Gene knock-in method, method for producing gene knock-in cell, gene knock-in cell, canceration risk evaluation method, cancer cell production method, and kit for use in same

Abstract

A gene knock-in method that is for knocking in exogenous DNA into a target site of genomic DNA and that includes a step in which donor DNA including the exogenous DNA and a CRISPR-Cas9 system in which a Cas9 protein and guide RNA therefor serve as components are introduced into a cell. The donor DNA includes, in this order from the 5' side, a 5'-microhomology region comprising a 1'-th nucleotide sequence linkable with a first nucleotide sequence on the 5' side of the target site of the genomic DNA by microhomology-mediated joining, the exogenous DNA, and a 3'-microhomology region comprising a 2'-th nucleotide sequence linkable with a second nucleotide sequence on the 3' side of the target site of the genomic DNA by microhomology-mediated joining. The CRISPR-Cas9 system cleaves the following: a first cleaved region between the target site and the first nucleotide sequence and a second cleaved region between the target site and the second nucleotide sequence in the genomic DNA; and a third cleaved region on the 5' side of the 5'-microhomology region, a fourth cleaved region between the exogenous DNA and the 5'-microhomology region, a fifth cleaved region between the exogenous DNA and the 3'-microhomology region, and a sixth cleaved region on the 3' side of the 3'-microhomology region in the donor DNA. The exogenous DNA is inserted between the first nucleotide sequence and the second nucleotide sequence of the genomic DNA.

Description

Gene knock-in method, method for producing gene knock-in cells, gene knock-in cells, cancerization risk evaluation method, cancer cell production method, and kit for use in these

[0001] The present invention relates to a gene knock-in method, a method for producing gene knock-in cells, gene knock-in cells, a cancerization risk evaluation method, a cancer cell production method, and a kit for use in these.

[0002] Since it was reported in 2012 that the CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats / CRISPR associated proteins 9) can be used as a restriction enzyme, the CRISPR-Cas9 system has been shown to function in various species and has become a general-purpose gene modification tool used by researchers around the world. In the DNA editing technology using the CRISPR-Cas9 system, typically, the target DNA is cleaved by the CRISPR-Cas9 system and the repair mechanism during its repair is utilized. However, with current technology, the mutations that can be generated by this technology are mainly deletions or insertions of several bases, and thus it is not suitable for DNA editing that requires substitution of long-chain DNA.

[0003] Furthermore, the CRISPR-Cas9 system has been considered for use in various applications. For example, International Publication No. 2017 / 090724 (Patent Document 1) describes methylating target DNA using a fusion protein of an inactivated CRISPR-associated endonuclease Cas9 (dCas9) that does not possess nuclease activity and a tag peptide array, a fusion protein of a tag peptide binding site and a methyltransferase, and guide RNA. Methylation of genomic DNA is particularly useful because it can suppress the expression of a specific gene by methylating all or part of its promoter. However, it has been reported that in order to completely suppress the expression of such a gene, 100% of the DNA up to 500 bp upstream of the transcription start site must be methylated, whereas the region that can be methylated by the method described in Patent Document 1 is only about 100 bp, and no method has been reported to date that can methylate such long-chain target DNA.

[0004] For example, while many factors related to the carcinogenesis of cells have been reported to be gene sequence abnormalities, factors that do not involve such sequence abnormalities include the methylation of genomic DNA (i.e., cytosine methylation) and its accumulation. However, until now, it has been difficult to methylate long chains of genomic DNA within cells as described above, so sufficient research on genomic DNA methylation has not yet progressed. Regarding factors related to the carcinogenesis of cells, for example, Japanese Patent Publication No. 2019-60749 (Patent Document 2) describes a method for evaluating the risk of cancer using a group of evaluation elements in serum as one of the indicators. However, such indicators are set based on statistical data calculated from a positive group that was truly diagnosed with cancer and a negative group that was not diagnosed, and conventionally, there has been no method to directly evaluate the risk of cancer based on the factors related to cancer.

[0005] Furthermore, other DNA editing techniques that allow for the deletion, insertion, or substitution of relatively long bases in target DNA include gene targeting (knockout or knock-in) techniques that rewrite the sequence of target DNA using exogenous DNA. In addition, methods combining this with the CRISPR-Cas9 system are also being investigated, for example, Sakuma et al., Nat protocol, Vol. 11 No. 1, 2016, pp. 118-133 (Non-patent Literature 1), Katayama et al., Angel Chem Int Ed, 55(22), 2016, pp. 6452-6456 (Non-patent Literature 2), and Katayama et al., Life Science Alliance, Vol. 3 No. Non-Patent Literature 3, etc., describes a method that combines a mechanism called microhomology, which recognizes and repairs short sequences (5-25 bp) at the ends of DNA after breaks (microhomology-mediated end joining, MMEJ), with the CRISPR-Cas9 system to knock in relatively long (e.g., 700 bp or more) exogenous DNA at a target site in genomic DNA. However, the gene knock-in efficiency of the methods described in these documents has not yet been sufficient (for example, the method in Non-Patent Literature 3 has an efficiency of about 20%).

[0006] International Publication No. 2017 / 090724, Japanese Patent Publication No. 2019-60749

[0007] Sakuma et al. , Nat protocol, Vol. 11 No. 1, 2016, p. 118-133 Katayama et al. , Angew Chem Int Ed, 55(22), 2016, p. 6452-6456Katayama et al. , Life Sci Alliance, Vol. 3 No. 1,2019,e201900528

[0008] This invention has been made in view of the problems of the prior art described above, and aims to provide a novel gene knock-in method, a gene knock-in cell production method, gene knock-in cells, and a kit for use therein, which can insert long chains of exogenous DNA into target sites of genomic DNA with higher efficiency than conventional methods.

[0009] Furthermore, the objective is to provide a novel method for assessing the risk of cancer development in cells, which can be directly evaluated by methylating the promoter of the cell's genomic DNA, and a method for producing cancer cells that does not involve gene sequence abnormalities due to methylation of the cell's genomic DNA promoter.

[0010] As a result of diligent research to achieve the above objective, the present inventors have developed a method for knocking in exogenous DNA into a target site of genomic DNA, in which a donor DNA containing exogenous DNA and a CRISPR-Cas9 system comprising Cas9 protein and its guide RNA are introduced into a cell, and the donor DNA is composed of, in order from the 5' side, a first nucleotide sequence (5'-microhomology region) that can be linked by microhomology-mediated linkage to the first nucleotide sequence on the 5' side of the target site of the genomic DNA, the exogenous DNA, and a second nucleotide sequence (3'-microhomology region) that can be linked by microhomology-mediated linkage to the second nucleotide sequence on the 3' side of the target site of the genomic DNA, and the CRISPR-Cas The 9 system was found to cleave a total of six locations in the genomic DNA: between the target site and the first nucleotide sequence (first cleavage region), between the target site and the second nucleotide sequence (second cleavage region), and on the donor DNA: the 5' side of the 5'-microhomology region (third cleavage region), between the exogenous DNA and the 5'-microhomology region (fourth cleavage region), between the exogenous DNA and the 3'-microhomology region (fifth cleavage region), and on the 3' side of the 3'-microhomology region (sixth cleavage region). This allowed for the insertion of relatively long exogenous DNA (e.g., 700 bp or more) between the first and second nucleotide sequences of the genomic DNA with higher efficiency than conventional methods.

[0011] Furthermore, we discovered that the risk of a cell becoming cancerous can be directly assessed by introducing methylation into the promoter of genomic DNA within the cell, thereby completely suppressing the expression of genes under the control of that promoter, and confirming whether or not the cell becomes cancerous as a result. We also found that this method allows us to evaluate whether or not the genes under the control of the promoter are involved in the cancer, using whether or not the cell becomes cancerous as an indicator. Moreover, we found that by using cells derived from a subject and the promoter as the promoter of a gene associated with a specific cancer, it is possible to directly assess the future risk of cancer (risk of developing the specific cancer) in the subject without relying on statistical indicators. Furthermore, we discovered that it is possible to produce cancer cells without gene sequence abnormalities by introducing methylation into such a promoter, thus completing the present invention.

[0012] The embodiments of the present invention obtained from these findings are as follows:

[0013] [1] A method for knocking in exogenous DNA into a target site of genomic DNA, comprising the steps of introducing into a cell donor DNA containing the exogenous DNA and a CRISPR-Cas9 system comprising Cas9 protein and its guide RNA, wherein the donor DNA comprises, in order from the 5' side, a 5'-microhomology region consisting of a first nucleotide sequence that can be linked by microhomology-mediated linkage to a first nucleotide sequence on the 5' side of the target site of the genomic DNA, the exogenous DNA, and a 3'-microhomology region consisting of a second nucleotide sequence that can be linked by microhomology-mediated linkage to a second nucleotide sequence on the 3' side of the target site of the genomic DNA, and the CRISPR-Cas9 system cleaves a first cleavage region between the target site and the first nucleotide sequence, and a second cleavage region between the target site and the second nucleotide sequence in the genomic DNA, and A gene knock-in method comprising cleaving the donor DNA at a third cleavage region on the 5' side of the 5'-microhomology region, a fourth cleavage region between the exogenous DNA and the 5'-microhomology region, a fifth cleavage region between the exogenous DNA and the 3'-microhomology region, and a sixth cleavage region on the 3' side of the 3'-microhomology region, wherein the exogenous DNA is inserted between a first nucleotide sequence and a second nucleotide sequence of the genomic DNA.

[0014] [2] The gene knock-in method according to [1], wherein the first cleavage region and the second cleavage region of the genomic DNA, and the third cleavage region, the fourth cleavage region, the fifth cleavage region and the sixth cleavage region of the donor DNA, are each recognized by the guide RNA, and the first guide RNA target sequence, the second guide RNA target sequence, the third guide RNA target sequence, the fourth guide RNA target sequence, the fifth guide RNA target sequence and the sixth guide RNA target sequence are respectively included.

[0015] [3] The gene knock-in method according to [1] or [2], wherein the Cas9 protein is a protein containing point mutations N692A, M694A, Q695A, and H698A in the wild-type Streptococcus pyogenes Cas9 (SpCas9) protein.

[0016] [4] The gene knock-in method according to any one of [1] to [3], wherein the exogenous DNA of the donor DNA is 700 bases or more.

[0017] [5] The gene knock-in method according to any one of [1] to [4], wherein the exogenous DNA of the donor DNA is methylated.

[0018] [6] The gene knock-in method according to [5], wherein the target site of the genomic DNA is included in the promoter and the exogenous DNA is the methylated target site.

[0019] A method for producing gene knock-in cells, comprising producing cells in which the exogenous DNA is inserted into a target site of genomic DNA using a gene knock-in method described in any one of items [7], [1], to [6].

[0020] [8] Gene knock-in cells into which exogenous DNA containing 700 bp or more methylated base pairs has been introduced.

[0021] [9] A method for evaluating the cancer risk of target cells, comprising: a methylation step of methylating a target promoter site included in the promoter of genomic DNA in the target cells; a detection step of detecting cancer in the target cells; and a cell evaluation step of evaluating the cancer risk of the cells using the cancer infestation of the target cells as an indicator.

[0022]

[10] The method for assessing the risk of carcinogenesis according to [9], wherein the length of the target promoter region is 700 bp or more.

