Method for producing a cell containing a deleted modified genomic DNA, method for producing an organism containing the cell

Abstract

The introduction and removal of foreign DNA, and the deletion of genomic DNA can be confirmed by a simple method, and a means for constructing a modified genomic DNA by deletion of a region on the genomic DNA that does not contain foreign DNA is provided. 【Solution means】(i) A step of cleaving the genomic DNA of a cell with a sequence-specific endonuclease and inserting, by homologous recombination repair, a foreign DNA containing a marker gene inside the deletion region, adjacent to the deletion region, or replacing the deletion region; (ii) A step of cleaving the genomic DNA at at least two positions with a sequence-specific endonuclease to obtain a genomic DNA that does not contain foreign DNA by DNA double-strand break repair of the cell; and (iii) A step of selecting cells based on the presence or absence of a marker gene, wherein a part of the genomic DNA is excised in (i), (ii), or both, and (iii) is performed after (i), after (ii), or after both of them.

Description

Technical Field

[0001] The present invention relates to a method for producing a cell containing a deleted modified genomic DNA, and a method for producing an organism containing the cell.

Background Art

[0002] Techniques for modifying the genome of a cell or an organism are known by genome editing techniques that directly manipulate genomic DNA. For example, by using the CRISPR / Cas system, zinc finger nuclease (ZFN), TALEN (transcription activator-like effector nuclease), meganuclease, etc., a specific sequence on the genome can be recognized and selectively cleaved. It is known that both ends of the cleaved genomic DNA are religated by the DNA double-strand break (DSB) repair mechanism originally possessed by the cell. When genomic DNA is cleaved at two locations, the region in between is excised and religated, and genomic DNA lacking the excised region can be artificially produced. It is known that several base deletions, insertions, substitutions, etc. can occur at the cleaved and ligated sites during the DSB repair process.

[0003] The deletion operation of genomic DNA by genome editing and the mutations introduced by DSB repair are essentially indistinguishable from the mutations caused by natural DNA double-strand breaks and repair in cells. Therefore, among the organisms (including cells) obtained by genome editing, those that do not transfer foreign nucleotides may be treated as organisms that do not fall under the "Genetically Modified Organisms" under the Cartagena Protocol on Biosafety to the Convention on Biological Diversity (Cartagena Protocol) (referred to as "non-genetically modified organisms"). Non-genetically modified organisms are subject to less regulation than genetically modified organisms and can be advantageous in terms of distribution, management, and commercial use.

[0004] On the other hand, in order to suppress the introduction of mutations during the DSB repair process, means for replacing or deleting a specific sequence on genomic DNA using homologous recombination repair (HDR) are also disclosed. In Non-Patent Documents 1 and 2, means for replacing a partial region of genomic DNA by a genome editing operation involving two HDRs are disclosed (see Fig. 1 of Non-Patent Document 1). In the first HDR, foreign DNA containing a GFP gene and a LoxP site or SNP is introduced, and cells containing the foreign DNA are separated by fluorescence-activated cell sorting (FACS). Next, as the second recombination, the GFP gene portion is removed by recombination with Cre recombinase or insertion of another foreign DNA. Cells from which the marker gene has been removed are obtained by separating cells that do not express GFP by FACS. In Non-Patent Document 3 as well, means for replacing a partial region of genomic DNA by a genome editing operation involving two HDRs are disclosed, and it is described that it is a genome editing method without "scars", which are undesirable secondary mutations.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] The methods disclosed in Non-Patent Documents 1 to 3 are intended for base substitution and insertion that do not exist on genomic DNA. However, in these methods, even if the mutations introduced by the second recombination repair are the same as those such as SNPs that can occur naturally on genomic DNA, since the modified genomic DNA is obtained by introducing a foreign DNA fragment, it corresponds to "one that transfers foreign nucleotides". Furthermore, when using LoxP, a foreign LoxP site will remain in the genomic DNA. Therefore, there is a problem that cells having the modified genome obtained by the methods of these documents correspond to genetically modified organisms under the Cartagena Protocol.

[0007] On the other hand, an object of the present invention is to provide a new means for constructing a genomic DNA modified by deletion of a region on the genomic DNA that does not contain foreign DNA, which can simply confirm the introduction of foreign DNA, the subsequent removal of foreign DNA, and the deletion of genomic DNA.

