Methods for preparing cells containing deleted modified genome, and methods for preparing organisms comprising the cells

Abstract

To provide means for constructing a modified genome not containing heterogenous DNA by deleting a region in genomic DNA, which enables confirmation of introduction and elimination of heterogenous DNA as well as deletion in the genomic DNA by a simple procedure.SOLUTION: The method comprises the steps of: (i) cleaving the genomic DNA in a cell by a sequence-specific endonuclease and inserting a heterogenous DNA with a marker gene at a position inside of, or adjacent to the deletion region (region to be deleted), or so as to substitute the deletion region by homologous recombination repair; (ii) cleaving the genomic DNA by the sequence-specific endonuclease at, at least two positions, to obtain genomic DNA not containing the heterogenous DNA by double stranded break repair of the cell; and (iii)selecting cells on the basis of the presence or absence of the marker gene, where in (i) or (ii) or both a part of the genomic DNA is cut out, and (iii) is carried out after (i), or after (ii) or after both of them.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to methods for producing cells containing deleted, modified genomic DNA, and to methods for producing organisms containing such cells. [Background technology]

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

[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, which may offer advantages in terms of distribution, management, and commercial use.

[0004] On the other hand, methods for replacing or deleting specific sequences in genomic DNA using homologous recombination repair (HDR) to suppress the introduction of mutations during DSB repair have also been disclosed. Non-Patent Documents 1 and 2 disclose a method for replacing a partial region of genomic DNA by a genome editing procedure involving two rounds of HDR (see Fig. 1 in Non-Patent Document 1). In the first round of 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, in the second round of recombination, the GFP gene portion is removed by recombination using Cre recombinase or by insertion of another foreign DNA. Cells from which the marker gene has been removed are obtained by separating cells that do not express GFP using FACS. Non-Patent Document 3 also discloses a method for replacing a partial region of genomic DNA by a genome editing procedure involving two rounds of HDR, and describes this genome editing method as being free of "scars," which are undesirable secondary mutations. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kuhn R. and Chu T. Genome Biology, 2015, Vol.16, Article number 244 [Non-patent document 2] Xi L. et al. Genome Biology, 2015, Vol.16, Article number 231 [Non-patent document 3] Lamb AM et al., Fly, 2017, Vol.11, No.1, pp.53-64 Summary of the Invention [Problem to be solved by the invention]

[0006] The methods disclosed in Non-Patent Documents 1 to 3 aim to substitute or insert bases that do not exist in genomic DNA. However, even if the mutations introduced by the second recombinational repair in these methods are similar to SNPs or other mutations that can naturally occur in genomic DNA, the modified genomic DNA is obtained by introducing an exogenous DNA fragment, and therefore falls under the category of "introducing an exogenous nucleotide." Furthermore, when LoxP is used, the exogenous LoxP site remains in the genomic DNA. Therefore, there is a problem in that cells with modified genomes obtained by the methods described in these documents fall under the category of genetically modified organisms under the Cartagena Protocol.

[0007] On the other hand, the present invention aims to provide a new means for constructing genomic DNA that has been modified by deleting a region on the genomic DNA that does not contain foreign DNA, which 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. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have found that the removal of foreign DNA and deletion of genomic DNA can be confirmed in a simple manner by the following method, and that genomic DNA that is modified by a deletion manipulation and does not contain foreign DNA can be constructed, thereby completing the present invention: (i) cleaving the genomic DNA of a cell with a sequence-specific endonuclease and inserting a foreign DNA containing a marker gene into a specific region of the genomic DNA to be deleted (deletion region) by homologous recombination (HDR) repair, or adjacent to the deletion region, or replacing the deletion region; (ii) 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 then repairing the DNA double-strand break (DSB) in the cell to obtain genomic DNA free of the foreign DNA; and (iii) selecting cells based on the presence or absence of the marker gene Including, a portion 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.