[0023]

[11] The carcinogenic risk assessment method according to [9] or

[10] , wherein the methylation step is a step of knocking in an exogenous methylation promoter site into the target promoter site of the genomic DNA of the target cell using a gene knock-in method according to any one of [1] to [6], wherein the target site is the target promoter site and the exogenous DNA is the methylation promoter site.

[0024]

[12] A method for assessing the risk of cancer according to any one of [9] to

[11] , further comprising a gene evaluation step of evaluating the carcinogenic potential of a gene controlled by a promoter containing the target promoter site, using the carcinogenicity of the target cell as an indicator.

[0025]

[13] A method for assessing the risk of cancer according to any one of [9] to

[11] , wherein the target cells are cells derived from a subject, the promoter containing the target promoter region is the promoter of at least one gene selected from the group consisting of SP3, CDKN2A, p53, p21, and TERT1, and further comprises a subject assessment step of assessing the subject's risk of cancer using the cancerous transformation of the target cells as an indicator.

[0026]

[14] The method for assessing the risk of cancer according to

[13] , wherein the target cell is at least one selected from the group consisting of B cells, hepatocytes, gastric epithelial cells, and pancreatic duct mucosal cells.

[0027]

[15] A method for producing cancer cells, comprising the step of methylating a target promoter site contained in the promoter of genomic DNA in cells to obtain cancer cells.

[0028]

[16] The method for producing cancer cells according to

[15] , wherein the length of the target promoter region is 700 bp or more.

[0029]

[17] The method for producing cancer cells according to

[15] or

[16] , wherein the methylation is a methylation in which an exogenous methylation promoter site is knocked into the target promoter site of the genomic DNA of the cell using a gene knock-in method according to any one of [1] to [6], the target site is the target promoter site and the exogenous DNA is the methylation promoter site.

[0030] A kit for use in a gene knock-in method described in any one of paragraphs

[18] [1] to [6], comprising: (a) a Cas9 protein that cleaves a first cleavage region and a second cleavage region of the genomic DNA, and a third cleavage region, a fourth cleavage region, a fifth cleavage region and a sixth cleavage region of the donor DNA, a polynucleotide encoding the Cas9 protein, or a vector expressing the Cas9 protein; and (b) a guide RNA for the Cas9 protein, a polynucleotide encoding the guide RNA, or a vector expressing the guide RNA.

[0031]

[19] The gene knock-in kit according to

[18] , comprising a first guide RNA, a second guide RNA, a third guide RNA, a fourth guide RNA, a fifth guide RNA, and a sixth guide RNA, each of which recognizes a first guide RNA target sequence and a second guide RNA target sequence of the genomic DNA, and a third guide RNA target sequence, a fourth guide RNA target sequence, a fifth guide RNA target sequence, and a sixth guide RNA target sequence of the donor DNA, respectively.

[0032]

[20] The gene knock-in kit according to

[18] or

[19] , wherein the Cas9 protein is a protein containing point mutations N692A, M694A, Q695A, and H698A in the wild-type Streptococcus pyogenes Cas9 (SpCas9) protein.

[0033] According to the present invention, it is possible to provide a novel gene knock-in method, a method for producing gene knock-in cells, gene knock-in cells, and a kit for use therein, which can insert long chains of exogenous DNA into target sites of genomic DNA with higher efficiency than conventional methods.

[0034] Furthermore, according to the present invention, it is possible to efficiently insert long-chain (e.g., 700 bp or more) exogenous DNA and / or exogenous DNA modified extracellularly into target sites of genomic DNA. For example, by inserting exogenous DNA containing 700 bp or more of 100% methylated base pairs into the promoter of a specific gene, it becomes possible to suppress the expression of that gene with high precision.

[0035] Furthermore, according to the present invention, it is possible to provide a novel method for assessing the risk of cancer in cells, which can be directly evaluated by methylating the promoter of the cell's genomic DNA, and a method for producing cancer cells without gene sequence abnormalities caused by methylation of the promoter of the cell's genomic DNA.

[0036] Therefore, according to the present invention, it is also possible to provide a method (gene evaluation method) that can evaluate whether a gene under the control of the promoter is a gene involved in carcinogenesis, and, if the cells are cells derived from a subject and the promoter is a promoter of a gene related to a specific cancer, a method (subject evaluation method) that can directly evaluate the future risk of cancer (risk of developing the specific cancer) in the subject without relying on statistical indicators.

[0037] This is a schematic diagram showing an example of the target DNA region (a) and donor DNA (b) of the genomic DNA according to the present invention. This is a graph showing the relationship between puromycin concentration and the number of viable cells (relative cell number) on the second day after the start of puromycin addition. This is a graph showing the relationship between puromycin concentration and the number of viable cells (relative cell number) on the third day after the start of puromycin addition. This is an electrophoresis image after T7EI treatment. This is an electrophoresis image of the RT-PCR product after gene knock-in. This is a graph showing the percentage of methylated cytosine in the hSP3 promoter after gene knock-in. This is a photograph showing the appearance of B cells derived from Person C after methylation introduction and Crystal Violet staining. This is a photograph showing the appearance of B cells derived from Person D after methylation introduction and Crystal Violet staining. This is a photograph showing the appearance of B cells derived from Person E after methylation introduction and Crystal Violet staining. This is a photograph showing the appearance of Person F-derived B cells after methylation induction and Crystal Violet staining.

[0038] The present invention will be described in detail below with reference to its preferred embodiments.

[0039] <Gene Knock-in Method> The present invention is a method for knocking in exogenous DNA into a target site of genomic DNA, comprising the steps of introducing into a cell donor DNA containing the exogenous DNA and a CRISPR-Cas9 system comprising Cas9 protein and its guide RNA, wherein the donor DNA comprises, in order from the 5' side, a 5'-microhomology region consisting of a first nucleotide sequence that can be linked by microhomology-mediated linkage to a first nucleotide sequence on the 5' side of the target site of the genomic DNA, the exogenous DNA, and a 3'-microhomology region consisting of a second nucleotide sequence that can be linked by microhomology-mediated linkage to a second nucleotide sequence on the 3' side of the target site of the genomic DNA, and the CRISPR-Cas9 system cleaves a first cleavage region between the target site and the first nucleotide sequence, and a second cleavage region between the target site and the second nucleotide sequence in the genomic DNA, and The present invention provides a gene knock-in method in which the donor DNA is cleaved at a third cleavage region on the 5' side of the 5'-microhomology region, a fourth cleavage region between the exogenous DNA and the 5'-microhomology region, a fifth cleavage region between the exogenous DNA and the 3'-microhomology region, and a sixth cleavage region on the 3' side of the 3'-microhomology region, thereby inserting the exogenous DNA between a first nucleotide sequence and a second nucleotide sequence of the genomic DNA.

[0040] It has been reported that there is a high probability of microhomology-mediated bonding (MMEJ) occurring at the cleaved sites, which are repaired by the linking of complementary sequences at the cleaved ends, when artificial DNA-cutting enzymes such as Cas9 are used. Against this backdrop, the methods described in Non-Patent Documents 1-3 include adding sequences recognized by the CRISPR-Cas9 guide RNA to both ends of the exogenous DNA in the donor DNA introduced into the cells. This causes Cas9 to cleave a total of three or four sites: the cleaved site on the genomic DNA (one site or two sites at both ends of the target site (the site to be knocked in with exogenous DNA)) and the two sites at both ends of the exogenous DNA on the donor DNA. Furthermore, in the methods described in Non-Patent Documents 1 to 3, the donor DNA is designed to be linked to the cleavage ends on the genomic DNA at the time of double-strand breaks by microhomology-mediated linkage. Since the cleavage ends on the genomic DNA generated by Cas9 are linked to the cleavage ends on both ends of the exogenous DNA also generated by Cas9 by microhomology-mediated linkage, it becomes possible to insert (knock in) the exogenous DNA at the cleavage site on the genomic DNA with high efficiency.

[0041] In contrast, in the gene knock-in method of the present invention, two additional sequences recognized by the CRISPR-Cas9 guide RNA are added to the donor DNA introduced into cells, outside the sequences linked by microhomology-mediated binding (5'-microhomology region, 3'-microhomology region) (on the 5' and 3' sides relative to the exogenous DNA). This causes Cas9 to cleave a total of six locations: the cleavage sites on the genomic DNA (two locations at both ends of the target site) and four locations on the donor DNA, two each on the 5' and 3' sides of the exogenous DNA. Therefore, when the exogenous DNA and the target site are aligned, at least three Cas9s and one pair of microhomology regions can be in close proximity on the 5' and 3' sides, respectively (in the conventional method described above, at least two Cas9s and one pair of microhomology regions can be in close proximity on the 5' and 3' sides, respectively). Conversely, the proximity of the three Cas9 molecules to a pair of microhomology regions facilitates alignment between the exogenous DNA and the target site. Therefore, in the gene knock-in method of the present invention, in which the sequences recognized by the CRISPR-Cas9 guide RNA are increased on the donor DNA, although the mechanism is not clear, it is presumed that the Cas9 molecules become more likely to interact with each other or form complexes, and as a result, the exogenous DNA and the target site can be aligned with a higher probability, making it possible to insert the exogenous DNA into the target site of the genomic DNA with higher efficiency than in conventional methods.

[0042] (Cell) In the present invention, the "cell" into which exogenous DNA is inserted for DNA editing, that is, the donor DNA and the CRISPR-Cas9 system are introduced, is not particularly limited, and eukaryotic cells and prokaryotic cells (eubacterial cells, archaeal cells) are included. Examples of the eukaryotic cells include animal cells (cells of mammals, fish, birds, reptiles, amphibians, insects, etc.), plant cells, algal cells, and yeast. Examples of the eubacterial cells include Escherichia coli, Salmonella, Bacillus subtilis, lactic acid bacteria, and hyperthermophilic bacteria. Examples of the archaeal cells include methanogens, halophilic archaea, hyperthermophilic archaea, and thermoacidophilic archaea.

[0043] The type of the cell is also not particularly limited. In animal cells, for example, germ cells such as oocytes and sperm; embryonic cells of embryos at each stage (e.g., 1-cell stage embryo, 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, 16-cell stage embryo, morula stage embryo, etc.); stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, and pancreatic cells. These cells may be in a state of being cultured in vitro (e.g., cells growing in a medium or on a culture medium) or in a state of existing in vivo.

[0044] (Genomic DNA) In the present invention, the region containing the target site for the target gene knock-in on the genomic DNA is referred to as the "target DNA region". For the sake of convenience in showing the correspondence with the following donor DNA and the CRISPR-Cas9 system, the target DNA region is assumed to include, in order from the 5'-side, the first nucleotide sequence, the first cleavage region, the target site, the second cleavage region, and the second nucleotide sequence. The first cleavage region consists of the first cleavage part (indicating between a certain base and the adjacent base cleaved by the CRISPR-Cas9 system; the same applies hereinafter) and the first guide RNA target sequence, and the second cleavage region consists of the second cleavage part and the second guide RNA target sequence. In Fig. 1(a), a schematic diagram showing an example of the mode of the target DNA region of the genomic DNA according to the present invention is shown.

[0045] In (a) of FIG. 1, each region within the target DNA region (100) is described as being directly adjacent in sequence from the 5'-side. However, the first nucleotide sequence (131) and the first cleavage region (121), the first cleavage region (121) and the target site (111), the target site (111) and the second cleavage region (122), and the second cleavage region (122) and the second nucleotide sequence (132) may each independently be directly adjacent as shown in (a) of FIG. 1, may have some overlapping bases (duplicated bases), or may be adjacent via some intervening bases.