Means for Solving the Problems

[0008] As a result of intensive studies, the present inventors have found that by the following method, it is possible to simply confirm the removal of foreign DNA and the deletion of genomic DNA, and to construct a genomic DNA modified by a deletion operation that does not contain foreign DNA, and have completed the present invention: (i) A step of cleaving the genomic DNA of a cell with a sequence-specific endonuclease and inserting, by homologous recombination (HDR) repair, a foreign DNA containing a marker gene inside a specific region (deletion region) on the genomic DNA to be deleted, adjacent to the deletion region, or replacing the deletion region; (ii) A step of cutting out the region containing the foreign DNA from the genomic DNA by cleaving the genomic DNA at at least two positions with a sequence-specific endonuclease and obtaining a genomic DNA that does not contain the foreign DNA by DNA double-strand break (DSB) repair of the cell; and (iii) A step of selecting cells based on the presence or absence of the marker gene comprising A method in which a part of the genomic DNA is excised in step (i), step (ii), or both, and step (iii) is performed after step (i), after step (ii), or both.

[0009] The present invention includes, but is not limited to, the following aspects. [1] A method for producing a cell containing a modified genomic DNA that does not contain foreign DNA, comprising: (i-1a) A step of co-existing with the genomic DNA of the cell a foreign DNA to which a first homology arm and a second homology arm, which are sequences homologous to the genomic DNA of the cell, are added, wherein the foreign DNA contains a marker gene, and the first homology arm and the second homology arm are configured to be insertable into or adjacent to a specific region (deletion region) on the genomic DNA by homologous recombination (HDR) repair; (i-2a) A step of cleaving at least one end or the interior of the deletion region with a sequence-specific endonuclease and inserting it into or adjacent to the interior of the deletion region by homologous recombination repair of the cell; and (iia) A step of cleaving the genomic DNA at at least two positions with a sequence-specific endonuclease, excising the deletion region and the foreign DNA together from the genomic DNA, and generating a modified genomic DNA in which the deletion region is deleted by DNA double-strand break (DSB) repair of the cell; and (iii) A step of selecting the cell based on the presence or absence of the marker gene comprising and step (iii) is performed after step (i-2a), after step (iia), or both. [2] The method according to [1], wherein a part of the deletion region is deleted upon insertion of the foreign DNA. [3] A method for producing a cell containing a modified genomic DNA that does not contain foreign DNA, comprising: (i-1b) a step of allowing a foreign DNA to which a first homology arm and a second homology arm, which are sequences homologous to the genomic DNA of a cell, are added to coexist with the genomic DNA of the cell, the foreign DNA comprises a marker gene, a step in which 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 a sequence-specific endonuclease to excise the foreign DNA from the genomic DNA, and generating a modified genomic DNA in which the deleted region is deleted by DNA double-strand break (DSB) repair in the cell; and (iii) selecting the cells based on the presence or absence of the marker gene. Including, The process, wherein step (iii) is carried out after step (i-2b), after step (iib), or both. [4] The method according to any one of [1] to [3], wherein the length of the deleted region is 1 to 60 bp. [5] A method for producing an organism, comprising the step of including in an organism a cell containing modified genomic DNA obtained by the production method according to any one of [1] to [4]. Effect of the Invention

[0010] According to the method for producing cells and organisms of the present invention, the insertion and removal of foreign DNA, as well as the deletion of specific regions in genomic DNA, can be determined by the presence or absence of marker genes, and cells having genomic DNA that does not contain foreign DNA can be easily selected. [Brief description of the drawings]

[0011]

Figure 1A

Figure 1B

Figure 1C

Figure 2

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate. However, the drawings are merely illustrative, and the present invention is not limited to the embodiments shown in the drawings.

[0013] In this specification, unless otherwise specified, nucleotide sequences are described from the 5'-end side to the 3'-end side. In this specification, unless otherwise specified, amino acid sequences are described from the N-terminal side to the C-terminal side.

[0014] In this specification, "N" in a nucleotide sequence represents any one of adenine, guanine, cytosine, and uracil in the case of RNA, and any one of adenine, guanine, cytosine, and thymine in the case of DNA.

[0015] In this specification, "5'-end side" or "5'-end" representing a position on a gene or a position relative to a gene represents the 5'-end side or the 5'-end on the sense strand of the gene, unless otherwise specified. In this specification, "3'-end side" or "3'-end" representing a position on a gene or a position relative to a gene represents the 3'-end side or the 3'-end on the sense strand of the gene, unless otherwise specified.

[0016] As used herein, the term "exogenous" or "foreign" gene or nucleotide refers to a gene or nucleotide that is not found in a cell before genetic manipulation and is introduced or to be introduced into the cell by genetic manipulation.

[0017] As used herein, the term "genetically modified organism" refers to an organism (including organisms and cells) having a nucleic acid or a replica thereof obtained by a technique for processing nucleic acids extracellularly (so-called recombinant DNA technique) or a technique for fusing cells of an organism in the Cartagena Protocol on Biosafety. Therefore, for example, even if a foreign nucleotide is inserted into genomic DNA or a foreign nucleotide such as a foreign plasmid is introduced into a cell, if the foreign nucleotide is completely removed in the final cell and the organism composed of the cell, it does not fall under the category of genetically modified organisms.

