Method for inducing large-scale deletions in genomic DNA and method for analyzing genomic DNA

Abstract

The present invention provides a method for causing large-scale deletions of a genome. The present invention provides a method for identifying a gene on a genome that affects the survival of a cell (e.g., a gene essential for survival). The present invention also provides a method for reintroducing a gene essential for survival at another location on a genome and a method for causing even larger deletions to the genome thereby.

Description

[Technical Field]

[0001] This invention relates to a method for inducing large-scale deletions in genomic DNA and a method for analyzing genomic DNA. [Background technology]

[0002] In higher organisms, only 1-2% of the genome sequence is used to encode proteins, whereas in mammals, large intergeneric sequences are allocated to proximal (promoter) or long-range (enhancer, suppressor, insulator, etc.) regulatory elements. Therefore, in designing genomes for creating functional artificial cells, the design of intergeneric regions, in addition to gene selection, is key. The Encyclopedia of DNA Elements (ENCODE) project suggests that in human cultured cells, the majority of intergeneric regions exhibit specific transcription factor binding and histone modification, suggesting that this may provide guidance for rational intergeneric region design. However, it remains unclear whether all of these transcriptional and epigenetic activities are necessary for normal cellular function. Mammalian cell networks have become evolutionarily robust due to the expansion of gene families and the increasing redundancy of gene regulatory networks. Therefore, it is possible that not only non-essential genes, but also the majority of intergeneric regions, are not necessarily required for cell survival or maintenance (Non-patent documents 1-3). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Xavier, JC, Patil, KR & Rocha, I., Microbiol Mol Biol Rev, 78, 487-509 (2014). [Non-Patent Document 2] Posfai, G., Science, 312, 1044-1046 (2006). [Non-Patent Document 3] Hutchison, CA et al., Science, 351, aad6253-aad6253 (2016). [Overview of the project]

[0004] This invention provides a method for inducing deletions in genomic DNA and a method for analyzing genomic DNA.

[0005] The inventors have developed a method for inducing deletion of a DNA region between two target sequences using a sequence-specific nucleic acid cleavage molecule that can sequence-specifically cleave target sequences. The inventors have also found that when the size of the DNA region to be deleted in genomic DNA is increased, deleting genes necessary for cell survival and / or proliferation negatively impacts cell survival and / or proliferation. Based on this, the inventors have identified the location of genes necessary for cell survival and / or proliferation and a method for identifying such genes. Furthermore, the inventors have found a method for creating cells capable of survival and / or proliferation despite having a DNA region deletion by ectopically inserting genes necessary for cell survival and / or proliferation into genomic DNA. The inventors have also found a method for deleting a DNA region into which a negative selection marker gene has been inserted. This invention is based on these findings.

[0006] According to the present invention, the following inventions may be provided. (1) an in vitro method, (a) Prepare a cell population containing isolated cells, (b) Applying sequence-specific nucleic acid cleavage molecules capable of sequence-specifically cleaving two target sequences on the genomic DNA to the genomic DNA of the cells in the cell population, thereby causing cleavage at the two target sequences on the genomic DNA, and thereby causing deletion of a DNA region in the region between the two cleavage sites in at least some of the cells in the cell population, (c) Subsequently, the obtained cell population is cultured to determine the effect of the deletion of the DNA region on cell proliferation or survival, including, method. (2) The method according to (1) above, wherein the determination in (c) above is made by determining the DNA region deletion efficiency or an estimate thereof and comparing the percentage of cells having DNA region deletions in the cell population after subsequent culture with the determined deletion efficiency or an estimate thereof. (3) The method according to (2) above, wherein in (c) above, the deletion efficiency or an estimated value thereof and the percentage of cells having deletions in the DNA region after culture are determined as the percentage of cells having deletions relative to the total number of cells contained in a suspension containing a cell population. (4) The method according to (3) above, wherein the above proportion is determined by a counting technique for genomic DNA with and without deletions contained in the suspension. (5) A method according to any of (1) to (4) above, (d) Based on the presence or magnitude of the effect on cell survival and / or proliferation in (c) compared to a control cell population, determine whether the deleted DNA region contains genes that control cell survival and / or proliferation compared to the control genomic DNA. Methods that further include this. (6) The method according to (5) above, wherein the control cell population is a cell population that includes cells having deletions of a larger DNA region, a smaller DNA region, or a different DNA region as described in (b) above. (7) A method according to (5) or (6) above, further comprising identifying at least one gene that controls cell survival and / or proliferation from genes present in the deleted DNA region compared to control genomic DNA. (8) The method described in any of the above () to (7), (f) Identify at least one gene from the deleted DNA region that controls cell survival and / or proliferation. Methods that further include this. (9)(g) Further comprising introducing at least one gene that controls the survival and / or proliferation of cells operably linked to a control array into genomic DNA having a deletion in the DNA region, the method according to (7) or (8) above. (10) The method according to any one of (1) to (9) above, wherein the size of the DNA region deleted in (b) is 0.5 Mbp or more. (11) The method according to any one of (1) to (10) above, wherein the size of the DNA region deleted in (b) is 1 Mbp or more. (12) An in vitro method comprising: (α) preparing a cell population containing isolated cells, wherein the isolated cells contain a negative selection marker gene in the region to be deleted; (β) respectively acting on the genomic DNA of the cells in the cell population with sequence-specific nucleic acid cleavage molecules capable of sequence-specifically cleaving two target sequences on the genomic DNA to cause cleavage at the two target sequences on the genomic DNA, thereby causing a deletion of a DNA region in the region between the two cleavage sites in at least some of the cells in the cell population, wherein the two sites are designed at positions sandwiching the negative selection marker gene; (γ) selecting cells having no negative selection marker gene; and comprising a method. (13) The method according to (12) above, wherein the negative selection marker gene is inserted exogenously into the region to be deleted. (14) The method according to (12) above, wherein the negative selection marker gene is an endogenous gene in the genomic DNA. (15) The method according to any one of (1) to (14) above, wherein the cells are eukaryotic cells. (16) The method according to any one of (1) to (11) above, wherein step (c) is carried out without passing through the step of screening some cells from the cell population obtained in step (b).