[0046] When there are overlapping bases, the number of overlapping bases is, independently for each, 1 to 5 bases, preferably 1 to 4 bases, more preferably 1 to 3 bases. Also, when adjacent via some intervening bases, the number of intervening bases is, independently for each, 1 to 5 bases, preferably 1 to 4 bases, more preferably 1 to 3 bases.

[0047] The first nucleotide sequence and the second nucleotide sequence are sequences to which the first' nucleotide sequence and the second' nucleotide sequence of the following donor DNA can be ligated by microhomology-mediated binding, respectively. The number of bases of the first nucleotide sequence and the second nucleotide sequence is, independently for each, 5 to 40 bases, preferably 5 to 30 bases, more preferably 5 to 20 bases.

[0048] In the first cleavage region and the second cleavage region, the first cleavage site and the second cleavage site may or may not be on the first guide RNA target sequence and the second guide RNA target sequence, respectively, but are preferably on each guide RNA target sequence.

[0049] The first guide RNA target sequence and the second guide RNA target sequence are sequences recognized by the first guide RNA and the second guide RNA of the CRISPR-Cas9 system described below (more specifically, complementary sequences (complementary strands) of sequences recognized and bound by the first guide RNA and the second guide RNA). The number of bases of the first guide RNA target sequence and the second guide RNA target sequence is 23 to 40 bases, preferably 23 to 30 bases, and more preferably 23 to 24 bases, independently of each other. It is preferable that the first guide RNA target sequence and the second guide RNA target sequence are selected such that there are no identical sequences other than the target DNA region on the target genomic DNA (no sequences with more than 50% identity).

[0050] The target site is a site where insertion of exogenous DNA is desired, and can be selected from all or part of a gene encoding sequence, all or part of a functional region such as a promoter, etc. The target site is not particularly limited and may be a single point between a base and an adjacent base, but is usually a region of 500 bases or more, preferably a region consisting of 500 to 1000 bases, and more preferably a region consisting of 500 to 700 bases.

[0051] The target DNA region is not particularly limited, and the CRISPR-Cas9 system is designed to include the following Cas9 protein PAM sequence capable of cleaving the first and second cleavage regions. Preferably, the first and second cleavage regions are selected from regions adjacent to the PAM (proto-spacer adjust motif) sequence at the 3' end. As described later, it is also possible to modify the recognition specificity of the PAM depending on the base sequence of the target DNA region by modifying the Cas9 protein (e.g., by introducing mutations).

[0052] Various methods are known for selecting target DNA regions containing the PAM sequence of such Cas9 proteins, for example, CRISPR Design Tool (http: / / crispr.mit.edu / ) (Massachusetts Institute of Technology), E-CRISP (http: / / www.e-crisp.org / E-CRISP / ), ZiFit Targeter (http: / / zifit.partners.org / ZiFiT / ) (Zing Finger Consortium), Cas9 This can be determined using tools such as design (http: / / cas9.cbi.pku.edu.cn / ) (Peking University), CRISPRdirect (http: / / crispr.dbcls.jp / ) (University of Tokyo), CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / ) (China Central Agricultural University), and Guide RNA Target Design Tool (https: / / wwws.blueheronbio.com / external / tools / gRNASrc.jsp) (Blue Heron Biotech).

[0053] (Donor DNA) In the present invention, donor DNA is DNA used to insert (knock in) a desired exogenous DNA into the target DNA region that has been cleaved by the Cas9 protein.

[0054] The donor DNA is designed to match the target site and includes, in order from the 5' end, a 5'-microhomology region consisting of a first nucleotide sequence that can be linked to the first nucleotide sequence of the genomic DNA by microhomology-mediated linkage, the exogenous DNA, and a 3'-microhomology region consisting of a second nucleotide sequence that can be linked to the second nucleotide sequence of the genomic DNA by microhomology-mediated linkage. More specifically, as shown below in correspondence with the CRISPR-Cas9 system, the donor DNA includes, in order from the 5' end, a third cleavage region, the 5'-microhomology region, a fourth cleavage region, the exogenous DNA, a fifth cleavage region, the 3'-microhomology region, a sixth cleavage region, and the PAM sequence of the Cas9 protein. Furthermore, the third cleavage region consists of a third cleavage portion and a third guide RNA target sequence, the fourth cleavage region consists of a fourth cleavage portion and a fourth guide RNA target sequence, the fifth cleavage region consists of a fifth cleavage portion and a fifth guide RNA target sequence, and the sixth cleavage region consists of a sixth cleavage portion and a sixth guide RNA target sequence. Figure 1(b) shows a schematic diagram illustrating an example of a genomic DNA embodiment according to the present invention.

[0055] In Figure 1(b), the regions within the donor DNA (200) are arranged to be directly adjacent to each other in order from the 5' side. However, the third cleavage region (223) and the 5'-microhomology region (231), the 5'-microhomology region (231) and the fourth cleavage region (224), the fourth cleavage region (224) and the exogenous DNA (112), the exogenous DNA (112) and the fifth cleavage region (225), the fifth cleavage region (225) and the 3'-microhomology region (232), and the 3'-microhomology region (232) and the sixth cleavage region (226) may be directly adjacent to each other as shown in Figure 1(b), or they may partially overlap by a few bases (repeated bases), or they may be adjacent via a few bases (intervening bases).

[0056] When several bases are duplicated, the number of bases in each of the duplicated bases is preferably 1 to 17 independently. Furthermore, when several bases are separated and adjacent, the number of bases in each of the intervening bases is preferably 1 to 23 independently.

[0057] The 5'-microhomology region consists of a first' nucleotide sequence that can be linked to the first nucleotide sequence of the genomic DNA by microhomology-mediated linkage, and the 3'-microhomology region consists of a second' nucleotide sequence that can be linked to the second nucleotide sequence of the genomic DNA by microhomology-mediated linkage. In the present invention, "linkable by microhomology-mediated linkage" of specific sequences A and B means that the complementary sequence of sequence A and sequence B can form a complementary chain, that is, sequences A and B are identical. Therefore, it is preferable that the first nucleotide sequence and the first' nucleotide sequence are identical, and independently of this, it is preferable that the second nucleotide sequence and the second' nucleotide sequence are identical. Furthermore, these do not necessarily have to be 100% identical; for example, when calculated using BLAST or the like (for example, default i.e., initial parameters), they may be 95% or more, preferably 97% or more, more preferably 99% or more, and even more preferably 99.9% or more identical. Furthermore, the nucleotide sequence of the first' and the nucleotide sequence of the second' may be the same or different, but from the viewpoint of efficiency, it is preferable that they be different (for example, the identity is 50% or less).

[0058] The number of bases in the nucleotide sequence 1' and 2' should be such that microhomology-mediated bonding is possible, and each should independently be 5 to 40 bases, preferably 5 to 30 bases, and more preferably 5 to 20 bases.

[0059] In the third to sixth cleavage regions, the third to sixth cleavage regions may or may not be located on the third to sixth guide RNA target sequences, but it is preferable that they be located on the respective guide RNA target sequences.

[0060] The third to sixth guide RNA target sequences are, respectively, sequences recognized by the third to sixth guide RNAs of the CRISPR-Cas9 system described below (more specifically, complementary sequences (complementary strands) of the sequences recognized and bound by the third to sixth guide RNAs). The number of bases for each of the third to sixth guide RNA target sequences is 23 to 40 bases, preferably 23 to 30 bases, and more preferably 23 to 24 bases.

[0061] Furthermore, it is preferable that the fourth and fifth guide RNA target sequences are identical to the first and second guide RNA target sequences on the target DNA region (sequences with 100%, 97%, 99% or more, or 99.9% or more identity), respectively. However, it is preferable that the third and sixth guide RNA target sequences do not have identical sequences on the target DNA region (no sequences with identity exceeding 50%). If sequences identical to the third and sixth guide RNA target sequences exist on the target DNA region, the gene knock-in mechanism by microhomology-mediated binding may not function. In addition, it is preferable that the third to sixth guide RNA target sequences are designed so that there are no identical sequences on the target genomic DNA other than the target DNA region (no sequences with identity exceeding 50%).

[0062] The exogenous DNA is not particularly limited and can be of various sizes. Examples of the number of bases (number of bases per strand) of such exogenous DNA include 100 bases or more, 200 bases or more, 300 bases or more, 400 bases or more, 500 bases or more, 600 bases or more, 700 bases or more, 800 bases or more, 900 bases or more, and 1k base or more. According to the gene knock-in method of the present invention, even long-chain exogenous DNA can be inserted into the target site of genomic DNA with high efficiency. The upper limit of the size of the exogenous DNA is not particularly limited, but examples include 5k bases or less, 4.5k bases or less, and 4k bases or less. Each of these upper limits can be arbitrarily combined with the lower limits.

[0063] The exogenous DNA can encode any gene and may be codon-optimized as appropriate depending on the cell into which it is introduced. Furthermore, the sequence of the target site to be replaced may be identical to (or identical to 100%, 97%, 99% or more, or 99.9% or more) and / or the number of bases may be identical or different. In addition, the exogenous DNA may be modified extracellularly (methylation, demethylation, etc.), for example, DNA that is methylated 100%, 99% or more, 98% or more, or 95% or more for the purpose of insertion into the promoter of a specific gene can be used. There are no particular limitations on the method of such DNA modification, and known methods can be used as appropriate. According to the gene knock-in method of the present invention, even long chain exogenous DNA can be inserted into the target site of genomic DNA, so long chain DNA can be modified by modifying the exogenous DNA extracellularly beforehand and then introducing it into cells.

[0064] Furthermore, if there are base sequences in the exogenous DNA that you wish to remove afterward, you can add, for example, loxP sequences or FRT sequences to both ends of those base sequences. The base sequences sandwiched between loxP sequences or FRT sequences can be removed by acting with Cre recombinase or FLP recombinase. You can also add, for example, a selection marker sequence (e.g., a fluorescent protein or a drug resistance gene) to confirm the success of the knock-in procedure.

[0065] Furthermore, a promoter and / or other regulatory sequence can be operably linked to the exogenous DNA. "Operatably linked" means that the promoter and the desired DNA are linked so that the desired DNA (gene) linked downstream of the promoter can be expressed. The promoter and / or other regulatory sequence are not particularly limited, but constitutive promoters, tissue-specific promoters, time-specific promoters, inducible promoters, CMV promoters, and other regulatory elements (e.g., terminator sequences) can be appropriately selected.

[0066] The donor DNA used in the gene knock-in method of the present invention may be a single type, or, if multiple types of guide RNA with different target DNA regions are used, there may be multiple types, each having exogenous DNA and microhomology regions corresponding to the respective target DNA regions. In this case, the exogenous DNA contained in the multiple types of donor DNA may be DNA with different base sequences.

[0067] The donor DNA is not particularly limited and can be prepared as appropriate by conventionally known methods or similar methods. Such preparation methods include, for example, chemical synthesis using a commercially available synthesizer from the 1' nucleotide sequence, the 2' nucleotide sequence, and the sequences of the 3rd to 6th cleavage regions based on the base sequence of the target DNA region; and preparation using PCR, with the target DNA region as a template and the 1' nucleotide sequence, the 2' nucleotide sequence, and / or the 3rd to 6th cleavage regions added as needed, and further modification as needed (for example, methylation of cytosine (-CH) using CpG methyltransferase). ３ Methods such as adding (or otherwise) are mentioned.