[0018] As used herein, the term "non-genetically modified organism" represents an organism that does not fall under the category of genetically modified organisms.

[0019] As used herein, the term "functional analog" refers to a polypeptide having an amino acid sequence similar to a certain polypeptide (for example, 95% or more, 98% or more, 99% or more, or 99.9% or more amino acid identity) and exhibiting substantially the same function. Specific examples include polypeptides into which amino acid mutations that do not affect the activity have been introduced with respect to a certain polypeptide.

[0020] As used herein, the identity (%) of an amino acid sequence or nucleotide sequence is the numerical value of "identity" in an alignment performed with the default parameters by selecting "Align two or more sequences" in Protein BLAST or Nucleotide BLAST of NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0021] [Method for Producing a Cell Containing Modified Genomic DNA] A method for producing a cell containing modified genomic DNA by deletion, which is one embodiment of the present invention (sometimes referred to herein as the "production method of the present invention"), comprises: (i) a first step of cleaving the genomic DNA of a cell with a sequence-specific endonuclease and inserting a foreign DNA (referred to herein simply as "foreign DNA" or "first foreign DNA") containing a marker gene into a specific region (deletion region) on the genomic DNA to be deleted by homologous recombination (HDR) repair, or adjacent to the deletion region, or replacing the deletion region; (ii) a second step of excising the region containing the foreign DNA from the genomic DNA by cleaving the genomic DNA at at least two sites with a sequence-specific endonuclease, and obtaining a genomic DNA free of foreign DNA by DNA double-strand break (DSB) repair; and (iii) a third step of selecting the cells based on the presence or absence of a marker gene; In the first step, the second step, or both, a portion of the genomic DNA is excised by a sequence-specific endonuclease; The third step is characterized in that it is carried out after the first step, after the second step, or after both of them.

[0022] (1st step) In the first step, the genomic DNA is site-specifically cleaved by a sequence-specific endonuclease. When a first foreign DNA having 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 in the vicinity of 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 repair mechanism inherent to the cell. As a result, the first foreign DNA can be inserted into the cleaved position of the genomic DNA.

[0023] By using an appropriate sequence-specific endonuclease, the cleavage position can be adjusted, and as a result, the insertion site of the foreign DNA can be adjusted. In the present invention, the first foreign DNA can be inserted into, adjacent to, or replacing a specific region (deletion region) on the genomic DNA to be deleted by modification.

[0024] In one embodiment, the foreign DNA is inserted into or adjacent to the deletion region (referred to as embodiment i-2a). In this embodiment, in the second step, the marker gene of the foreign DNA and the deletion region can be easily excised together, and by selecting cells without the marker gene, cells having genomic DNA not containing the deletion region can be easily selected. Figure 1A shows an example of a mode in which a foreign DNA containing a marker gene (GFP, ampicillin resistance gene) is inserted adjacent to the deletion region. Also, Figure 1B shows an example of a mode in which a foreign DNA containing a marker gene (RFP, kanamycin resistance gene) is inserted inside the deletion region. Also, as shown in Figure 1C, cleavage does not have to be performed at one location, and cleavage may be performed at two or more locations selected from both ends and the inside of the deletion region. Thereby, when inserting the foreign DNA in the first step, genomic DNA in which at least a part of the deletion region is deleted can be obtained. In this mode, the first homology arm or the second homology arm is not particularly limited as long as it is configured to be able to insert the foreign DNA into or adjacent to a specific region (deletion region) on the genomic DNA by homologous recombination repair. In this mode, at least one of the first homology arm or the second homology arm contains a part or all of the deletion region, but the homology arm may further contain a region adjacent to the deletion region.

[0025] In another embodiment, a sequence-specific endonuclease cleaves both ends of the deletion region, the entire deletion region is excised, and then foreign DNA is inserted so as to replace the deletion region (referred to as mode i-2b). An example thereof is shown in FIG. 2. In such a case, the first homology arm and the second homology arm are sequences capable of homologous recombination with sequences (referred to as "outer sequences") outside both ends of the deletion region (the side of the genomic DNA remaining after the deletion operation).

[0026] The first step may include a step of co-existing foreign DNA used for homologous recombination with the genomic DNA of the cell. As means for co-existing foreign DNA with the genomic DNA of the cell, for example, introducing the foreign DNA itself into the cell, introducing nucleotides that are precursors of the foreign DNA (for example, circular DNA or linear DNA containing the sequence of the foreign DNA), and the like can be mentioned.