Brief Description of the Drawings

[0007] [Figure 1] Figure 1 shows a method for generating human cells with a deletion in the DNA region containing the HPRT1 gene. Panel a shows a DNA region deletion scheme using a negative selection marker gene. When the DNA region containing the negative selection marker gene is deleted, the cells become more viable. Therefore, by selecting cells using the expression of the negative selection marker gene as an indicator, cells with a DNA region deletion can be obtained. Panel a illustrates an example using the CRISPR-Cas9 system as a sequence-specific nucleic acid cleavage molecule. Cells are transfected with a plasmid that expresses gRNAs for two target sequences positioned on either side of the negative selection marker gene, and Cas9 endonuclease. After two days, some cells are obtained and junction PCR of the genomic deletion region is performed using digital PCR. The cells are cultured in a selective medium until colonies are formed. As a result, cells with the negative selection marker gene, i.e., cells without deletion, die, and only cells with deletion survive, proliferate, and form colonies. The number of colonies is counted, and junction PCR is performed again using digital PCR to confirm the presence of the deletion. Panel b shows the results of deleting the region around the endogenous HPRT1 gene locus on the X chromosome, followed by PCR of the junction region using digital PCR. In digital PCR, 100 genomes were used as the template for 1 PCR, and 48 PCRs were performed. Amplification products were obtained in 14 cases, so the deletion efficiency is 14 / 48 / 100×100 = approximately 0.3 (%). Panel c shows the colonies formed after selection with 6-thioguanine (6-TG) and their number. Panel d shows the sequence of the sequences before and after the deletion region using L1-R1. Generally, it can be seen that the DNA region between the two target sequences was deleted. [Figure 2]Figure 2 shows a map of the genomic region surrounding the HPRT1 gene and the creation of megascale deletions in that region. Panel a shows the Refseq genes surrounding the HPRT1 gene on the X chromosome. Essential genes for cell proliferation, as estimated by the OGEE database, are indicated by circles. Panels b and c show the results of experiments in which deletion regions were significantly altered in the centromere and telomere directions. Target sequences of sequence-specific nucleic acid cleavage molecules are indicated by vertical dashed lines with symbols L1-L5 and R1-R5. The number of colonies formed and deletion efficiency (%) after 6-TG selection of cell populations in which deletions of each DNA region were induced are shown. [Figure 3] Figure 3 shows the results of genomic analysis of clones with L4-R4 deletions. Panel a shows the PCR results revealing the presence of genes in HAP1 cells (WT) and clones with L4-R4 deletions, respectively. Panel b shows the relative proliferative capacity of clones with L4-R4 deletions. Relative proliferative capacity is determined as the ratio of cells with deletions to wild-type cells, measured at 0 and 16 days after deletion, and indicates that this ratio did not change. Panel c shows the results of microarray analysis of gene expression in clones with L4-R4 deletions. [Figure 4] Figure 4 shows megascale deletions involving essential genes and deletion rescue experiments. Panel a shows a scheme for gene insertion into the 3' untranslated region (UTR) of the GAPDH locus using a pHY262 vector containing 3kb upstream and downstream sequences flanking the essential gene. Panel b shows the results of real-time PCR analysis of the expression of the rescued RBMX2 and MMGT1 genes. Panels c and d show the number of colonies formed and deletion efficiency for strains lacking endogenous RBMX2 and MMGT1 genes and strains in which RBMX2 and MMGT1 were rescued, respectively. [Figure 5]Figure 5 shows the results of megascale deletions in regions containing essential genes. Panels a and c show the results of experiments in which regions containing the RBMX2 and MMGT1 genes were deleted, but the genes were reintroduced downstream of the GAPDH locus, further expanding the deleted region. Panel b shows the relative colony size measurements after the experiment in panel a. The p-value was determined from the results of three independent experiments. [Figure 6] Figure 6 shows the creation of megascale deletions in HCT116 cells using a negative selection marker gene. Panel a shows the scheme for inserting thymidine kinase (TK) downstream of the OCRL gene in HCT116 cells using a pHYT271 vector containing a TK expression cassette. Insertion was confirmed by the increased size of the amplified product by junction PCR. Panel b shows the gene map of the locus that causes deletion in HCT116 cells. Panel c shows the deletion of a megascale DNA region using the negative selection marker gene TK. The deletion was expanded in the centromere direction. The number of colonies formed after selection and the deletion efficiency for each deletion are shown. The numbers represent the mean and standard deviation of three independent experiments. [Figure 7] Figure 7 shows a method for inducing megascale deletions without negative selection. In HAP1 cells, three target regions, A to C, were deleted. Two and seventeen days after inducing genomic deletion, the presence or absence of deletions was determined by digital PCR on 100 genomes per well, and the percentage of cells with deletions in the cell population was calculated. As a result, when target regions B and C were deleted, the percentage of cells with deletions in the cell population decreased. This decrease indicates that the deleted regions contain genes necessary for cell survival and / or proliferation. [Figure 8] Figure 8 shows the results of an experiment in which regions on the human X chromosome that were previously considered not essential for cell survival were deleted using the method of the present invention, and the effect of each deletion on cell viability was investigated. Specific description of the invention