[0068] (CRISPR-Cas9 system) The CRISPR-Cas9 system used in the gene knock-in method of the present invention comprises, as components, at least a Cas9 protein and its guide RNA.

[0069] [Cas9 Protein] In the CRISPR-Cas9 system according to the present invention, the Cas9 protein is not particularly limited and includes, for example, Cas9 protein derived from Streptococcus pyogenes (SpCas9), Cpf1 protein derived from Francisella novicida (FnCpf1), Cas9 protein derived from Staphylococcus aureus (SaCas9), and Cas9 protein derived from Campylobacter jejuni (CjCas9). Furthermore, various Cas9 proteins of different origins are known, and the amino acid sequences and nucleotide sequences of Cas9 proteins are registered in publicly available databases, such as GenBank (http: / / www.ncbi.nlm.nih.gov), and these can be used as appropriate in the present invention.

[0070] Furthermore, the Cas9 protein may be a homolog of the known Cas9 protein, a mutant containing one or more mutations (e.g., addition, deletion, substitution, or combination thereof), a partial peptide, or a modified amino acid sequence, as long as it possesses endonuclease activity. Moreover, the Cas9 protein may have mutations to alter PAM recognition (e.g., Benjamin, P. et al., Nature 523, 481-485 (2015), Hirano, S. et al., Molecular Cell 61, 886-894 (2016)), or mutations introduced to suppress off-target activity or modify function. Additionally, the Cas9 protein may be encoded by a sequence contained within a commercially available vector.

[0071] The Cas9 protein used in the gene knock-in method of the present invention may be a combination of two or more types depending on the guide RNA described below, but from the viewpoint of simplicity, it is preferable to use one type alone.

[0072] Among these, the Cas9 protein used in the gene knock-in method of the present invention is preferably a protein containing point mutations N692A, M694A, Q695A, and H698A in the Cas9 protein (SpCas9) derived from wild-type Streptococcus pyogenes (hereinafter, sometimes referred to as "MSpCas9"). The base sequence of the polynucleotide encoding MSpCas9 is shown in Sequence ID No. 1. The PAM (proto-spacer adjust motif) sequence of SpCas9 and MSpCas9 is "5'-NGG (N is any base)-3'". However, it is also possible to modify the recognition specificity of the above PAM, thereby expanding the options for the target DNA region.

[0073] [Guide RNA] In the CRISPR-Cas9 system according to the present invention, the guide RNA is a combination of crRNA (CRISPR RNA) having a base sequence complementary to the sequence on the target DNA (complementary sequence of the guide RNA target sequence (complementary strand)) and tracrRNA (trans-activating crRNA).

[0074] The guide RNA for the Cas9 protein according to the present invention includes a crRNA with a base sequence complementary to the complementary sequence of each guide RNA target sequence of the target DNA region and the donor DNA (hereinafter, optionally referred to as the "targeted base sequence").

[0075] The crRNA further contains a nucleotide sequence on its 3' side that can interact (hybridize) with tracrRNA. On the other hand, since tracrRNA contains a nucleotide sequence on its 5' side that can interact (hybridize) with some of the nucleotide sequences of crRNA, the guide RNA forms a double-stranded RNA that interacts with the Cas9 protein through the interaction of these nucleotide sequences. For this reason, in the gene knock-in method of the present invention, by introducing the CRISPR-Cas9 system (Cas9 protein and its guide RNA) into cells, the guide RNA recognizes the respective guide RNA target sequences of the target DNA region and the donor DNA, respectively. That is, it binds to the complementary sequence (complementary strand) of each guide RNA target sequence and induces the Cas9 protein, which forms a complex with the guide RNA, to the respective cleavage regions of the target DNA region and the donor DNA. The induced Cas9 protein then cleaves the respective cleavage regions of the target DNA region and the donor DNA by its endonuclease activity.

[0076] The guide RNA for the CRISPR-Cas9 system according to the present invention may be a single-molecule guide RNA (sgRNA) containing crRNA and tracrRNA, or a bi-molecule guide RNA consisting of a crRNA fragment and a tracrRNA fragment.

[0077] In the target DNA region and the donor DNA, the desired DNA cleavage site (cleavage site) does not necessarily have to be included in the guide RNA target sequence, but it is preferable that it be included in the guide RNA target sequence. Typically, cleavage in the target DNA region and the donor DNA occurs at a position determined by both the complementarity of base pairing between the targeted base sequence of the guide RNA and the complementary sequence of the guide RNA target sequence, and the PAM sequence located on the 3' side. Furthermore, it is preferable that all guide RNA target sequences are configured such that cleavage by the Cas9 protein causes the loss of the PAM sequence and the sequence adjacent to the cleavage site, so as not to be subjected to further cleavage by the Cas9 protein present in the cell. In the case of SpCas9 protein and MSpCas9 protein, generally, the site where cleavage occurs (cleavage site) in the target DNA region and the donor DNA is three bases upstream (5' end) of the PAM sequence.

[0078] In the gene knock-in method of the present invention, the guide RNA includes a first guide RNA, a second guide RNA, a third guide RNA, a fourth guide RNA, a fifth guide RNA, and a sixth guide RNA, each recognizing a first guide RNA target sequence and a second guide RNA target sequence of the genomic DNA, and a third guide RNA target sequence, a fourth guide RNA target sequence, a fifth guide RNA target sequence, and a sixth guide RNA target sequence of the donor DNA, respectively.

[0079] The targeting base sequences of the first to sixth guide RNAs may be identical to each other (identity of 100%, 97%, 99% or more, or 99.9% or more) or different, but it is preferable that at least the targeting base sequences of the first to second guide RNAs and the targeting base sequences of the third and sixth guide RNAs are different (identity of 50% or less), more preferably the targeting base sequences of the first and second guide RNAs are different to each other (identity of 50% or less), and even more preferably the targeting base sequences of the first and fourth guide RNAs are identical to each other (identity of 100%, 97%, 99% or more, or 99.9% or more), and the targeting base sequences of the second and fifth guide RNAs are identical to each other (identity of 100%, 97%, 99% or more, or 99.9% or more).

[0080] (Introduction of donor DNA and CRISPR-Cas9 system into cells) In the gene knock-in method of the present invention, the donor DNA introduced into the cells may be in the form of the donor DNA itself (DNA fragment) or in the form of a vector containing said DNA fragment.

[0081] Furthermore, in the gene knock-in method of the present invention, the CRISPR-Cas9 system introduced into cells may be in the form of a protein, RNA or DNA (polynucleotide) encoding the protein, or a vector expressing the protein (expression vector). Independently thereafter, the guide RNA may be in the form of RNA, DNA (polynucleotide) encoding the RNA, or a vector expressing the RNA (expression vector).

[0082] When using an expression vector in the introduction of the CRISPR-Cas9 system, for example, a vector expressing the Cas9 protein and a vector expressing its guide RNA may be introduced into the cell, or a vector expressing both the Cas9 protein and its guide RNA may be introduced into the cell. When using multiple Cas9 proteins, the polynucleotides encoding each of these Cas9 proteins may be loaded onto the same expression vector or onto separate expression vectors. Similarly, the polynucleotides encoding each of the multiple guide RNAs may be loaded onto the same expression vector or onto separate expression vectors.

[0083] Furthermore, when an expression vector is used in the introduction of the CRISPR-Cas9 system, the polynucleotide encoding the Cas9 protein may be appropriately codon-optimized depending on the cell to which it is introduced. It is also preferable that the expression vector includes a promoter and / or other regulatory sequences operably linked to the polynucleotide to be expressed. Moreover, it is preferable that the expression vector can stably express the encoding protein without being incorporated into the host genome. Such an expression vector can be prepared according to conventionally known methods.

[0084] As a method for introducing the donor DNA and the CRISPR-Cas9 system into the cells, known methods for introducing proteins, DNA, RNA fragments, etc. into cells can be appropriately adopted depending on the type of cell. Examples of such methods include electroporation, microinjection, particle gun, calcium phosphate, polyethyleneimine (PEI), liposome (lipofection), DEAE-dextran, cationic lipid-mediated transfection, viruses (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium, lithium acetate, spheroplast, and heat shock (calcium chloride, rubidium chloride). Such methods are described in many standard laboratory manuals, such as "Leonard G. Daviset al., Basic methods in molecular biology, New York: Elsevier, 1986".

[0085] When the donor DNA and the CRISPR-Cas9 system are introduced into the cells, the first and second guide RNAs of the CRISPR-Cas9 system each bind to the complementary sequences (complementary strands) of the first and second guide RNA target sequences in the target DNA region, and the third to sixth guide RNAs each bind to the complementary sequences (complementary strands) of the third to sixth guide RNA target sequences in the donor DNA. Furthermore, the first to sixth guide RNAs each form a complex with the Cas9 protein, thereby guiding the Cas9 protein to the first to sixth guide RNA target sequences (the first to sixth cleavage regions). The guided Cas9 protein, through its endonuclease activity, cleaves the first to sixth cleavage sites within the first to sixth cleavage regions. At this time, the first and second nucleotide sequences, which become the cleavage ends of the target DNA region, are aligned with the first' and second' nucleotide sequences of the donor DNA, respectively, by microhomology-mediated binding. Furthermore, three Cas9 proteins are induced around these microhomologies by the first, third, and fourth guide RNAs on the 5' side (first and first' nucleotide sequences) and by the second, fifth, and sixth guide RNAs on the 3' side (second and second' nucleotide sequences), so that exogenous DNA is inserted between the first and second nucleotide sequences of the target DNA region.

[0086] In the gene knock-in method of the present invention, although the mechanism is not clear, it has been confirmed that the exogenous DNA is inserted between the first and second nucleotide sequences of the target DNA region with higher efficiency than in conventional methods (for example, Non-Patent Literature 3 states that the knock-in efficiency is about 20%, whereas in the example below, the knock-in efficiency is 31.6%). This is thought to be partly due to the fact that increasing the number of sequences recognized by the guide RNA of CRISPR-Cas9 (guide RNA target sequences) makes it easier for Cas9 proteins to interact with each other or to form complexes.

[0087] <Method for producing gene knock-in cells, method for producing non-human organisms, gene knock-in cells> The present invention provides a method for producing cells (gene knock-in cells) in which the exogenous DNA is inserted into the target site. This method includes the step of obtaining the gene knock-in cells by the gene knock-in method described above.

[0088] The present invention also provides a method for producing a non-human individual, which includes cells into which the exogenous DNA has been inserted at the target site. This method includes the step of producing a non-human individual from cells obtained by the gene knock-in method or the gene knock-in cell production method described above. Examples of the non-human individual include non-human animals and plants. Examples of the non-human animals include mammals (mice, rats, guinea pigs, hamsters, rabbits, humans, monkeys, pigs, cows, goats, sheep, etc.), fish, birds, reptiles, amphibians, and insects, but are preferably rodents such as mice, rats, guinea pigs, and hamsters, and are particularly preferably mice. Examples of the plants include grains, oil crops, fodder crops, fruits, and vegetables. Specific examples of crops include rice, corn, bananas, peanuts, sunflowers, tomatoes, rapeseed, tobacco, wheat, barley, potatoes, soybeans, cotton, and carnations.