[0027] (Foreign DNA, homology arm) The length of the first or second homology arm is not particularly limited as long as it is a length capable of homologous recombination with the sequence of the first end or the second end on the genomic DNA, and each 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, for example, 10000 bp or less, 5000 bp or less, 2000 bp or less, or 1000 bp or less.

[0028] In one embodiment, the first and second homology arms are sequences homologous to the sequence of the first end or the second end on the genomic DNA and do not include sequence substitution, insertion or gap on the genomic DNA. Such an embodiment is preferable in that it can reduce the risk that the cell containing the finally obtained modified genomic DNA corresponds to a genetically modified organism without introducing substitution, insertion or gap derived from the homology arm during the insertion of foreign DNA by homologous recombination.

[0029] As another embodiment, as long as homologous recombination is possible, the first or second homology arm may contain substitutions, insertions or gaps with respect to the sequences of the first or second end portions described above. In such an embodiment, mutations derived from the homology arm will be introduced into the genomic DNA. Therefore, in order to finally obtain a cell as a non-genetically modified organism, it is necessary to separately go through a step of removing the region containing the mutation from the genomic DNA.

[0030] The foreign DNA contains a marker gene for cell selection. Thereby, the presence or absence of the foreign DNA in the genomic DNA of the cell can be discriminated by the presence or absence of the product of the marker gene. As the marker gene, either a positive selection marker gene or a negative selection marker gene can be used. The positive selection marker gene is a gene that enables cells to be selected based on its presence. 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-based resistance gene (ALS), glyphosate resistance gene (EPSPS), etc.), reporter enzyme genes (luciferase, β-galactosidase, β-glucuronidase (GUS), dihydrofolate reductase (DHFR) gene, etc.). The negative selection marker gene is a gene that enables cells to be selected based on its absence. Examples include genes encoding toxic proteins, suicide genes (HSV-TK, iCasp9, etc.).

[0031] When a fluorescent protein is used as a marker gene, cells can be selected on a large scale and easily by a cell sorter or the like. Also, when a drug resistance gene is used as a marker gene, cells having the resistance gene are selected by culturing the cells in a medium containing a drug. Therefore, as the marker gene for cell selection used in the production method of the present invention, a fluorescent protein or a drug resistance gene is more preferable. Among them, when the marker gene is a fluorescent protein, it is possible to confirm the absence thereof by the fluorescence intensity of the cells, and it is particularly preferable because both the presence and absence of the foreign DNA can be confirmed relatively easily.

[0032] In one embodiment, one foreign DNA contains one marker gene. In another embodiment, as also illustrated in FIGS. 1A to 1C, one foreign DNA contains two or more marker genes. In such an embodiment, it is preferable in that the selection accuracy of the target cells can be further increased by combining the selections by a plurality of marker genes.

[0033] The foreign DNA may contain a sequence essential for cleavage of the target sequence, such as a PAM sequence in the CRISPR / Cas system, so that the foreign DNA can be cleaved in the second step.

[0034] (Deletion region) The position, function, and sequence of the genomic DNA in the deletion region are not particularly limited as long as both ends can be cleaved by the presence of the target sequence of the sequence-specific endonuclease, and can be appropriately selected according to the purpose of modification of the genomic DNA.

[0035] The length of the deletion region is not particularly limited and can 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 by a simple method such as the polymerase chain reaction (PCR) method. On the other hand, the production method of the present invention can easily determine whether the target region has been correctly removed by combining the labeling of the deletion region with a marker gene and the cleavage by a sequence-specific endonuclease. From the viewpoint of more significantly demonstrating the effects of the present invention, the length of the deletion region is preferably a length at which it is difficult to distinguish the difference in base length by the PCR method, for example, 1 to 60 bp, more preferably 1 to 50 bp, still more preferably 1 to 40 bp, and even more preferably 1 to 30 bp.

[0036] (Step 2) In the second step, the genomic DNA is cleaved at at least two positions by a sequence-specific endonuclease to excise the region containing the foreign DNA from the genomic DNA, and then genomic DNA not containing the foreign DNA is obtained by DNA double-strand break (DSB) repair. The DSB repair is carried out by either non-homologous end joining (NHEJ) repair in which ends without homologous regions are ligated or microhomology-mediated end joining (MMEJ) repair in which ends having complementary sequences of about 5 to 20 bases are ligated. However, from the viewpoint of high freedom of the target sequence, it is more preferable to use ligation by NHEJ repair.

[0037] During DSB repair, deletions and insertions of several nucleotides may occur at the ends generated by cleavage. Even if such mutations associated with DSB repair are present in the modified genomic DNA, since they do not involve the insertion of foreign DNA, the cell or organism containing the modified genomic DNA does not correspond to a genetically modified organism.