[0008] In this specification, "cell" refers to the basic unit of life that has at least genomic DNA, cytoplasm, and a membrane structure enclosing them. Cells are not particularly limited, but examples include prokaryotic cells and eukaryotic cells. Genomic DNA includes the cell's endogenous DNA, but is not necessarily composed solely of the cell's endogenous factors.

[0009] Cells contain their own genomic DNA, but may also contain the genomic DNA of foreign invaders (e.g., pathogens). In this specification, the genomic DNA of the cell itself is referred to as "host genomic DNA." Invader genomic DNA may exist within the cell separately from the host genomic DNA, but it may also be incorporated into the host genomic DNA. Host genomic DNA may contain foreign elements (e.g., insertions of all or part of the genomic DNA of viruses, etc.).

[0010] In this specification, "cell population" means a composition containing multiple cells.

[0011] In this specification, “isolation” means separating a cell of interest from at least one other component. Isolation may be performed, for example, by separating and removing a cell in its natural state from other components that coexist with it in its natural state. Isolation may be performed, for example, by separating and removing some cells from a multicellular organism. The technique for handling isolated cells is referred to herein as an in vitro technique.

[0012] In this specification, “purification” means further separating isolated cells of interest from other coexisting components. Purification may be carried out, for example, by separating cells of interest from other components based on morphology or surface markers. Purification may be carried out by ultradilution and / or cloning of cells. Purification may be carried out by cell line formation of the cells of interest. Purification may be carried out based on the expression of marker genes, such as drug resistance genes or genes encoding fluorescent proteins, if the cells of interest have such marker genes. In this specification, “enrichment” means increasing the density of cells of interest.

[0013] In this specification, the terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to nucleotide polymers in which nucleotides are linked by phosphodiester bonds. “Polynucleotide” and “nucleic acid” may be DNA, RNA, or a combination of DNA and RNA. Furthermore, “polynucleotide” and “nucleic acid” may be polymers of natural nucleotides, polymers of natural nucleotides and non-natural nucleotides (analogs of natural nucleotides, nucleotides in which at least one of the base, sugar, and phosphate parts is modified (e.g., a phosphorothioate skeleton)), or polymers of non-natural nucleotides.

[0014] In this specification, the base sequences of "polynucleotides" or "nucleic acids" are described using commonly accepted single-letter codes unless otherwise specified. Unless otherwise specified, base sequences are described from the 5' end to the 3' end. The nucleotide residues constituting "polynucleotides" or "nucleic acids" may be described simply as adenine, thymine, cytosine, guanine, or uracil, or by their single-letter codes.

[0015] In this specification, the term “gene” refers to a polynucleotide containing at least one open reading frame that codes for a particular protein. A gene may contain both exons and introns.

[0016] In this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to polymers of amino acids linked by amide bonds. A "polypeptide," "peptide," or "protein" may be a polymer of natural amino acids, a polymer of natural amino acids and non-natural amino acids (chemical analogs, modified derivatives, etc. of natural amino acids), or a polymer of non-natural amino acids. Unless otherwise specified, amino acid sequences are described from the N-terminus to the C-terminus.