[0089] Known methods can be used to produce non-human individuals from the aforementioned gene knock-in cells. When producing non-human individuals from cells in animals, germ cells or pluripotent stem cells are usually used. For example, the donor DNA and the CRISPR-Cas9 system can be microinjected into oocytes, and the resulting oocytes can be transplanted into the uterus of a female non-human mammal in a pseudopregnancy state, after which offspring can be produced. In plants, it has long been known that their somatic cells possess totipotency, and for example, the donor DNA and the CRISPR-Cas9 system can be microinjected into plant cells, and a plant body can be regenerated from the resulting plant cells to obtain a plant body in which the desired DNA has been knocked in. Furthermore, offspring or clones in which the desired DNA has been knocked in can also be obtained from the resulting non-human individuals.

[0090] The presence or absence of genetic modification and the determination of the genotype can be performed using conventionally known methods, such as PCR, sequencing, and Southern blotting.

[0091] According to the gene knock-in method of the present invention, it is possible to efficiently insert long chains (e.g., 700 bp or more) exogenous DNA, and / or exogenous DNA modified extracellularly, into target sites of genomic DNA. For example, by inserting exogenous DNA containing 700 bp or more 100% methylated base pairs into the promoter of a specific gene, it is possible to obtain gene knock-in cells in which the expression of that gene is suppressed with high precision.

[0092] <Method for Assessing the Risk of Cancer Transformation> The present invention provides a method for assessing the risk of cancer transformation of target cells, comprising: a methylation step of methylating a target promoter site included in the promoter of genomic DNA in the target cells; a detection step of detecting cancer in the target cells; and a cell evaluation step of evaluating the risk of cancer transformation of the cells using the cancer initiation of the target cells as an indicator (hereinafter, sometimes simply referred to as the "evaluation method").

[0093] (Target Cells) The "target cells" in the evaluation method of the present invention refer to the cells that are the target of the evaluation method of the present invention, and include the cells listed above as "cells" into which exogenous DNA is inserted and DNA is edited. Among these, for example, cells of humans and non-human animals (e.g., mammals (mice, rats, guinea pigs, hamsters, rabbits, monkeys, pigs, cows, goats, sheep, etc.), fish, birds, reptiles, amphibians, insects) are preferred, and cells of humans and non-human mammals are more preferred. The types of target cells in the evaluation method of the present invention are also as described above, but among them, cells collected from living organisms and / or cells cultured outside the body (e.g., cells growing in or on a culture medium) are preferred. The evaluation method of the present invention can be performed in vitro on these cells.

[0094] (Methylation step) In the evaluation method of the present invention, first, the target promoter site contained in the promoter of genomic DNA in the target cell is methylated (methylation step). In the evaluation method of the present invention, "DNA methylation" refers to cytosine methylation.

[0095] In the evaluation method of the present invention, the "target promoter site" refers to a site on the genomic DNA of the target cell that is subject to methylation and is included in any promoter. This site may be the entire sequence of the promoter or a part thereof. However, the length of the sequence to be methylated in the promoter (i.e., the length of the target promoter site) is preferably 500 bp or more, and more preferably 700 bp or more.

[0096] Furthermore, in the evaluation method of the present invention, it is preferable that the target promoter region to be methylated includes 100% of the region up to 500 bp upstream of the transcription start site of the gene controlled by the promoter containing the target promoter region. That is, in the methylation according to the evaluation method of the present invention, it is preferable that 100% of the promoter up to 500 bp upstream of the transcription start site is methylated. If the range of the target promoter region to be methylated is less than the lower limit, the expression of the gene controlled by the promoter containing the target promoter region cannot be completely controlled, and the evaluation accuracy tends to decrease. There is no particular limit to the upper limit of the length of the target promoter region according to the evaluation method of the present invention, but for example, it may be 5 kbp or less, 4.5 kbp or less, 4 kbp or less, or 1 kb or less. Each of the upper limits can be arbitrarily combined with the lower limits.

[0097] The methylation method (methylation method) related to the evaluation method of the present invention is not particularly limited as long as it is a method that can methylate a long chain target promoter site that satisfies the above conditions in the target cell. Examples of such methods include Sakuma et al., Nat protocol, Vol. 11 No. 1, 2016, pp. 118-133 (Non-patent Literature 1), Katayama et al., Angel Chem Int Ed, 55(22), 2016, pp. 6452-6456 (Non-patent Literature 2), and Katayama et al., Life Science Alliance, Vol. 3 No. Examples include: a method of knocking in an exogenous methylation promoter site into the target promoter site using the method described in 1,2019,e201900528 (Non-Patent Literature 3), etc., or a method similar thereto; a method of knocking in an exogenous methylation promoter site into the target promoter site using the gene knock-in method of the present invention described above; and a method using Microhomology-mediated end joining (MMEJ).

[0098] Among these, as the methylation method for the evaluation method of the present invention, it is preferable to use the gene knock-in method of the present invention described above, from the viewpoint that it is possible to insert all or part of a longer and methylated promoter (hereinafter referred to as "methylated promoter site" in this specification) as exogenous DNA into the target promoter site of the genomic DNA with higher efficiency than conventional methods. That is, as the evaluation method of the present invention, it is preferable that the methylation step is a step of knocking in an exogenous methylated promoter site into the target promoter site of the genomic DNA of the target cell using the gene knock-in method of the present invention, wherein the target site is the target promoter site and the exogenous DNA is the methylated promoter site.

[0099] In the evaluation method of the present invention, if the methylation step is a step using the gene knock-in method of the present invention, then in the methylation step, the "cell" in the gene knock-in method is the target cell; the "genomic DNA" is the genomic DNA of the target cell; the "target site" is the target promoter site; the "exogenous DNA" is the "methylation promoter site"; and the "donor DNA" is DNA containing the methylation promoter site.

[0100] In the evaluation method of the present invention, if the methylation step is a step using the gene knock-in method of the present invention, the target promoter site is the site that replaces the methylated promoter site. Furthermore, the methylated promoter site represents a sequence in which the same sequence as the target promoter site is methylated, or a sequence in which a part of the sequence of the target promoter site is methylated, and is DNA that is inserted into the genomic DNA in place of the target promoter site by the gene knock-in method.

[0101] According to the gene knock-in method of the present invention, it is possible to efficiently insert a long-chain (e.g., 700 bp or more) methylation promoter site (exogenous DNA) into a target promoter site (target site) of genomic DNA. For example, it is possible to insert a methylation promoter site consisting of 700 bp or more base pairs that are 100% methylated into the target promoter site.

[0102] The methylation promoter site may be appropriately codon-optimized depending on the cells to which it is introduced. The sequence of the methylation promoter site may differ from that of the target promoter site to be replaced, but it is preferable that they be identical (100%, 97%, 99% or more, or 99.9% or more). The length of the methylation promoter site is preferably the same as that of the target promoter site to be replaced, more preferably 500 bp or more, and even more preferably 700 bp or more. Furthermore, it is preferable that 98% or more, more preferably 100%, of the methylation promoter site is methylated. There are no particular upper limits on the length of the methylation promoter site, but for example, they may be 5 kbp or less, 4.5 kbp or less, 4 kbp or less, or 1 kb or less. Each of these upper limits can be arbitrarily combined with the lower limits.

[0103] The methylation promoter site can be prepared by, for example, chemically synthesizing it using a commercially available synthesizer from the target promoter site and the first nucleotide sequence on its 5' side and the second nucleotide sequence on its 3' side, as well as the sequences of the third to sixth cleavage regions; or by preparing it using the PCR method, adding the first nucleotide sequence, the second nucleotide sequence, and / or the third to sixth cleavage regions as needed, using the target promoter site as a template; and then methylating it (for example, methylating cytosine using CpG methyltransferase (-CH4). ３ This can be obtained by adding (or otherwise) something.

[0104] (Detection step) In the evaluation method of the present invention, the next step is to detect the cancerous transformation of the target cells. In the present invention, "cancerous transformation of cells" means that cells become abnormally proliferated (cancer), and in particular in the present invention, this means that such a state is achieved by the accumulation of DNA methylation on the promoter or the suppression of the expression of genes controlled by the promoter. An example of a detection method for detecting such cancerous transformation of cells is a method for detecting cancer cells after the methylation step. The method for detecting cancer cells includes not only a method for detecting the presence or absence of cancer cells, but also a method for quantifying the cancer cells.

[0105] The method for detecting the cancer cells is not particularly limited, and a method similar to conventionally known methods can be adopted as appropriate depending on the type of target cell. For example, if the target cell is a B cell, a method can be used in which the target cells after the methylation step are cultured in an agar gel medium and viable cells are detected. When the target cells after the methylation step are cultured in an agar gel medium, if the B cells are not cancerous, they will not be able to survive in the gel and will die. However, if the B cells become cancerous and turn into cancer cells, they will form spheroids and be able to survive. Therefore, by detecting such viable cells, the cancerous transformation of the target cells can be detected. The method for detecting the viable cells is not particularly limited, and for example, they can be detected by staining with a crystal violet, and they may also be quantified as needed.

[0106] Other detection methods include, for example, immunohistochemical staining methods using anti-CD20 antibody, anti-CD10 antibody, anti-BCL-6 antibody, anti-MUM-1 antibody, and combinations of these antibodies.

[0107] (Evaluation step) In the evaluation method of the present invention, the risk of the target cells becoming cancerous is then evaluated using the cancerous transformation of the target cells as an indicator (cell evaluation step).

[0108] In the present invention, "risk of cell carcinogenesis" refers to the possibility that the target cells will become cancerous (preferably due to the accumulation of DNA methylation on the promoter or the suppression of gene expression controlled by the promoter). In the present invention, the evaluation of the risk of carcinogenesis involves determining that the target cells have a high or no possibility of becoming carcinogenic if carcinogenesis is detected, and selecting them from the group with no or no possibility. On the other hand, if carcinogenesis is not detected in the target cells, the cells are determined to have no or no possibility of becoming carcinogenic, and selecting them from the group with or with a high possibility. In the present invention, the determination of possibility includes not only determining whether or not there is a possibility, but also evaluating the degree of possibility if there is a possibility (e.g., high / moderate / low). Furthermore, the evaluation method of the present invention also includes screening to select subjects with a high or no risk of carcinogenesis from a normal group with no or low risk of carcinogenesis.

[0109] (Gene evaluation method) The evaluation method of the present invention may further include a gene evaluation step (hereinafter referred to as the "gene evaluation method") in which the carcinogenic potential of a gene controlled by a promoter containing the target promoter site is evaluated using the carcinogenicity of the target cell as an indicator.

[0110] In the present invention, "oncogenic potential of a gene" indicates that the gene may be involved in carcinogenesis, and includes the possibility that cells may become cancerous due to the accumulation of DNA methylation in the promoter of the gene, and the possibility that cells may become cancerous due to the suppression of the expression of the gene. In the present invention, the evaluation of the oncogenic potential of the gene is performed by, when carcinogenesis of the target cell is detected, determining that the gene controlled by the promoter containing the target promoter site in the target cell has a high or low possibility of being involved in carcinogenesis, and selecting it from the group with no or low possibility. On the other hand, if carcinogenesis of the target cell is not detected, determining that the gene has no or low possibility of being involved in carcinogenesis, and selecting it from the group with or with high possibility.