[0038] In one embodiment, mutations associated with DSB repair are included in the modified genomic DNA. The number of mutated bases is defined by the number of substitutions or gaps in the 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 at both ends of at least one cleavage site.

[0039] Depending on the embodiment of the first step, in the second step, the deletion region on the genomic DNA is also excised together with the foreign DNA and removed from the genomic DNA. For example, when the first step is the above embodiment (i-2a), it is preferable to excise the deletion region and the foreign DNA together in a form that does not include other regions (referred to as embodiment iia). In such an embodiment, the fact that the deletion region has been removed from the genomic DNA can be easily confirmed by the absence of the marker gene of the foreign DNA.

[0040] Also, as in the case of the above embodiment (1-2b), when the deletion region has already been removed in the first step, both ends of the foreign DNA may be cleaved to remove the foreign DNA (referred to as embodiment iib).

[0041] The first step and the second step can be performed on two or more deletion regions. In that case, in the first step, foreign DNA containing the same marker gene or different markers can be inserted in two or more deletion regions.

[0042] (Step 3) The third step is a step of selecting cells based on the presence or absence of a marker gene. By performing the third step after the first step and selecting cells in which the marker gene is present, cells into which foreign DNA has been inserted into genomic DNA can be selected. Further, by performing the third step after the second step and selecting cells in which the marker gene is absent, cells from which the foreign DNA containing the marker gene has been removed by the second step can be selected. The third step can be performed after the first step, after the second step, or both. However, from the viewpoint of easily discriminating both the insertion and removal of foreign DNA and the removal of the deletion region, it is preferable to perform it after both the first step and the second step. As illustrated in FIGS. 1A to C and FIG. 2, since the insertion of foreign DNA in the first step or the removal of foreign DNA in the second step is associated with the removal of the deletion region, by performing the third step after the first step and the second step, cells from which foreign DNA and the deletion region have been removed can be easily selected.

[0043] When the first step and the second step are performed for two or more deletion regions, in one embodiment, the third step is performed after performing the first step and the second step for each deletion region. In another embodiment, the third step is performed after performing the first step or the second step for two or more deletion regions, preferably all deletion regions. Among them, it is preferable to perform the third step after performing the first step and the second step for all deletion regions. When selection is performed after collectively inserting or removing foreign DNA in this way, the foreign DNA inserted into each deletion region may be labeled with the same marker gene or with different marker genes.

[0044] (Cell) The cell is a eukaryotic or prokaryotic cell, preferably a eukaryotic cell. In the present specification, the cells produced by the production method of the present invention include proliferating cells (replicas) obtained by culturing or the like after being produced.

[0045] Examples of eukaryotes include animals, plants, fungi, protists, etc., but are preferably animals or plants.

[0046] 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, hamsters, etc.; birds such as budgerigars, parrots, etc.; reptiles such as lizards, snakes, etc.; amphibians such as frogs, Japanese common toads, etc.; fish such as salmon, tuna, bonito, sea bream, yellowtail, eels, medaka, etc.; and arthropods such as insects and crustaceans.

[0047] Examples of plants include gramineous plants such as wheat, rice, barley, oats, rye, corn, sugarcane, millet, and foxtail millet; leguminous plants such as soybeans, azuki beans, peas, and kidney beans; solanaceous plants such as tobacco, tomatoes, eggplants, peppers, and potatoes; cucurbitaceous plants such as pumpkins, watermelons, cucumbers, melons, and wax gourds; seed plants such as Arabidopsis thaliana, buckwheat, cassava, sweet potatoes, taro, mulberry, pine, cedar, cypress, ginkgo, and eucalyptus; fern plants; and moss plants.

[0048] Examples of fungi include yeasts such as the genus Saccharomyces, Pichia, Schizosaccharomyces, and Candida; filamentous fungi such as the genus Rhizopus and Aspergillus; dimorphic fungi such as the genus Penicillium; and mushrooms.

[0049] Examples of protists include algae (green algae, red algae, brown algae, cyanobacteria, Euglenophyta, Haptophyta, Cryptophyta, etc.), ciliates, amoebas, etc.

[0050] Examples of prokaryotes include bacteria (Escherichia coli, Bacillus subtilis, thermophilic bacteria such as the genus Thermus, rhizobia, cyanobacteria, etc.) or archaea.

[0051] The cell is not particularly limited as long as it is genome - editable. In one embodiment, the cell is a cell in a tissue isolated from a living body of a human or a non - human organism (ex vivo cell; for example, a cell derived from an excised organ, a leaf or a stem of a plant, etc.), or an in vitro cell (a primary cultured cell, a sub - cultured cell, a cultured cell differentiated from a stem cell such as an iPS cell, etc.). In one embodiment, the cell is an in vitro cell. In another embodiment, the cell is a cell in a living body (in vivo) of a non - human organism. In yet another embodiment, the cell is a cell in a human living body.