[0017] In this specification, the term "allele" refers to a set of nucleotide sequences located at the same locus on a chromosomal genome. In some embodiments, diploid cells may have two alleles at the same locus, and triploid cells may have three alleles at the same locus. In other embodiments, additional alleles may be formed by abnormal copies of the chromosome or abnormal additional copies of the locus.

[0018] In this specification, the terms “genome modification” and “genome editing” are used interchangeably and refer to inducing mutations at a desired location (target region) on the genome. Genome modification may include the use of sequence-specific nucleic acid cleavage molecules (e.g., sequence-specific or sequence-dependent endonucleases) designed to cleave target region DNA. In a preferred embodiment, genome modification may include the use of nucleases engineered to cleave target region DNA. In a preferred embodiment, genome modification may include the use of nucleases engineered to cleave target sequences having a specific base sequence within the target region (e.g., TALENs or zinc finger nucleases (ZFNs)). In a particularly preferred embodiment, genome modification may include the use of nucleases engineered to cleave target sequences having a specific base sequence within the target region (e.g., the CRISPR-Cas9 system). In a preferred embodiment, genome modification may include the use of restriction enzymes that have only one cleavage site in the genome, such as meganucleases (e.g., 16-base sequence-specific restriction enzymes (theoretically 4)) to cleave target sequences having a specific base sequence within the target region. 16 Restriction enzymes with 17-base sequence specificity (theoretically 4) (present at a ratio of one per base) 17 Restriction enzymes that have 18-base sequence specificity (theoretically 4 18Sequence-specific endonucleases, such as those present at a rate of one per base, can also be used. Typically, the use of site-specific nucleases induces double-strand breaks (DSBs) in the DNA of the target region, after which the genome is repaired by endogenous cellular processes such as homologous directed repair (HDR) and non-homologous end-joining repair (NHEJ). NHEJ is a repair method that joins the ends of double-strand breaks without using donor DNA, and insertions and / or deletions (indels) are frequently induced during repair. HDR is a repair mechanism that uses donor DNA, and it is also possible to introduce desired mutations into the target region. As for genome modification technologies, the CRISPR / Cas system is a preferred example.Examples of meganucleases include I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI , I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-Na nI, I-NclIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-Pg rIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp68031, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I- TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-Mtul, PI-MtuHIP A meganuclease and its cleavage site (or recognition site) selected from the group consisting of PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, and PI-TliII, and functional derivative restriction enzymes thereof, preferably a meganuclease and its cleavage site (or recognition site) that is a restriction enzyme having sequence specificity of 18 bases or more, in particular a meganuclease and its cleavage site that does not cleave the cell genome at one or more locations.

[0019] The term "target region" refers to a region targeted by a genome modification system. In this invention, a DNA region on the genome located between two target regions (for example, a first target region and a second target region) can be deleted.

[0020] The term "sequence-specific nucleic acid cleavage molecule" refers to a molecule that recognizes a specific nucleic acid sequence and can cleave the nucleic acid at that specific sequence. A sequence-specific nucleic acid cleavage molecule is a molecule that has the activity to cleave nucleic acids in a sequence-specific manner (sequence-specific nucleic acid cleavage activity).

[0021] The term "target sequence" refers to the DNA sequence in the genome that is targeted for cleavage by a sequence-specific nucleic acid cleavage molecule. When the sequence-specific nucleic acid cleavage molecule is a Cas protein, the target sequence refers to the DNA sequence in the genome that is targeted for cleavage by the Cas protein. When using the Cas9 protein as the Cas protein, the target sequence must be a sequence adjacent to the 5' end of a protospacer adjacent motif (PAM). Typically, the target sequence is selected from a sequence of 17 to 30 bases (preferably 18 to 25 bases, more preferably 19 to 22 bases, and even more preferably 20 bases) adjacent to the 5' end of a PAM. Known design tools such as CRISPR DESIGN (crispr.mit.edu / ) can be used to design the target sequence.

[0022] The term "Cas protein" refers to a CRISPR-associated protein. In a preferred embodiment, the Cas protein forms a complex with guide RNA and exhibits endonuclease activity or nickase activity. Examples of Cas proteins are not particularly limited, but include the Cas9 protein. The term "Cas protein" includes wild-type Cas proteins and their homologs (paralogs and orthologs), as well as their variants, insofar as they cooperate with guide RNA to exhibit endonuclease activity or nickase activity. In a preferred embodiment, the Cas protein is involved in a class 2 CRISPR / Cas system, and more preferably in a type II CRISPR / Cas system. A preferred example of a Cas protein is the Cas9 protein.

