express a polypeptide, a functional RNA, or both.

36. Modified DNA comprising, compared to wild-type DNA of a cell, at least two deleted regions (deleted regions) in an existing gene and its adjacent non-coding regions of the wild-type DNA, at least one linked fragment corresponding to a region in the existing gene between the deleted regions (deleted regions), and a gene whose nucleotide sequence is modified relative to the existing gene due to the deletions.

37. An organism (excluding humans) comprising a cell produced by the method of any one of claims 1 to 21 or a cell according to any one of claims 22 to 32.

38. A method for producing a gene product, comprising the step of expressing a gene product encoded by the modified gene in a cell produced by the method of any one of claims 1 to 21, a cell of any one of claims 22 to 32, or an organism (excluding humans) containing such a cell.

39. A marker for genetic testing comprising a modified gene in DNA produced by the method of claim 33 or 34, or part or all of the modified gene described in claim 35 or 36.

40. A method for producing cells (excluding human cells) containing modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, comprising performing the steps of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deleted site at least twice, thereby deleting at least two regions from an existing gene and the non-coding regions adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not included in the modified DNA due to the deletions; and at least one linked fragment that is a region sandwiched between the two deleted regions and remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and a step of designing an artificially modified gene configured to express a polypeptide, functional RNA, or both from the modified gene; (Ia3) a step of preparing the artificially modified gene; and (c) a step of introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.

41. A cell (excluding human cells) containing a modified gene, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deletion site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and (Ia3) a step of preparing the artificially modified gene: (c) introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.

42. A method for producing modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificially modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deletion site at least twice, thereby deleting at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and (Ia3) a step of preparing the artificially modified gene: (c) introducing the artificially modified gene obtained by step (Ia3) into a cell of the same species as the organism from which the first cell is derived, or into a second cell derived from an organism that can mate with the organism from which the first cell is derived, or into a second cell that can infect the first cell.

43. A modified gene obtained from modified DNA that does not contain foreign DNA, comprising: (Ia2) a step of designing an artificial modified gene, the step of specifically deleting a target sequence from the DNA of a first cell and ligating both outer ends of the deleted site at least twice to delete at least two regions from an existing gene and a non-coding region adjacent to the existing gene, thereby designing a modified gene that differs from the nucleotide sequence of the existing gene, wherein the DNA of the cell in step (Ia2) comprises: at least two deleted regions that are not included in the modified DNA due to the deletion; and at least one linked fragment that is a region sandwiched between the two deleted regions and is a portion that remains in the modified DNA; at least one of the linked fragments is 1 to 150 base pairs in length; the promoter region, the transcribed region, or both of the modified gene comprises the entire length or a portion of at least one of the linked fragments; and a step of designing an artificial modified gene configured to express a polypeptide, a functional RNA, or both of the modified gene from the modified gene; and (Ia3) A modified gene obtained by a method comprising the step of preparing the artificially modified gene.

44. An organism (excluding humans) comprising a cell produced by the method of claim 40, or a cell according to claim 41, a modified DNA obtained by the method of claim 42, or a modified gene according to claim 43.

45. A method for producing a gene product, comprising the step of expressing a gene product encoded by a modified gene in an organism (excluding humans) containing a cell produced by the method of claim 40, a cell of claim 41, a modified DNA obtained by the method of claim 42, or a modified gene of claim 43.

46. ​​A marker for genetic testing comprising modified DNA produced by the method of claim 42 or part or all of the modified gene of claim 43.

47. A method for producing a cell containing modified DNA that is free of foreign DNA, comprising: (i-1a) allowing foreign DNA to coexist with the DNA of the cell, wherein the foreign DNA comprises a marker gene and is configured so that the foreign DNA can be inserted into or adjacent to a specific region (deleted region) in the DNA of the cell; (i-2a) cleaving at least one end or the interior of the deleted region with a sequence-specific endonuclease and inserting the foreign DNA into or adjacent to the deleted region; and (iia) cleaving the DNA of the cell at least two sites with the sequence-specific endonuclease, excising the deleted region and the foreign DNA together from the DNA of the cell, and generating modified DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) selecting the cell based on the presence or absence of the marker gene, wherein step (iii) is performed after step (i-2a), after step (iia), or both.

48. The method of claim 47, wherein a portion of the deleted region is deleted upon insertion of the foreign DNA.

49. A method for producing a cell containing modified DNA that is free of foreign DNA, comprising: (ib) a step of allowing foreign DNA to coexist with the genomic DNA of the cell, wherein the foreign DNA contains a marker gene, and a step of cleaving both ends of a specific region (deleted region) in the DNA of the cell with a sequence-specific endonuclease and inserting the foreign DNA to replace the deleted region; and (iib) a step of cleaving the genomic DNA at at least two sites with the sequence-specific endonuclease to excise the foreign DNA from the DNA of the cell, and generating modified DNA in which the deleted region has been deleted by DNA double-strand break (DSB) repair in the cell; and (iii) a step of selecting the cell based on the presence or absence of the marker gene, wherein step (iii) is performed after step (ib), after step (iib), or both.

50. The method of claim 47 or 49, wherein the length of the deleted region is 1 to 60 bp.

51. A method for producing an organism, comprising the step of incorporating cells containing modified genomic DNA obtained by the method of claim 47 or 49 into the organism.

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