[0052] Also, the cell type is not particularly limited as long as it is genome - editable. When producing a genome - edited organism, germ cells, pluripotent cells (such as iPS cells, ES cells), or dedifferentiable cells (such as plant cells) are preferred.

[0053] In one embodiment, the cell is a cell used in food or used in the production of food. Such cells are preferably, for example, (i) those contained in food, such as those generally served for eating and drinking as food according to Article 7, Paragraph 2 of the Food Sanitation Law of Japan, or (ii) those generally used in the production of said food. Here, "food" includes so - called general foods as well as food additives. Specific examples of (ii) include cells used in the production of fermented foods such as soy sauce and sake, and cells for fermentative production of amino acids, enzymes for food production, and other food additives.

[0054] (Sequence - specific endonuclease, target sequence) To delete a region on genomic DNA in the first step and the second step, the genomic DNA is cleaved by a sequence - specific endonuclease specific to the target sequence. In this specification, the target sequence refers to the sequence necessary for the sequence - specific endonuclease to identify and cleave the cleavage site.

[0055] The sequence-specific endonuclease is not particularly limited as long as it can cleave the target sequence in the target cell, but it preferably recognizes and cleaves a target sequence that exists only once on the genomic DNA of the cell (for example, a specific target sequence of 16 bases or more or 20 bases or more). Examples of such endonucleases include the CRISPR / Cas system, zinc finger nuclease (ZFN), TALEN (transcription activator-like effector nuclease), meganuclease, and the like. The sequence-specific endonuclease may be a wild-type enzyme or a modified mutant.

[0056] For each target sequence, different types or the same type of sequence-specific endonuclease may be used. Therefore, different sequence-specific endonucleases can also be used in each step of the production method of the present invention. However, from the viewpoint of simplifying the operation, it is preferable that all the sequence-specific endonucleases are of the same type.

[0057] When the cell is a eukaryotic cell, the sequence-specific endonuclease preferably contains at least one nuclear localization signal (NLS).

[0058] Among them, the CRISPR / Cas system is preferable as the sequence-specific endonuclease because specificity for a specific target sequence can be easily imparted. The CRISPR / Cas system includes a Cas protein having endonuclease activity and a guide RNA that specifies the target sequence. Cas pairs with the guide RNA and cleaves a nucleotide having a protospacer adjacent motif (PAM) sequence at a specific position. The Cas protein and the guide RNA may be naturally occurring or a non-naturally occurring combination.

[0059] As the Cas protein, Cas9 or Cas12a (Cpf1) is preferred in that it has DNA cleavage activity and has the activity of precisely cleaving at the target sequence. CRISPR / Cas9 forms blunt ends whether the PAM sequence is included on the sense strand or the antisense strand. Therefore, Cas9 has the advantage that the constraints on the target sequence are relatively few compared to other Cas proteins.

[0060] Naturally, CRISPR / Cas9 includes crRNA as the guide RNA and further tracrRNA as components. However, in the production method of the present invention, a system using a single-stranded guide RNA (sgRNA) in which Cas, tracrRNA, and the target sequence are a single RNA is more preferably used.

[0061] When using the CRISPR / Cas9 system, for Cas9 protein, guide RNA, etc., those that can be used in the target cells may be selected based on known literature. The Cas9 protein is preferably Cas9 of the genus Staphylococcus, and more preferably Cas9 derived from Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus. These Cas9s may be wild-type or mutant as long as they have target sequence specificity and DNA cleavage activity.

[0062] The limitation of nucleotides by the PAM sequence complicates the design of guide RNAs. Therefore, Cas9 with a small number of positions limited to specific nucleotides by the PAM sequence is particularly preferably used. As such Cas9, for example, Cas9 derived from Streptococcus pyogenes that recognizes NGG as the PAM sequence (SpCas9), a variant of SpCas9 that recognizes NG as the PAM sequence (SpCas-NG) (Nishimasu, H. et al., 2018, Science, Vol.361, pp.1259-1262), xCas9-3.7 (Hu, J.H. et al., 2018, Nature, Vol.556, pp.57-63), ScCas9 that recognizes NNG, (Chatterjee, P. et al., 2018, Sci. Adv. Vol.4, eaau0766), Sc ++ (Chatterjee, P. et al. Nat. Biotechnol., 2020, Vol.38, pp.1154-1158), SpG that recognizes NGN (Walton R.T. et al., 2020, Science, Vol.368, pp.290-296), or functional analogs thereof, etc. can be mentioned.