[0023] The term "Cas9 protein" refers to the Cas protein involved in the type II CRISPR / Cas system. The Cas9 protein forms a complex with guide RNA and exhibits activity in cooperation with the guide RNA to cleave DNA in a target region. The Cas9 protein includes the wild-type Cas9 protein and its homologs (paralogs and orthologs), as well as their variants, as long as they possess the aforementioned activity. The wild-type Cas9 protein has a RuvC domain and an HNH domain as nuclease domains, but the Cas9 protein used herein may have either the RuvC domain or the HNH domain inactivated. Cas9 with either the RuvC domain or the HNH domain inactivated introduces single-strand breaks (nicks) into double-stranded DNA. Therefore, when using Cas9 with either the RuvC domain or the HNH domain inactivated to cleave double-stranded DNA, a modified system can be constructed in which target sequences for Cas9 are set for both the sense strand and the antisense strand, so that nicks in the sense strand and antisense difference occur at sufficiently close positions, thereby inducing double-strand breaks. The species from which the Cas9 protein originates is not particularly limited, but bacteria belonging to the genera Streptococcus, Staphylococcus, Neisseria, or Treponema are preferred examples. More specifically, Cas9 proteins derived from S. pyogenes, S. thermophilus, S. aureus, N. meningitidis, or T. denticola are preferred examples. In a preferred embodiment, the Cas9 protein is a Cas9 protein derived from S. pyogenes.

[0024] The terms “guide RNA” and “gRNA” are used interchangeably and refer to RNA that can form a complex with the Cas protein and guide the Cas protein to a target region. In a preferred embodiment, the guide RNA includes CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). crRNA is involved in binding to the target region on the genome, and tracrRNA is involved in binding to the Cas protein. In a preferred embodiment, the crRNA includes a spacer sequence and a repeat sequence, the spacer sequence binding to the complementary strand of the target sequence in the target region. In a preferred embodiment, the tracrRNA includes an anti-repeat sequence and a 3' tail sequence. The anti-repeat sequence has a sequence complementary to the repeat sequence of the crRNA and forms base pairs with the repeat sequence, and the 3' tail sequence typically forms three stem-loops. The guide RNA may be a single guide RNA (sgRNA) formed by ligating the 5' end of tracrRNA to the 3' end of crRNA, or it may be a separate RNA molecule with base pairings formed by repeat and anti-repeat sequences. In a preferred embodiment, the guide RNA is sgRNA.

[0025] The repeat sequence of crRNA and the sequence of tracrRNA can be appropriately selected depending on the type of Cas protein, and those derived from the same bacterial species as the Cas protein can be used. For the CRISPR-Cas9 system, Cas9 protein, crRNA, and tracrRNA (or sgRNA) derived from S. pyogenes can be used. Various crRNA repeat sequences and tracrRNA sequences have been proposed for sgRNA design, and those skilled in the art can design sgRNAs based on known techniques (e.g., Jinek et al. (2012) Science, 337, 816-21; Mali et al. (2013) Science, 339: 6121, 823-6; Cong et al. (2013) Science, 339: 6121, 819-23; Hwang et al. (2013) Nat. Biotechnol. 31: 3, 227-9; Jinek et al. (2013) eLife, 2, e00471).

[0026] The term "functionally ligated" as used with respect to polynucleotides means that the first base sequence is positioned close enough to the second base sequence that the first base sequence can influence the second base sequence or a region under the control of the second base sequence. For example, functionally ligated polynucleotides to a promoter means that the polynucleotide is ligated in such a way that it is expressed under the control of the promoter.

[0027] The term "expression-ready state" refers to a state in which a polynucleotide can be transcribed within a cell into which the polynucleotide has been introduced. The term "expression vector" refers to a vector containing a target polynucleotide and equipped with a system that enables the expression of the target polynucleotide within the cell into which the vector is introduced. For example, a "Cas protein expression vector" means a vector that enables the expression of the Cas protein within the cell into which the vector is introduced. Similarly, a "guide RNA expression vector" means a vector that enables the expression of guide RNA within the cell into which the vector is introduced.

[0028] In this specification, sequence identity (or homology) between nucleotide sequences or amino acid sequences is determined by juxtaposing the two nucleotide sequences or amino acid sequences, inserting gaps in the areas corresponding to insertions and deletions so that the corresponding nucleotides or amino acids are most likely to match, and then determining the proportion of matching nucleotides or amino acids relative to the entire nucleotide sequence or amino acid sequence, excluding the gaps in the resulting alignment. Sequence identity between nucleotide sequences or amino acid sequences can be determined using various homology search software known in the art. For example, the sequence identity value of a nucleotide sequence can be obtained by calculation based on the alignment obtained by the known homology search software BLASTN, and the sequence identity value of an amino acid sequence can be obtained by calculation based on the alignment obtained by the known homology search software BLASTP.