[0009] The present invention includes, but is not limited to, the following aspects. [1] 1. A method for producing a cell containing modified genomic DNA that is free of exogenous DNA, comprising: (i-1a) a step of allowing foreign DNA to coexist with the genomic DNA of a cell, the foreign DNA having a first homology arm and a second homology arm added thereto, the first homology arm and the second homology arm being sequences homologous to the genomic DNA of the cell, the foreign DNA comprises a marker gene, a step in which 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 the interior of the deleted region with a sequence-specific endonuclease and inserting the fragment into or adjacent to the deleted region by homologous recombination repair in the cell; and (iia) cleaving the genomic DNA at least two times with a sequence-specific endonuclease to excise the deleted region and the foreign DNA together from the genomic DNA, thereby generating 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, wherein 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 when the foreign DNA is inserted. [3] 1. A method for producing a cell containing modified genomic DNA that is free of exogenous DNA, comprising: (i-1b) a step of allowing 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 the 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 least two times with a sequence-specific endonuclease to excise the foreign DNA from the genomic DNA, and generating 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, wherein step (iii) is performed 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 incorporating cells containing modified genomic DNA obtained by the production method according to any one of [1] to [4] into an organism. [Effects of the Invention]

[0010] According to the method for producing cells and organisms of the present invention, the insertion and removal of foreign DNA and the deletion of specific regions on 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 explanation of the drawings]

[0011] [Figure 1A] FIG. 1 shows an example of an embodiment in which foreign DNA is inserted adjacent to a deleted region, and then the foreign DNA and the deleted region are excised. [Figure 1B] FIG. 1 shows an example of an embodiment in which foreign DNA is inserted into a deleted region and then the foreign DNA and the deleted region are excised. [Figure 1C] FIG. 1 shows an example of an embodiment in which foreign DNA is inserted so as to replace part of the deleted region by cutting at two points within the deleted region, and then the foreign DNA and the deleted region are excised. [Figure 2] FIG. 1 shows an example of an embodiment in which foreign DNA is inserted to replace a deleted region and then excised. DETAILED DESCRIPTION OF THE INVENTION

[0012] 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.

[0013] 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.

[0014] As used herein, "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.

[0015] As used herein, 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, unless otherwise specified. As used herein, 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.

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

[0017] As used herein, "genetically modified organisms" refers to organisms (including organisms and cells) that contain nucleic acids or replicates thereof obtained by techniques for processing nucleic acids extracellularly (so-called recombinant DNA techniques) or techniques for fusing living organism cells, as defined in the Cartagena Protocol. Therefore, even if foreign nucleotides are inserted into genomic DNA or foreign nucleotides such as foreign plasmids are introduced into cells during the process, they do not fall under the category of genetically modified organisms as long as those foreign nucleotides are completely removed in the final cells and organisms comprising those cells.

[0018] As used herein, the term "non-genetically modified organism" refers to 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 that has an amino acid sequence similar to that of a given polypeptide (e.g., amino acid identity of 95% or more, 98% or more, 99% or more, or 99.9% or more) and exhibits the same qualitative function. Specific examples include polypeptides into which amino acid mutations that do not affect activity have been introduced into a given polypeptide.

[0020] 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.

[0021] [Method for producing cells containing modified genomic DNA] One embodiment of the present invention is a method for producing a cell containing modified genomic DNA by deletion (sometimes referred to herein as the "production method of the present invention"), which 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 of the genomic DNA to be deleted (deleted region) by homologous recombination (HDR) repair, or adjacent to the deleted region, or replacing the deleted 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 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 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, 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, using the cell's inherent repair mechanism. As a result, the first foreign DNA can be inserted into the cleaved site of the genomic DNA.

[0023] By using an appropriate sequence-specific endonuclease, the cleavage site 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.