[0063] In SpCas9 and SpCas-NG, the target sequence consists of 5′-N(17)-(Cas cleavage site)-NNN-PAM sequence-3′. Here, N(17) represents any 17 nucleotides. In this specification, the 3 nucleotides immediately before the PAM sequence are called the spacer sequence.

[0064] When the sequence-specific nuclease is a zinc finger nuclease (ZFN), TALEN (transcription activator-like effector nuclease), or meganuclease, two nucleases with different target sequences can coexist in the cell to perform cleavage in the first step or the second step.

[0065] When performing target sequence-specific cleavage with the CRISPR / Cas system, when performing cleavage at multiple sites, for example, one type of Cas and two types of guide RNAs complementary to the target sequences at each cleavage site can be used inside the cell. However, it is not necessary for Cas to be the same in each cleavage. When using multiple Cas, target sequences containing appropriate PAM sequences can be selected according to Cas.

[0066] (Step of introducing a sequence-specific endonuclease and / or a factor related to its function) In one embodiment, the production method of the present invention may further include a step of introducing a second foreign DNA containing a target sequence-specific endonuclease or its gene into the cell and a step of removing the second foreign DNA in order to make the target sequence-specific endonuclease function inside the cell.

[0067] When the target sequence-specific endonuclease is a CRISPR / Cas system, it may further include a step of introducing an appropriate guide RNA or a second foreign DNA capable of expressing the guide RNA inside the cell. These steps may be performed simultaneously or separately.

[0068] When directly introducing a sequence-specific endonuclease and a guide RNA into the cell, it is preferably introduced into the cell before cleavage.

[0069] The first foreign DNA and the second foreign DNA may be the same DNA molecule or different DNA molecules.

[0070] In one embodiment, from the viewpoint of facilitating complete removal of the second foreign DNA from the cell, the second foreign DNA exists inside the cell in a form separated from genomic DNA. Such a second foreign DNA can be introduced into the cell as a vector such as a plasmid, cosmid, artificial chromosome, etc. When the second foreign DNA is separated from genomic DNA, the second foreign DNA may be naturally lost, and the second foreign DNA can be removed by selecting cells that do not contain the second foreign DNA.

[0071] In another embodiment, a second foreign DNA is inserted into the genomic DNA. Such a second foreign DNA can be introduced into cells, for example, as linear DNA, a viral vector, or the like. In this embodiment, the production method of the present invention further includes a step of removing the second foreign DNA. Optionally, it may also include a step of selecting cells from which the second foreign DNA has been removed.

[0072] To confirm the absence of the second foreign DNA, for example, primers capable of specifically PCR-amplifying the second foreign DNA can be designed and detected by various PCR methods. In addition, the absence of the second foreign DNA on the genomic DNA can also be confirmed, for example, by genomic sequencing.

[0073] From the perspective of improving the expression in cells, the gene of the above-mentioned second foreign DNA target sequence-specific endonuclease may be adjusted to a codon usage frequency similar to that of the cells (so-called codon optimization).

[0074] In addition to the target gene, for example, a promoter, an enhancer, an insulator, an intron, a terminator, a polyA addition signal, a selection marker gene, etc. can be ligated to the vector.

[0075] The target gene inserted into the vector may be one or a plurality of different types per vector.

[0076] 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 in a living state of the cells. For example, it can be carried out by the liposome method (such as lipofection), the particle gun (gene gun) method, the electroporation method, the polyethylene glycol (PEG) method, the plasma method (see, for example, WO2018016217), the whisker method, the laser injection method, etc. When the cell is a plant cell, the particle gun method is preferred.

[0077] (Step of selecting cells without off-target mutations, step of removing off-target mutations) After going through the above first to third steps, the production method of the present invention preferably further includes a step of further selecting cells without off-target mutations. In this specification, "off-target mutation" refers to a mutation that occurs as a result of cleavage and DSB repair at a portion that is not originally intended to be cleaved.

[0078] The presence or absence of off-target mutations is determined, for example, by performing genome sequencing on the modified genomic DNA.

[0079] In addition to the above first to third steps, the production method of the present invention may further include a step of removing off-target mutations. Specific examples of methods for removing off-target mutations include the step of producing an organism containing the modified genome described later, and mating (backcrossing) the organism containing the modified genome with a homologous organism that is a non-recombinant organism without the off-target mutation (for example, a wild-type organism having the cells before modification).

[0080] (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 screening cells having the target modified genomic DNA based on the determined sequence. By including those steps, cells having the target modified genomic DNA can be isolated and concentrated.