[0029] <Method of the present invention> According to the present invention, (a) Prepare a cell population containing cells, (b) Applying sequence-specific nucleic acid cleavage molecules capable of sequence-specifically cleaving two target sequences on the genomic DNA to the genomic DNA of the cells in the cell population, thereby causing cleavage at the two target sequences on the genomic DNA, and thereby causing deletion of a DNA region in the region between the two cleavage sites in at least some of the cells in the cell population, Methods including The present invention provides a method that can be performed in vitro. The present invention may also preferably use isolated cells.

[0030] Therefore, according to the present invention, an in vitro method is provided, (a) Prepare a cell population containing isolated cells, (b) Applying sequence-specific nucleic acid cleavage molecules capable of sequence-specifically cleaving two target sequences on the genomic DNA to the genomic DNA of the cells in the cell population, thereby causing cleavage at the two target sequences on the genomic DNA, and thereby causing deletion of a DNA region in the region between the two cleavage sites in at least some of the cells in the cell population, A method including this is provided.

[0031] According to one aspect of the present invention, I, the present inventor, (c) In a cell population that includes cells that have lost the DNA region, determine the proportion of cells that have lost the DNA region relative to the total number of cells. It also includes.

[0032] In (a) above, the cells included in the cell population may be of a single type or of multiple types. Preferably, the cells included in the cell population may be of a single type (e.g., a cell line, cloned cells). The cell population may be a population of suspension cells or a population of adherent cells. The cell population may be a population of single-celled cells or a cell population containing cell clumps. The cell population may include cells and physiologically acceptable excipients. Physiologically acceptable excipients are excipients that have appropriate conditions for cell maintenance, such as water, salt, pH buffers, isotonic agents, etc. The cell population may be, for example, 10 2 The above 10 3 The above 10 4 The above 10 5 or more, or 10 6 It may contain the above cells.

[0033] In some aspects of the present invention, the cells may be isolated cells. The cells may be purified cells. The cells may be cells of a single-celled organism. The cells may be a cell line. The cells may be cloned cells (cell clones). The cells may be single-celled cells. The cells may be suspension cells. The cells may be adherent cells. The cells may form cell aggregates. The cells may form colonies. The cells are placed under conditions suitable for their maintenance or proliferation.

[0034] In some embodiments, cells may be selected from a group consisting of pluripotent cells and pluripotent stem cells (such as embryonic stem cells and induced pluripotent stem cells). In some embodiments, cells may be tissue stem cells. In some embodiments, cells may be somatic cells. In some embodiments, cells may be tissue progenitor cells. In some embodiments, cells may be germline cells (e.g., germ cells). In some embodiments, cells may be cell lines. In some embodiments, cells may be immortalized cells. In some embodiments, cells may be cancer cells. In some embodiments, cells may be non-cancerous cells. In some embodiments, cells may be cells from diseased patients. In some embodiments, cells may be cells from healthy individuals. In some embodiments, cells may be cells infected with foreign pathogens. In some embodiments, cells may be uninfected cells. In some embodiments, the cells may be animal cells (e.g., bird cells, fish cells, amphibian cells, reptile cells, mammalian cells, rodent cells, primate cells, human cells). In some embodiments, the cells may be selected from the group consisting of, for example, insect cells (e.g., silkworm cells), HEK293 cells, HEK293T cells, Expi293F® cells, FreeStyle® 293F cells, Chinese hamster ovary cells (CHO cells), CHO-S cells, CHO-K1 cells, and ExpiCHO cells, as well as cells derived from these cells (in particular, recombinant protein-producing cells). In some embodiments, the cells may be plant cells. In some embodiments, the cells may also be microbial cells, such as filamentous fungi, Gram-positive bacteria such as actinomycetes, Gram-negative bacteria such as Escherichia coli, and fungi such as yeast.

[0035] The cells used in the present invention may preferably be haploid or diploid. Other polyploid cells may also be used without particular limitation. In all embodiments of the present invention, haploid cells may be preferably used.

[0036] In (b) above, sequence-specific nucleic acid cleavage molecules capable of sequence-specifically cleaving two target sequences on the genomic DNA can be applied to the cellular genomic DNA (particularly the host genomic DNA). The target sequences can be appropriately set by those skilled in the art in light of the cleavage characteristics of the sequence-specific nucleic acid cleavage molecules used. The target sequences are present in the base sequence of at least one allele, and preferably are sequences that are common to two or more or all alleles. To cleave multiple target sequences on the genome, for example, the CRISPR-Cas9 system, TALEN, and zinc finger nucleases can all be preferably used. Specifically, this is because multiple cleavage sites can be achieved simply by increasing the amount of guide RNA used (a combination of crRNA and tracrRNA, or sgRNA) in accordance with the increase in the number of target sequences (see, for example, WO2014 / 093661). When using a genome modification system other than the CRISPR-Cas9 system for cleavage, a cleavage molecule is prepared for each target sequence.