[0111] For example, if methylation is introduced into the promoter of any gene A in any target cell, and the target cell becomes cancerous (cancer cells are detected), then it can be determined that the target cell has a high or high potential for cancer development, and that gene A has a high or high potential for involvement in cancer development. Furthermore, if the method for detecting cancer is a quantitative method, then, depending on the degree of cancer development, it may be determined that the target cell has a high or low potential for cancer development, and that gene A has a high or low potential for involvement in cancer development. The criteria for the above determination can be appropriately set depending on the purpose of the evaluation, the type of cell and promoter, etc.

[0112] If the evaluation method of the present invention is the gene evaluation method, the target cell and the promoter can be any and independent depending on the purpose of the evaluation. For example, by selecting multiple promoters of any gene as promoters that include the target promoter site that performs methylation, it becomes possible to search among them for genes that are involved in carcinogenesis due to the accumulation of DNA methylation in the promoter or carcinogenesis due to expression suppression, using the carcinogenesis of the target cell as an indicator.

[0113] (Method for evaluating the risk of cancer in subjects) Furthermore, the evaluation method of the present invention may also include a subject evaluation step in which the target cells are cells derived from the subject, and the risk of cancer in the subject is evaluated using the cancerous transformation of the target cells as an indicator (hereinafter, depending on the case, referred to as the "subject evaluation method").

[0114] In the present invention, "the subject's risk of developing cancer" refers to the possibility that the subject will develop cancer (preferably, cancer caused by the accumulation of DNA methylation on the promoter including the target promoter site, or cancer caused by the suppression of gene expression controlled by the promoter including the target promoter site). Alternatively, if the subject has already developed cancer, it may refer to the possibility that the cancer is caused by the accumulation of DNA methylation on the promoter including the target promoter site, or cancer caused by the suppression of gene expression controlled by the promoter including the target promoter site. Such cancers are not particularly limited, but examples include blood cancers, liver cancers, stomach cancers, and pancreatic cancers. Furthermore, in the present invention, "subject" is not particularly limited as long as it is a human being who is the subject of the evaluation method of the present invention. For example, it may refer to a healthy person or a person without symptoms for the purpose of predicting or screening the risk of developing cancer, or it may refer to a patient who is already known to have cancer or the like.

[0115] In the present invention, the evaluation of the cancer risk of the subject is as follows: If cancerous transformation of the target cells is detected, the subject from which the target cells originate is determined to have a high probability of developing (or having developed) the cancer, and is selected from the group with no or low probability. On the other hand, if cancerous transformation of the target cells is not detected, the subject is determined to have no or low probability of developing (or having developed) the cancer, and is selected from the group with or with high probability.

[0116] When the evaluation method of the present invention is the subject evaluation method, the promoter containing the target promoter site must be the promoter of a gene known to cause cancer in cells due to the accumulation of methylation in the promoter or suppression of expression. Examples of such promoters include the promoter of at least one gene selected from the group consisting of SP3, CDKN2A, p53, p21, and TERT1, and among these, the promoter of at least one gene selected from the group consisting of SP3 and CDKN2A is preferred.

[0117] Furthermore, the target cells in this case may be, for example, at least one selected from the group consisting of B cells, hepatocytes, gastric epithelial cells, and pancreatic duct mucosal cells, and among these, at least one selected from the group consisting of B cells and hepatocytes is preferred. In the subject evaluation method described above, the target cells may be obtained from a specimen (blood, tissue fragments, etc.) collected from the subject by a known method as appropriate, depending on the purpose of the evaluation method.

[0118] Furthermore, examples of such combinations of target cells and genes controlled by promoters include at least one selected from the group consisting of B cells and SP3, hepatocytes and CDKN2A, gastric epithelial cells and p53, p21, etc., and pancreatic ductal mucosal cells and TERT1, and among these, at least one selected from the group consisting of B cells and SP3 and hepatocytes and CDKN2A is preferred.

[0119] For example, if methylation is introduced into the target promoter site contained in the SP3 gene promoter in B cells derived from a subject, and the B cells become cancerous (cancer cells are detected), then it can be determined that the B cells have a high or no possibility of becoming cancerous, and that the subject has a high or no possibility of developing cancer involving B cells and / or the SP3 gene (e.g., B-cell lymphoma). Furthermore, if the method for detecting cancer is a quantitative method, the subject may be determined to have a high or low probability of becoming cancerous depending on the degree of the cancer. The criteria for the above determination can be appropriately set depending on the purpose of the evaluation, the type of cells and promoter, etc.

[0120] According to the subject evaluation method described above, the risk of developing cancer due to the accumulation of DNA methylation can be directly evaluated using the subject's own cells. Furthermore, by identifying subjects at high risk of developing the aforementioned cancer and conducting regular checkups and management, it becomes possible to detect cancer early or prevent its onset. The subject evaluation method of the present invention can also be described as a method to assist in diagnosis by physicians, etc., or a method to provide information for diagnosis by physicians, etc.

[0121] <Method for Producing Cancer Cells> The present invention also provides a method for producing cancer cells, comprising the step of obtaining cancer cells by methylating a target promoter site contained in the promoter of genomic DNA in cells. This method includes a methylation step of methylating the target promoter site by the above-mentioned methylation step, and the methylation step, including its preferred embodiments, is as described in the evaluation method of the present invention.

[0122] In the cancer cell production method of the present invention, if the target cells and the target promoter site are to be selected arbitrarily, the method preferably further includes the above-described detection step, and cells in which carcinogenesis is detected by the detection step can be obtained as the cancer cells. Furthermore, in the cancer cell production method of the present invention, by using a promoter containing the target cells and / or the target promoter site as a promoter of cells and / or genes known to become carcinogenic through the accumulation of DNA methylation, cancer cells in which the promoter of the target gene has been methylated and become carcinogenic can be obtained. In this case as well, the method may further include the above-described detection step.

[0123] The present invention also provides a method for producing a non-human organism containing cancer cells in which the target promoter site has been methylated. This method includes the step of producing a non-human organism having cells obtained by the cancer cell production method described above. The method for producing a non-human organism is as described above. Examples of the non-human organism include the non-human animal, preferably a rodent such as a mouse, rat, guinea pig, or hamster, and particularly preferably a mouse.

[0124] <Kit> The present invention also provides a kit for use in the gene knock-in method, the gene knock-in cell production method, the carcinogenic risk assessment method, the cancer cell production method, or the non-human individual production method of the present invention, that is, a kit for use in the gene knock-in method of the present invention. The kit of the present invention comprises at least one selected from the group consisting of: (a) a Cas9 protein that cleaves the first and second cleavage regions of the genomic DNA, and the third, fourth, fifth, and sixth cleavage regions of the donor DNA, a polynucleotide encoding the Cas9 protein, or a vector expressing the Cas9 protein; and (b) a guide RNA for the Cas9 protein, a polynucleotide encoding the guide RNA, or a vector expressing the guide RNA. The kit of the present invention may also comprise (c) the donor DNA. Furthermore, (b) the guide RNA for the Cas9 protein, the polynucleotide encoding the guide RNA, or the vector expressing the guide RNA may be (b') a vector containing an insertion site for inserting a guide RNA that has been appropriately designed and prepared to match the sequence of the target DNA region. These proteins, polynucleotides, and vectors are each independently as described in the gene knock-in method of the present invention described above.

[0125] The kit of the present invention may further include one or more additional reagents. Such additional reagents include, but are not limited to, dilution buffers, reconstitution solutions, wash buffers, nucleic acid delivery reagents, protein delivery reagents, and control reagents (e.g., control guide RNA). The kit may also further include instructions for use in carrying out the method of the present invention.

[0126] Each element included in the kit may be contained in a separate container or in the same container. Each element may be contained in a single-use portion or in a single container containing multiple uses. Each element may be contained in a dry form or in a dissolved form in a suitable solvent.

[0127] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. Furthermore, each operation such as PCR described below was performed by conventional methods as appropriate.

[0128] (Example 1) (1) CRISPR-Cas9 System [MSpCas9] Using PX459 plasma (Gen Script) expressing wild-type SpCas9 protein as a template, the following primers were designed to introduce point mutations N692A, M694A, Q695A, and H698A into the wild-type SpCas9 protein: MSpCas9 forward primer (SEQ ID NO: 2) MSpCas9 reverse primer (SEQ ID NO: 3) and KOD ONE PCR Master Mix (Toyobo) were used to perform PCR. The obtained PCR product was phosphorylated using T4 polynucleotide kinase (Toyobo) and ligated using Mighty Ligation mix (Takara) to obtain a plasmid (PX459-MSpCas9) expressing a modified Cas9 protein (MSpCas9) containing the point mutation. Sanger sequencing confirmed the introduction of the mutation, and it was confirmed that the wild-type SpCas9 protein had indeed acquired a nucleotide sequence encoding a protein containing the point mutation (nucleotide sequence encoding MSpCas9 protein: SEQ ID NO: 1).

[0129] [Guide RNA] Guide RNA 1 (sgRNA1) targets the 700 bp on the 3' side of the promoter of the human SP3 gene (hSP3), and consists of a nucleotide sequence complementary to the complementary sequence of guide RNA target sequence 1 located upstream of the PAM sequence (5'-NGG) of the MSpCas9 protein in the 5' region of the leading strand (targeted nucleotide sequence, SEQ ID NO: 4); Guide RNA target sequence 2 is located upstream of the PAM sequence of the MSpCas9 protein in the 3' region of the leading strand of the target site. Polynucleotides encoding the following were synthesized: Guide RNA 2 (sgRNA2) consisting of a complementary sequence and a complementary base sequence (targeted base sequence, SEQ ID NO: 5); Guide RNA 3 (sgRNA3) consisting of a complementary sequence and a complementary base sequence (targeted base sequence, SEQ ID NO: 6) of the guide RNA target sequence 3 of the donor DNA below; and Guide RNA 6 (sgRNA6) consisting of a complementary sequence and a complementary base sequence (targeted base sequence, SEQ ID NO: 7) of the guide RNA target sequence 6 of the donor DNA below.

[0130] Plasmid CRISPR1 (PX459-MSpCas9-sgRNA1-sgRNA3), which expresses MSpCas9 protein, guide RNA1, and guide RNA3, was constructed by inserting polynucleotides (DNA) encoding guide RNA1 and guide RNA3 into the guide RNA insertion site of plasmid PX459-MSpCas9.

[0131] Furthermore, the polynucleotide encoding guide RNA 2 and the polynucleotide encoding guide RNA 6 were inserted into the guide RNA insertion site of another plasmid, PX459-MSpCas9, to construct plasmid CRISPR2 (PX459-MSpCas9-sgRNA2-sgRNA6), which expresses the MSpCas9 protein, guide RNA 2, and guide RNA 6.