[0081] The production method of the present invention may further include a step of growing cells containing the modified genomic DNA. For cell growth, known means used in the growth of the original cells (for example, in vitro cell culture using a medium that can be used for culturing the cells) can be used. The step of growing cells containing the modified genomic DNA can be performed either before, during, or after the production of cells containing the modified genomic DNA, but it is preferably performed after the production of cells.

[0082] In the production method of the present invention, the cells may appropriately undergo a dedifferentiation process, a differentiation process, etc. according to their intended use. These processes can be carried out either before, during, or after the production of the modified genomic DNA.

[0083] [Method for producing an organism] One embodiment of the method for producing an organism of the present invention includes a step of incorporating a cell containing modified genomic DNA obtained by the above [method for producing a cell containing modified genomic DNA] into an organism.

[0084] Examples of the step of incorporating a cell containing modified genomic DNA into an organism include the following cases, and each can be produced using a known method used for the production of an organism: (I) Introduce a cell containing modified genomic DNA into an organism or its embryo; (II) Induce germ cells from a cell containing modified genomic DNA and generate an organism; (III) Apply the method for producing a cell containing modified genomic DNA directly to the cells of the organism itself; (IV) Propagate the organism obtained by any of (I) to (III).

Claims

1. 1. A method for producing a cell (other than a human cell) that contains modified genomic DNA that is free of foreign DNA, comprising: (i-1a) a step of allowing a foreign DNA to which a first homology arm and a second homology arm, which are sequences homologous to the genomic DNA of a cell, are added to coexist with the genomic DNA of the cell, the foreign DNA comprises a marker gene, the first homology arm and the second homology arm are configured to allow the foreign DNA to 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 within the deleted region with a sequence-specific endonuclease and inserting the foreign DNA within or adjacent to the deleted region by homologous recombination repair in the cell; and (iia) cleaving the genomic DNA at at least two sites with a sequence-specific endonuclease to excise the deleted region and the foreign DNA together from the genomic DNA, thereby generating a modified genomic DNA in which the deleted region is deleted by DNA double-strand break (DSB) repair in the cell; and (iii) selecting the cells based on the presence or absence of the marker gene. Including, The method of claim 1, wherein step (iii) is performed once each after step (i-2a) and after step (iia), and 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.

2. The method of claim 1, wherein a portion of the deleted region is deleted upon insertion of the foreign DNA.

3. 1. A method for producing a cell (other than a human cell) that contains modified genomic DNA that is free of foreign DNA, comprising: (i-1b) a step of allowing a foreign DNA to which a first homology arm and a second homology arm, which are sequences homologous to the genomic DNA of a cell, are added to coexist with the genomic DNA of the cell, the foreign DNA comprises a marker gene, a step in which 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 a sequence-specific endonuclease to excise the foreign DNA from the genomic DNA, and generating a modified genomic DNA lacking the deleted region by DNA double-strand break (DSB) repair in the cell; and (iii) selecting the cells based on the presence or absence of the marker gene. Including, The method of claim 1, wherein step (iii) is performed once each after step (i-2b) and after step (iib), and 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.

4. The method according to claim 1 or 3, wherein the length of the deleted region is 1 to 1000 bp.

5. A method for producing an organism (excluding humans), comprising the steps of: (i-1a) a step of allowing a foreign DNA to which a first homology arm and a second homology arm, which are sequences homologous to the genomic DNA of a cell, are added to coexist with the genomic DNA of the cell, the foreign DNA comprises a marker gene, the first homology arm and the second homology arm are configured to allow the foreign DNA to 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 within the deleted region with a sequence-specific endonuclease and inserting the foreign DNA within or adjacent to the deleted region by homologous recombination repair in the cell; and (iia) cleaving the genomic DNA at at least two sites with a sequence-specific endonuclease to excise the deleted region and the foreign DNA together from the genomic DNA, thereby generating a modified genomic DNA in which the deleted region is 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, which is performed once each after step (i-2a) and after step (iia), in which after step (i-2a), the cells are selected based on the presence of the marker gene, and after step (iia), the cells are selected based on the absence of the marker gene; and (iv) including cells selected based on the absence of said marker gene after step (iia) in an organism. A method for producing an organism, comprising:

6. A method for producing an organism (excluding humans), comprising the steps of: (i-1b) a step of allowing a foreign DNA to which a first homology arm and a second homology arm, which are sequences homologous to the genomic DNA of a cell, are added to coexist with the genomic DNA of the cell, the foreign DNA comprises a marker gene, a step in which 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 a sequence-specific endonuclease to excise the foreign DNA from the genomic DNA, and generating a modified genomic DNA lacking the deleted region 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, which is performed once each after step (i-2b) and after step (iib), in which 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; and (iv) including cells selected based on the absence of said marker gene after step (iib) in an organism. A method for producing an organism, comprising:

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