[0024] In one embodiment, the foreign DNA is inserted within or adjacent to the deleted region (referred to as embodiment i-2a). In this embodiment, in the second step, the marker gene of the foreign DNA and the deleted region can be easily excised together, and cells having genomic DNA that does not contain the deleted region can be easily selected by selecting cells that do not contain the marker gene. Figure 1A shows an example of an embodiment in which foreign DNA containing a marker gene (GFP, ampicillin resistance gene) is inserted adjacent to the deleted region. Figure 1B shows an example of an embodiment in which foreign DNA containing a marker gene (RFP, kanamycin resistance gene) is inserted within the deleted region. Furthermore, as shown in Figure 1C, cleavage does not need to be performed at a single site; cleavage may be performed at two or more sites selected from both ends and within the deleted region. This allows genomic DNA in which at least a portion of the deleted region is deleted to be obtained during the insertion of foreign DNA in the first step. In this embodiment, the first homology arm or the second homology arm is not particularly limited as long as it is configured so that foreign DNA can be inserted into or adjacent to a specific region (deleted region) on the genomic DNA by homologous recombination repair. In this embodiment, 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.

[0025] In another embodiment, a sequence-specific endonuclease cleaves 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 this 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 (toward the genomic DNA remaining after the deletion manipulation).

[0026] 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, or introducing nucleotides that serve as precursors of the foreign DNA (e.g., circular or linear DNA containing the sequence of the foreign DNA) into the cell.

[0027] (foreign DNA, homology arms) 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.

[0028] In one embodiment, the first and second homology arms have sequences homologous to the sequences at the first end or second end of the genomic DNA, but do not contain sequence substitutions, insertions, or gaps in 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 cells containing the modified genomic DNA are genetically modified organisms.

[0029] 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 final cell a non-genetically modified organism, a separate step of removing the region containing the mutation from the genomic DNA is required.

[0030] The foreign DNA contains a marker gene for cell selection. This allows the presence or absence of the foreign DNA in the genomic DNA of a cell to be determined by the presence or absence of the marker gene product. Both positive and negative selection marker genes can be used as marker genes. Positive selection marker genes are genes that allow cells to be selected based on their presence, such as 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, such as genes encoding toxic proteins and suicide genes (HSV-TK, iCasp9, etc.).

[0031] 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.

[0032] In one embodiment, one exogenous DNA comprises one marker gene. In another embodiment, one foreign DNA contains two or more marker genes, as illustrated in Figures 1A to 1C. This embodiment is preferable because the accuracy of selecting the target cells can be further improved by combining selection using multiple marker genes.

[0033] 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.

[0034] (deleted region) The location, function, and sequence of the deletion region on the genomic DNA are not particularly limited as long as both ends can be cleaved by the presence of a target sequence for the sequence-specific endonuclease, and can be selected appropriately depending on the purpose of modifying the genomic DNA.

[0035] The length of the deleted region is not particularly limited and may be, for example, 10,000 to 100,000 base pairs (bp), 10,000 to 10,000 bp, 1 to 1,000 bp, or 1 to 100 bp. The shorter the deleted region, the more difficult it is to determine whether the deleted 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 deleted 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. To more significantly achieve the effects of the present invention, the length of the deleted 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.

[0036] (2nd process) In the second step, the genomic DNA is cleaved at least two times with 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 free of foreign DNA. DSB repair can be performed by either non-homologous end joining (NHEJ), which joins ends that do not have homologous regions, or microhomology-mediated end joining (MMEJ), which joins ends that share a complementary sequence of about 5 to 20 bases. However, NHEJ repair is more preferred because of its high degree of flexibility in target sequence selection.

[0037] During DSB repair, several additional nucleotides may be deleted or inserted at the ends of the resulting break. Even if such DSB repair-related mutations 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.

[0038] 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 break site.

[0039] 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.

[0040] Furthermore, when the deleted region has already been removed in the first step, as in the case of 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).

[0041] Steps 1 and 2 can be performed on two or more deletion regions. In this case, in step 1, foreign DNA containing the same marker gene or different markers can be inserted into two or more deletion regions.

[0042] (3rd step) The third step 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 with the marker gene 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 without the marker gene, cells in which foreign DNA, including 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 1A-C and 2, 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.

[0043] 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.