[0132] (2) The 3' end 700 bp of the donor DNA hSP3 promoter was amplified using the following primers: donor hSP3pro forward primer (SEQ ID NO: 8) and donor hSP3pro reverse primer (SEQ ID NO: 9). The PCR product obtained using the primers includes, from the 5' end of the leading strand: guide RNA target sequence 3 recognized by guide RNA 3; a first nucleotide sequence (5'-microhomology region, 20 bp, including the PAM sequence of the MSpCas9 protein) identical to the first nucleotide sequence on the 5' end of the target site (linkable by microhomology-mediated binding); guide RNA target sequence 1; 700 bases on the 3' end of the hSP3 promoter (exogenous DNA, including the PAM sequence of the MSpCas9 protein); guide RNA target sequence 2; a second nucleotide sequence (3'-microhomology region, 20 bp, including the PAM sequence of the MSpCas9 protein) identical to the second nucleotide sequence on the 3' end of the target site (linkable by microhomology-mediated binding); guide RNA target sequence 6 recognized by guide RNA 6; and the PAM sequence of the MSpCas9 protein. Furthermore, the 5'-microhomology region and guide RNA target sequence 1, and the 3'-microhomology region and guide RNA target sequence 6, each overlap with each other. The PCR product was methylated using CpG methyltransferase (New England Biolabs) to obtain donor DNA.

[0133] (3) Cell Culture First, HEK293 cells (JCRB cell bank) were maintained in a 10% medium (DMEM (Nacalai Tesq) with 10% FBS (Hyclone serum, GE Healthcare) and 0.5% Penicillin-Streptomycin (Nacalai Tesq) added. Next, the maintained cells were detached using 0.25% Trypsin-EDTA (Nacalai Tesq) and 1 × 10⁶ cells were separated. ５ Seeds were sown in 24-well plates to achieve a cells / well ratio.

[0134] (4) Puromycin Selection After seeding in a 24-well plate, the cells were cultured in a medium supplemented with 0-5 μg / mL of puromycin starting 24 hours later. Cells were harvested on the second and third days after the start of supplementation, and the number of viable cells (unstained cells) was counted after adding Trypan Blue (Wako).

[0135] Figure 2 shows the relationship between puromycin concentration and the number of viable cells on day 2 after the start of administration, and Figure 3 shows the relationship between puromycin concentration and the number of viable cells on day 3 after the start of administration. In Figures 2 and 3, the number of viable cells (vertical axis) is the relative number of cells to the number of viable cells when the puromycin concentration is 0 μg / mL. As shown in Figures 2 and 3, the optimal concentration of puromycin that can kill more than 90% of HEK293 cells on day 2 and completely eliminate HEK293 cells on day 3 was 1 μg / mL.

[0136] (5) T7EI Assay HEK293 cells were seeded in a 24-well plate in the same manner as in (3) above. The day after seeding, the cells were transfected with either plasmid CRISPR1 or CRISPR2 constructed above, at a dose of 400 ng / well, using polyethyleneimine (PEI, Polyplus Transfection). The day after transfection, the medium was changed with the 10% medium mentioned above, and 48 hours after transfection, the cells were cultured in a medium supplemented with 1 μg / mL of puromycin.

[0137] Genomic DNA was extracted from cells (adherent cells) that survived 48 hours after the start of puromycin addition using the Qiaamp DNA mini kit (Qiagen). Using this as a template, the region containing the 700 bp (target site) on the 3' side of the hSP3 promoter was amplified by PCR using the following primers: hSP3pro forward primer (SEQ ID NO: 10) and hSP3pro reverse primer (SEQ ID NO: 11).

[0138] 200 ng of the obtained PCR product, 10×M buffer (Takara), and DDW were mixed to make a total of 10 μL of reaction mix, which was reacted under the following conditions: 95°C for 10 min, 85°C for 1 min, 75°C for 1 min, 65°C for 1 min, 55°C for 1 min, 45°C for 1 min, 35°C for 1 min, and 25°C for 1 min. To the 10 μL of reaction mix after the reaction, 1.5 μL of 10×NEB2 buffer (New England Biolabs), 0.5 μL of T7 Endonuclease I (T7EI, New England Biolabs), and 3 μL of DDW were added, and T7EI treatment was performed under the conditions of 37°C for 30 min. After T7EI treatment, electrophoresis was performed on a 3% agarose gel (Nippon Gene).

[0139] Figure 4 shows the electrophoresis images after the electrophoresis. Figure 4 also shows the results of electrophoresis of only the plasmid CRISPR1 or CRISPR2 fragment (Control). As shown in Figure 4, in HEK293 cells into which plasmid CRISPR1 or CRISPR2 was introduced, heteroduplex fragments formed between wild-type DNA and mutant DNA were observed, and it was confirmed that both the CRISPR-Cas9 systems introduced by plasmid CRISPR1 and CRISPR2 were able to correctly cleave the 5' guide RNA target sequence 1 and the 3' guide RNA target sequence 2 of the target site.

[0140] (6) Gene knock-in HEK293 cells were seeded in a 24-well plate in the same manner as in (3) above. The day after seeding, the cells were transfected using polyethyleneimine (PEI, Polyplus Transfection) with the plasmids CRISPR1 150 ng / well, CRISPR2 150 ng / well, and donor DNA 150 ng / well constructed above. The day after transfection, the medium was changed with the 10% medium described above, and 48 hours after transfection, the cells were cultured in a medium supplemented with 1 μg / mL of puromycin.

[0141] Forty-eight hours after the start of puromycin addition, surviving cells were detached using 0.25% Trypsin-EDTA (Nacalai Tesq), and the cells were diluted to a limiting dilution of 25 cells / 10 mL. 200 μL of the diluted cells were seeded into each well of a 96-well plate and cultured for 10–12 days to establish single-cell clones.

[0142] (7) RT-PCR First, RNA was extracted from the Single-cell clone established in (6) above using QIAZOL (Qiagen). Next, using ReverTraAce qPCR RT Master mix with gDNA remover (Toyobo), genomic DNA was removed and reverse transcription was performed from 1 μg of the extracted RNA to obtain cDNA. The obtained cDNA was diluted four-fold with DDW and used as a template, and RT-PCR was performed using the following primers: GAPDH forward primer (SEQ ID NO: 12) GAPDH reverse primer (SEQ ID NO: 13) hSP3 forward primer (SEQ ID NO: 14) hSP3 reverse primer (SEQ ID NO: 15). The obtained RT-PCR products were subjected to electrophoresis on a 2% agarose gel (Nippon Gene). Furthermore, a single-cell clone (control clone) was established in the same manner as in (6) above, except that donor DNA was not transfected, and RT-PCR was performed in the same manner.

[0143] Figure 5 shows electrophoresis images of the RT-PCR products obtained by performing RT-PCR on clones 1 to 3. Figure 5 also shows the electrophoresis image of the RT-PCR product (Control) obtained by performing RT-PCR on the control clone. As shown in Figure 5, in the plasmids CRISPR1 and CRISPR2, and in the clones (Clones 1 to 3) into which donor DNA containing methylated exogenous DNA was introduced, it was confirmed that hSP3 expression (transcription) was suppressed (no hSP3 band was observed), and that the donor DNA (methylated exogenous DNA) was inserted into the target site of the hSP3 promoter.

[0144] (8) Gene knock-in efficiency In the same manner as in (7) above, cDNA was obtained from the Single-cell clone established in (6) above, and RT-PCR was performed using this as a template, and the resulting RT-PCR products were subjected to electrophoresis. In addition, a Single-cell clone (control clone) was established in the same manner as in (6) above, except that donor DNA was not transfected, and RT-PCR was performed in the same manner.

[0145] In each electrophoresis result, the presence or absence of insertion of donor DNA into the target site was determined by confirming the hSP3 band. In the 19 control clones (Control 1-19) established without donor DNA introduction, no clones showed suppressed hSP3 expression (transcription) (hSP3 bands were confirmed in all control clones), so the knock-in efficiency was determined to be 0%. On the other hand, in the 19 clones (Clone 1-19) established using donor DNA, six clones showed suppressed hSP3 expression, so the knock-in efficiency was determined to be 31.6% (6 / 19 × 100). According to Non-Patent Literature 3, the knock-in efficiency using the method described in that document was approximately 20%, so a higher knock-in efficiency was achieved than conventional methods.

[0146] (9) Bisulfite Sequencing First, genomic DNA was extracted from the Single-cell clone established in (6) above using the Qiaamp DNA mini kit (Qiagen). Next, 200 ng of the extracted genomic DNA was treated with bisulfite using the EpiMark Bisulfite Conversion Kit (New England Biolabs).

[0147] Using 50 ng of bisulfite-treated genomic DNA as a template, PCR was performed using the following primers: hSP3pro forward primer 2 (SEQ ID NO: 16), hSP3pro reverse primer 2 (SEQ ID NO: 17), and EpiTaq (New England Biolabs) to amplify the region corresponding to the target site of the hSP3 promoter. Next, pUC19 plasmid (NipponGene) was cleaved with SmaI (New England Biolabs), and the resulting PCR product was cloned using Mighty ligation mix (Takara). The cloned sequence was determined by Sanger sequencing.

[0148] Figure 6 shows the percentage of cytosine in the determined sequence (i.e., the percentage of methylated cytosine. Unmethylated cytosine is converted to uracil by the bisulfite treatment: methylated cytosine (%) = cytosine / (uracil + cytosine) × 100). As shown in Figure 6, it was confirmed that the percentage of methylated cytosine was almost 100% in the region corresponding to the target site of the hSP3 promoter in plasmids CRISPR1 and CRISPR2, as well as in clones into which donor DNA was introduced (Clones 1-3).

[0149] As shown above, it was confirmed that the above knock-in method allows for highly efficient knock-in of methylated exogenous DNA (methylated hSP3 promoter) at 700 bp on the 3' end of the hSP3 promoter.

[0150] (Example 2) (1) Introduction of methylation into B cells First, four types of human B cells purchased from the JCRB Cell Bank (from JCRB Cell Bank, Persons C, D, E, and F) were maintained in a 10% medium (RPMI1640 (Nacalai Tesq) with 10% FBS (Hyclone serum, GE Healthcare) and 0.5% Penicillin-Streptomycin (Nacalai Tesq) added. Next, the maintained human B cells were cultured at a rate of 1 × 10⁶ ５Seeds were sown in 24-well plates to achieve a cells / well ratio.

[0151] Here, Persons C and E are B cells derived from humans (cancer patients) who died from cancer (B-cell lymphoma), while Persons D and F are B cells derived from humans who died from causes other than cancer. Persons C, D, E, and F do not possess any conventionally known genes or mutations (driver genes and driver mutations) associated with carcinogenesis.

[0152] The day after seeding, the combination of plasmid CRISPR1 150 ng / well, CRISPR2 150 ng / well, and donor DNA 150 ng / well, constructed in Example 1, was transfected using polyethyleneimine (PEI, Polyplus Transfection). The day after transfection, the medium was changed to the aforementioned 10% medium, and 48 hours after transfection, the cells were cultured in a medium supplemented with 1 μg / mL of puromycin. 48 hours after the start of puromycin supplementation, the medium was changed back to the aforementioned 10% medium, and the cells were maintained in culture for 4 days.

[0153] As a control, the following day after seeding, the plasmid PX459-MSpCas9 (without guide RNA) 150 ng / well and donor DNA 150 ng / well, constructed in Example 1, were transfected using polyethyleneimine (PEI, Polyplus Transfection), and the cultures were maintained for 4 days in the same manner as described above.