[0037] When genomic DNA (especially host genomic DNA) is broken, the cell's genome repair mechanism reconnects the broken sites. If two breaks occur in genomic DNA, the genome repair mechanism will, with a certain probability, directly connect the telomere end of the genomic DNA with the centromere end of the genomic DNA, and the DNA region between the two break sites may be deleted from the genomic DNA.

[0038] Therefore, in (b) above, a cell population is obtained that includes at least one cell (preferably more cells) having a deletion of the DNA region that occurred in the region between the two cleavage sites.

[0039] In (b) above, the two cleavage sites may be designed to sandwich a region containing one or more genes or candidate genes. The one or more genes or candidate genes contained in the two cleavage sites may include one or more genes that positively control the survival and / or proliferation of one or more cells (e.g., genes essential for cell survival and / or proliferation). The one or more genes or candidate genes contained in the two cleavage sites may include one or more genes whose deletion promotes cell proliferation, such as genes that suppress the proliferation of one or more cells. A person skilled in the art does not necessarily need to know in advance which genes positively control the survival and / or proliferation (e.g., genes essential for cell survival and / or proliferation). However, which genes positively control the survival and / or proliferation (e.g., genes essential for cell survival and / or proliferation) can be estimated in advance using publicly available databases such as the Online Gene Essentiality (OGEE) database (http: / / ogee.medgenius.info / browse / ).

[0040] In one embodiment, a region containing one or more genes or candidate genes may include genes that are presumed to positively regulate cell survival and / or proliferation (e.g., genes essential for cell survival and / or proliferation). In another embodiment, a region containing one or more genes or candidate genes may include genes that are presumed to negatively regulate cell survival and / or proliferation. In another embodiment, a region containing one or more genes or candidate genes may include genes that are presumed to positively regulate cell survival and / or proliferation (e.g., genes essential for cell survival and / or proliferation) and genes that are presumed to negatively regulate cell survival and / or proliferation.

[0041] In (b) above, the two cleavage sites are not particularly limited, but may be designed to sandwich regions of 0.1 Mb or more, 0.2 Mb or more, 0.3 Mb or more, 0.4 Mb or more, 0.5 Mb or more, 0.6 Mb or more, 0.7 Mb or more, 0.8 Mb or more, 0.9 Mb or more, 1 Mb or more, 2 Mb or more, 3 Mb or more, 4 Mb or more, or 5 Mb or more in length. That is, the DNA region deleted from the genomic DNA may have a length of 0.1 Mb or more, 0.2 Mb or more, 0.3 Mb or more, 0.4 Mb or more, 0.5 Mb or more, 0.6 Mb or more, 0.7 Mb or more, 0.8 Mb or more, 0.9 Mb or more, 1 Mb or more, 2 Mb or more, 3 Mb or more, 4 Mb or more, or 5 Mb or more.

[0042] As a result, in (b) above, a cell population can be obtained that includes cells having a DNA region deletion that occurred in the region between the two cleavage sites. DNA such as genomic DNA with two cleavage sites can be repaired within the cell, and this repair can, with a certain probability, produce cells having DNA such as genomic DNA that has a DNA region deletion that occurred in the region between the two cleavage sites. Therefore, as described above, a cell population can be obtained that includes cells having a DNA region deletion that occurred in the region between the two cleavage sites.

[0043] Cells containing a DNA region deletion occurring in the region between the two aforementioned cleavage sites can be obtained from a cell population. This can be done, for example, by ultradiluting the cell population and cloning it. In genomic DNA with a deleted DNA region, the regions before and after the deletion are considered to be directly linked. Therefore, by designing amplification primers to surround this junction, the presence or absence of a DNA region deletion can be determined by PCR using the genomic DNA with the deleted DNA region as a template (junction PCR). Alternatively, the presence or absence of a DNA region deletion can be determined by sequencing the junction. In this case, if the deleted DNA region does not contain a gene that positively regulates cell survival and / or proliferation (e.g., a gene essential for cell survival and / or proliferation), cells with the deletion in that DNA region can be obtained. If the deleted DNA region contains a gene that positively regulates cell survival and / or proliferation (e.g., a gene essential for cell survival and / or proliferation), the cells with the deletion will stop proliferating or die over time, reducing their proportion in the cell population. Alternatively, if the deleted DNA region contains a gene that negatively regulates cell survival and / or proliferation, the cells with the deletion will proliferate over time, increasing their proportion in the cell population.

[0044] In (c) above, the effect of the DNA region deletion on cell proliferation or survival can be determined based on the percentage of cells that have experienced the DNA region deletion. The effect of the deletion of the DNA region on cell proliferation or survival can be determined by various methods.