[0044] (cell) The cells are eukaryotic or prokaryotic cells, preferably eukaryotic cells. As used herein, the cells produced by the production method of the present invention include proliferated cells (replicates) obtained by culturing or the like after production.

[0045] Eukaryotes include, for example, animals, plants, fungi, protists, etc., and 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, 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.

[0047] Examples of plants include grasses such as wheat, rice, barley, oats, rye, corn, sugarcane, foxtail millet, and barnyard millet; legumes such as soybeans, adzuki beans, peas, and kidney beans; solanaceae plants such as tobacco, tomato, eggplant, chili pepper, and potato; cucurbits such as pumpkin, watermelon, cucumber, melon, and Japanese cantaloupe; seed plants such as Arabidopsis, buckwheat, cassava, sweet potato, taro, mulberry, pine, cedar, cypress, ginkgo, and eucalyptus; ferns; and mosses.

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

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

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

[0051] 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, a plant leaf, a stem, etc.), or an in vitro cell (a primary culture cell, a passaged 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 the living body of a non-human organism (in vivo). In yet another embodiment, the cell is a cell in the living body of a human.

[0052] The type of cell is also not particularly limited as long as it is genome-editable. When creating a genome-edited organism, germ cells, pluripotent cells (iPS cells, ES cells, etc.), or cells that can be dedifferentiated (e.g., plant cells) are preferred.

[0053] 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 under 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.

[0054] (sequence-specific endonuclease, target sequence) To delete a region on genomic DNA in steps 1 and 2, the 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.

[0055] 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 (e.g., 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 activator-like effector nucleases), meganucleases, etc. The sequence-specific endonuclease may be a wild-type enzyme or a modified mutant.

[0056] The sequence-specific endonuclease may be of a different type for each target sequence, or may be of the same type. Therefore, different sequence-specific endonucleases may 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] If the cell is a eukaryotic cell, the sequence-specific endonuclease preferably contains at least one nuclear localization signal (NLS).

[0058] 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 with endonuclease activity and a guide RNA that specifies the target sequence. The Cas pairs with the guide RNA and cleaves nucleotides containing 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 occur in nature.

[0059] Cas9 or Cas12a (Cpf1) are preferred Cas proteins because they have DNA cleavage activity and pinpoint cleavage in target sequences. CRISPR / Cas9 forms blunt ends regardless of whether the PAM sequence is on the sense or antisense side. Therefore, Cas9 has the advantage of being less restricted in target sequences than other Cas proteins.

[0060] 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 using single-stranded guide RNA (sgRNA), in which Cas, tracrRNA, and the target sequence are combined into a single RNA.

[0061] When using the CRISPR / Cas9 system, Cas9 proteins, guide RNAs, and other components can be selected based on published literature and are compatible with target cells. Cas9 proteins are preferably derived from Staphylococcus, and more preferably from Streptococcus pneumoniae, S. pyogenes, or Streptococcus thermophilus. These Cas9 proteins may be wild-type or mutant, as long as they have target sequence specificity and DNA cleavage activity.

[0062] The restriction of nucleotides by PAM sequences complicates the design of guide RNA. Therefore, Cas9 with a small number of positions restricted to specific nucleotides in the PAM sequence is particularly suitable for use. Examples of such Cas9 include Cas9 (SpCas9) derived from Streptococcus pyogenes that recognizes NGG as a PAM sequence, a variant of SpCas9 (SpCas-NG) that recognizes NG as a 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 that recognizes NNG (Chatterjee, P. et al., 2018, Sci. Adv. Vol. 4, eaau0766), and ScCas9. ++ (Chatterjee, P. et al. Nat. Biotechnol., 2020, Vol. 38, pp. 1154-1158), SpG, which recognizes NGN (Walton RT et al., 2020, Science, Vol. 368, pp. 290-296), or functional analogs thereof.

[0063] 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-nucleotide sequence. The three nucleotides immediately preceding the PAM sequence are referred to herein as the spacer sequence.