[0154] (2) Cell Culture Bottom Layer 0.8% agarose gel Low Melt Agarose Gel (Biorad) was added to 10% culture medium to make a 0.8% agarose gel solution, and the agarose gel was completely dissolved in an 80°C water bath. 5 mL was dispensed into 10 cm dishes. These were left to solidify at room temperature for 30 minutes to prepare the agar gel bottom layer. Furthermore, Low Melt Agara Gel was added to Cell-containing layer 0.3% Agara Gel 10% medium to make a 0.3% Agara Gel solution, and the Agara Gel was completely dissolved in an 80°C water bath. The solution was then left at room temperature and cooled to 37°C. To this, cells that had been maintained in culture for 4 days as described in (1) above were added at a rate of 1.25 × 10⁶ per 5 mL. ４ The cells were suspended and 5 mL each was dispensed onto the bottom layer described above to form cell-containing layers. Every three days, 2.5 mL of 10% medium was added per 10 cm dish, and the cells were maintained in culture for two weeks.

[0155] (3) Evaluation of carcinogenesis (Crystal Violet staining) First, the cell-containing layer cultured in (2) above was collected in a 50 mL tube, and an equal volume of 4% PFA (Wako) was added and left at room temperature for 20 minutes to fix the cells. Next, the volume was increased to 50 mL with D-PBS (Nacalai Tesq) containing 10% FBS, and the mixture was centrifuged at 1100 rpm for 10 minutes to precipitate the fixed cells, and the supernatant was removed.

[0156] Next, Crystal Violet (Sigma) was diluted with 20% ethanol (Wako) to make 0.005% Crystal Violet. 5 mL of the 0.005% Crystal Violet solution was added to the precipitated fixed cells and left at room temperature for 1 hour to stain them. Next, this was made up to 50 mL with D-PBS containing 10% FBS, and the solution was centrifuged at 1100 rpm for 10 minutes to precipitate, and the supernatant was removed. Then, the stained cells were lysed with 10 mL of D-PBS and the entire volume was dispensed into one 10 cm dish. This was left at room temperature overnight to dry completely.

[0157] Photographs showing the appearance of the 10 cm dish after drying are shown in Figures 7 (Person C), 8 (Person D), 9 (Person E), and 10 (Person F), respectively. In Figures 7 to 10, (a) shows the results (Methylated) in cells transfected with the plasmid CRISPR1, CRISPR2, and donor DNA combination to introduce methylation, and (b) shows the results (Control) in control cells transfected with the plasmid PX459-MSpCas9 (without guide RNA) and donor DNA combination. If B cells become cancerous, the cells can survive in the agar gel culture described in (2) above, and therefore turn blue by the Crystal Violet staining described above. As shown in Figures 7 to 10, it was confirmed that introducing methylation into the promoter of the SP3 gene in B cells derived from cancer patients specifically induces the cancerous transformation of those cells.

[0158] As described above, the present invention makes it possible to provide a novel gene knock-in method, a method for producing gene knock-in cells, gene knock-in cells, and kits for use therein, which enable the insertion of long chains of exogenous DNA into target sites of genomic DNA with higher efficiency than conventional methods.

[0159] Furthermore, according to the present invention, it is possible to efficiently insert long-chain (e.g., 700 bp or more) exogenous DNA and / or exogenous DNA modified extracellularly into target sites of genomic DNA. For example, by inserting exogenous DNA containing 700 bp or more 100% methylated base pairs into the promoter of a specific gene, it becomes possible to suppress the expression of that gene with high precision.

[0160] Furthermore, according to the present invention, it is possible to provide a novel method for assessing the risk of cancer in cells, which can be directly evaluated by methylating the promoter of the cell's genomic DNA, and a method for producing cancer cells without gene sequence abnormalities caused by methylation of the promoter of the cell's genomic DNA.

[0161] Therefore, according to the present invention, it is also possible to provide a method (gene evaluation method) that can evaluate whether a gene under the control of the promoter is a gene involved in carcinogenesis, and, if the cells are cells derived from a subject and the promoter is a promoter of a gene related to a specific cancer, a method (subject evaluation method) that can directly evaluate the future risk of cancer (risk of developing the specific cancer) in the subject without relying on statistical indicators.

[0162] 100...Target DNA region, 111...Target site (target promoter site), 121...First cleavage region, 122...Second cleavage region, 131...First nucleotide sequence, 132...Second nucleotide sequence, 101...First cleavage site, 102...Second cleavage site, 200...Donor DNA, 112...Exogenous DNA (methylated promoter site), 223...Third cleavage region, 224...Fourth cleavage region, 225...Fifth cleavage region, 226...Sixth cleavage region, 231...5'-microhomology region, 232...3'-microhomology region, 203...Third cleavage site, 204...Fourth cleavage site, 205...Fifth cleavage site, 206...Sixth cleavage site.

[0163] Sequence ID: 1 <223> MSpCas9 Sequence ID: 2 <223> MSpCas9 Forward Primer Sequence ID: 3 <223> MSpCas9 Reverse Primer Sequence ID: 4 <223> sgRNA1 Sequence ID: 5 <223> sgRNA2 Sequence ID: 6 <223> sgRNA3 Sequence ID: 7 <223> sgRNA6 Sequence ID: 8 <223> Donor hSP3pro Forward Primer Sequence ID: 9 <223> Donor hSP3pro Reverse Primer Sequence ID: 10 <223> hSP3pro Forward Primer Sequence ID: 11 <223> hSP3pro Reverse Primer Sequence ID: 12 <223> GAPDH Forward Primer Sequence ID: 13 <223> GAPDH Reverse Primer Sequence ID: 14 ​​<223> hSP3 Forward Primer Sequence ID: 15 <223> hSP3 Reverse Primer SEQ ID NO: 16 <223> hSP3pro Forward Primer 2 SEQ ID NO: 17 <223> hSP3pro Reverse Primer 2

Claims

1. A method for knocking in exogenous DNA into a target site in genomic DNA, comprising the step of introducing into a cell donor DNA containing the exogenous DNA and a CRISPR-Cas9 system comprising a Cas9 protein and its guide RNA, wherein the donor DNA comprises, in order from the 5' side, a 5'-microhomology region consisting of a first nucleotide sequence that can be linked by microhomology-mediated binding to a first nucleotide sequence on the 5' side of the target site in the genomic DNA, the exogenous DNA, and a 3'-microhomology region consisting of a second nucleotide sequence that can be linked by microhomology-mediated binding to a second nucleotide sequence on the 3' side of the target site in the genomic DNA, and the CRISPR-Cas9 system cleaves a first cleavage region between the target site and the first nucleotide sequence and a second cleavage region between the target site and the second nucleotide sequence in the genomic DNA, and a third cleavage region on the 5' side of the 5'-microhomology region, a fourth cleavage region between the exogenous DNA and the 5'-microhomology region, a fifth cleavage region between the exogenous DNA and the 3'-microhomology region, and a sixth cleavage region on the 3' side of the 3'-microhomology region in the donor DNA; and the exogenous DNA is inserted between a first nucleotide sequence and a second nucleotide sequence in the genomic DNA.

2. The gene knock-in method of claim 1, wherein the first cleavage region and second cleavage region of the genomic DNA, and the third cleavage region, fourth cleavage region, fifth cleavage region, and sixth cleavage region of the donor DNA comprise a first guide RNA target sequence, a second guide RNA target sequence, a third guide RNA target sequence, a fourth guide RNA target sequence, a fifth guide RNA target sequence, and a sixth guide RNA target sequence, respectively, that are recognized by the guide RNA.

3. The gene knock-in method according to claim 1 or 2, wherein the Cas9 protein is a wild-type Streptococcus pyogenes Cas9 (SpCas9) protein containing point mutations N692A, M694A, Q695A, and H698A.

4. A gene knock-in method according to any one of claims 1 to 3, wherein the length of the exogenous DNA of the donor DNA is 700 bp or more.

5. A gene knock-in method according to any one of claims 1 to 4, wherein the exogenous DNA of the donor DNA is methylated.

6. The gene knock-in method of claim 5, wherein the target site of the genomic DNA is contained in a promoter, and the exogenous DNA is the target site that has been methylated.

7. A method for producing gene knock-in cells, comprising using the gene knock-in method according to any one of claims 1 to 6 to produce cells in which the exogenous DNA has been inserted into a target site in genomic DNA.

8. Gene knock-in cells into which exogenous DNA containing 700 bp or more of methylated base pairs has been introduced.

9. A method for assessing the risk of cancer in a target cell, comprising: a methylation step of methylating a target promoter site contained in a promoter of genomic DNA in the target cell; a detection step of detecting cancer in the target cell; and a cell evaluation step of assessing the risk of cancer in the cell using the cancer in the target cell as an indicator.

10. A method for assessing the risk of cancer described in claim 9, wherein the length of the target promoter site is 700 bp or more.

11. The method for assessing cancer risk described in claim 9 or 10, wherein the methylation step is a step of knocking in an exogenous methylated promoter site into the target promoter site of the genomic DNA of the target cell using a gene knock-in method described in any one of claims 1 to 6, wherein the target site is the target promoter site and the exogenous DNA is the methylated promoter site.

12. A method for assessing the risk of cancer described in any one of claims 9 to 11, further comprising a gene evaluation step of evaluating the cancer potential of a gene controlled by a promoter containing the target promoter site, using canceration of the target cell as an indicator.

13. A method for assessing the risk of cancer according to any one of claims 9 to 11, wherein the target cells are cells derived from a subject, the promoter containing the target promoter site is a promoter of at least one gene selected from the group consisting of SP3, CDKN2A, p53, p21, and TERT1, and the method further comprises a subject evaluation step of assessing the risk of cancer in the subject using canceration of the target cells as an indicator.

14. The method for assessing the risk of cancer according to claim 13, wherein the target cells are at least one selected from the group consisting of B cells, hepatocytes, gastric epithelial cells, and pancreatic duct mucosal cells.

15. A method for producing cancer cells, comprising the step of methylating a target promoter site contained in a promoter of genomic DNA in a cell to obtain cancer cells.

16. The method for producing cancer cells described in claim 15, wherein the length of the target promoter region is 700 bp or more.

17. A method for producing cancer cells according to claim 15 or 16, wherein the methylation is performed by knocking in an exogenous methylated promoter site into the target promoter site of the genomic DNA of the cell using a gene knock-in method according to any one of claims 1 to 6, wherein the target site is the target promoter site, and the exogenous DNA is the methylated promoter site.

18. A kit for use in the gene knock-in method according to any one of claims 1 to 6, comprising at least one selected from the group consisting of the following (a) to (b): (a) a Cas9 protein that cleaves the first cleavage region and the second cleavage region of the genomic DNA, and the third cleavage region, the fourth cleavage region, the fifth cleavage region, and the sixth cleavage region of the donor DNA, a polynucleotide encoding the Cas9 protein, or a vector expressing the Cas9 protein; and (b) a guide RNA for the Cas9 protein, a polynucleotide encoding the guide RNA, or a vector expressing the guide RNA.

19. The gene knock-in kit of claim 18, wherein the guide RNAs comprise a first guide RNA, a second guide RNA, a third guide RNA, a fourth guide RNA, a fifth guide RNA, and a sixth guide RNA that recognize a first guide RNA target sequence and a second guide RNA target sequence of the genomic DNA, and a third guide RNA target sequence, a fourth guide RNA target sequence, a fifth guide RNA target sequence, and a sixth guide RNA target sequence of the donor DNA, respectively.

20. The gene knock-in kit according to claim 18 or 19, wherein the Cas9 protein is a wild-type Streptococcus pyogenes Cas9 (SpCas9) protein containing point mutations N692A, M694A, Q695A, and H698A.