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

[0065] When using the CRISPR / Cas system to perform target sequence-specific cleavage, for example, one type of Cas and two types of guide RNAs that pair with the target sequence at each cleavage site can be used in the cell. However, the Cas does not need to be the same for each cleavage. When using multiple Cas, target sequences containing appropriate PAM sequences can be selected depending on the Cas.

[0066] (Step of introducing sequence-specific endonuclease and / or its functionally associated factor) In one embodiment, the production method of the present invention can further include the steps of introducing a second foreign DNA containing a target sequence-specific endonuclease or its gene into the cell, and removing the second foreign DNA, in order to allow the above-mentioned target sequence-specific endonuclease to function in the cell.

[0067] 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 a second foreign DNA capable of expressing the guide RNA in the cell. These steps may be performed simultaneously or separately.

[0068] When the sequence-specific endonuclease and guide RNA are directly introduced into cells, they are preferably introduced into cells before cleavage is carried out.

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

[0070] In one embodiment, the second 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 second foreign DNA can be introduced into the cell as a vector, such as a plasmid, cosmid, or artificial chromosome. When the second foreign DNA is separated from the genomic DNA, the second foreign DNA is naturally lost, and the second foreign DNA can sometimes 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 second 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 second foreign DNA. Optionally, the method 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 amplifying the second foreign DNA can be designed and used for PCR amplification, and the absence of the second foreign DNA in the genomic DNA can also be confirmed by, for example, genome sequencing.

[0073] The gene encoding the target sequence-specific endonuclease of the second 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.

[0074] 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.

[0075] The target gene to be inserted into a vector may be one or more types per vector.

[0076] As used herein, 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.

[0077] (Step of selecting cells that do not contain off-target mutations, step of removing off-target mutations) Preferably, the production method of the present invention 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 is not originally intended to be cleaved.

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

[0079] 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 (for example, a wild-type organism having cells before modification).

[0080] (Other processes) 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.

[0081] 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.

[0082] 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 modified genomic DNA.

[0083] [Method of creating organisms] One embodiment of the present invention, a method for producing an organism, comprises the step of incorporating cells containing modified genomic DNA obtained by the above-mentioned "Method for producing cells containing modified genomic DNA" into an organism.

[0084] Examples of the step of incorporating cells containing modified genomic DNA into an organism include the following, which can be produced using known methods used to produce organisms: (I) introducing cells containing the modified genomic DNA into an organism or its embryo; (II) deriving germ cells from cells containing the modified genomic DNA to generate organisms; (III) applying the method for producing cells containing modified genomic DNA directly to the cells of the organism itself; (IV) Propagating the organism obtained by any one of (I) to (III).

Claims

1. 1. A method for producing a cell containing modified genomic DNA that is free of exogenous DNA, comprising: (i-1a) a step of allowing foreign DNA to coexist with the genomic DNA of a cell, the foreign DNA having a first homology arm and a second homology arm added thereto, the first homology arm and the second homology arm being sequences homologous to the genomic DNA of the cell, the foreign DNA comprises a marker gene, a step in which 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 the interior of the deleted region with a sequence-specific endonuclease and inserting the fragment into or adjacent to the deleted region by homologous recombination repair in the cell; and (iia) cleaving the genomic DNA at least two times with a sequence-specific endonuclease to excise the deleted region and the foreign DNA together from the genomic DNA, thereby generating 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, wherein step (iii) is performed after step (i-2a), after step (iia), or both.

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 containing modified genomic DNA that is free of exogenous DNA, comprising: (i-1b) a step of allowing 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 the 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 least two times with a sequence-specific endonuclease to excise the foreign DNA from the genomic DNA, and generating 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, wherein step (iii) is performed after step (i-2b), after step (iib), or both.

4. The method according to claim 1 or 3, wherein the length of the deleted region is 1 to 60 bp.

5. A method for producing an organism, comprising the step of incorporating cells containing modified genomic DNA obtained by the production method of claim 1 or 3 into an organism.

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