[0045] Step (c) can be carried out immediately following step (b). Here, cell sorting (isolation of specific cells from other cells, for example, sorting some cells from a cell population) may not be performed between steps (b) and (c). That is, the cell population obtained in step (b) can be cultured as is, and step (c) can be carried out. The culture can be carried out under conditions suitable for culturing (or growing) the cells before deletion. Alternatively, a cell sorting step may be performed between steps (b) and (c).

[0046] In (c) above, the determination may be made by determining the DNA region deletion efficiency or an estimate thereof, and then comparing the percentage of cells with DNA region deletions in the cell population after subsequent culture with the determined deletion efficiency or an estimate thereof.

[0047] In (c) above, the determination of the deletion efficiency or its estimate, and the percentage of cells with deletions in the DNA region after culture, can be determined as the percentage of cells with deletions relative to the total number of cells in the suspension containing the cell population. The above percentage can be determined by a counting technique for genomic DNA with and without deletions in the suspension.

[0048] The effect of DNA region deletion on cell proliferation or survival can be determined from the efficiency of deletion introduction (or an estimate thereof) and the percentage of cells with DNA region deletion after subsequent culture. For example, (c) above {the same applies to the process of determining the effect of DNA region deletion} may include calculating the efficiency of deletion introduction (or an estimate thereof). The efficiency of DNA region deletion introduction can be performed after the treatment in (b) above, but before the effect of the DNA region deletion occurs in the cells. Immediately after DNA region deletion, the effect of the DNA region deletion is small because transcripts and translation products derived from the deleted DNA region remain in the cells. Therefore, after the treatment in (b) above, the efficiency of introducing the DNA region deletion can be confirmed, for example, at 2 hours to 3 days, 4 hours to 3 days, 6 hours to 3 days, 8 hours to 3 days, 12 hours to 3 days, 18 hours to 3 days, 1 day to 2 days, 1 day to 3 days, 1 day to 60 hours, 4 hours to 60 hours, 4 hours to 48 hours, 4 hours to 36 hours, 4 hours to 30 hours, or 4 hours to 2 days. The same applies to the steps in (c) below, which determine the effect of the DNA region deletion, and may also include culturing the obtained cells. Culturing can be carried out under conditions suitable for culturing cells before the DNA region deletion treatment. For example, a cell population containing cells having the DNA region deletion obtained after (b) above can be cultured and evaluated by counting the number of proliferated cells per a certain number of cells, or the number of colonies formed by the proliferated cells. Specifically, a change in the number of proliferating cells per a given number of cells, or the number of colonies formed by the proliferating cells (or a decrease in the proportion of colonies or cells with deletions compared to the efficiency of deletion introduction), means that the deleted DNA region contains genes that control cell survival and / or proliferation. More specifically, a large or increased number of proliferating cells per a given number of cells, or the number of colonies formed by the proliferating cells, means that the deletion of the DNA region had a positive effect on cell proliferation or survival, or that the DNA region contained genes that negatively control cell survival and / or proliferation.Furthermore, a small or decreased number of proliferating cells per a given number of cells, or a small number of colonies formed by the proliferating cells, can be used to assess whether the deletion of the DNA region negatively affected cell proliferation or survival, or whether the DNA region contained a gene that positively regulates cell survival and / or proliferation (in particular, a gene essential for cell survival and / or proliferation). The culture may be performed under conditions suitable for culturing the cells before modification. The ratio of colonies with DNA region deletions to the total number of colonies formed may also be examined.

[0049] Furthermore, for example, the deletion introduction efficiency (or an estimated value thereof) and the proportion of cells in a cell population that have the deletion of the DNA region can also be determined using counting methods such as digital PCR or digital counting techniques for nucleic acids using molecular barcoding. The measurement method does not necessarily involve separating a cell from other cells and / or causing the cells to form colonies. That is, the deletion introduction efficiency (or an estimated value thereof) and the proportion of cells in a cell population that have the deletion of the DNA region can be calculated using a cell suspension (or using genomic DNA extracted from a cell suspension or its amplification product).

[0050] Digital PCR is a method for absolutely quantifying template nucleic acids by dispensing nucleic acids (in this case, genomic DNA) into multiple micro-compartments at concentrations and volumes that ensure a constant number of genomes per well, subjecting them to a PCR reaction, and counting the number of wells in which a PCR reaction occurs. Using microfluidic devices or droplet methods with droplets in oil, nucleic acids can be fractionated into multiple fractions containing a single molecule, and nucleic acid amplification reactions are induced in parallel in each fraction containing a single molecule. By utilizing the fact that no amplification product is produced when no amplification template is present, and an amplification product is produced when an amplification template is present, it is possible to digitally determine whether an amplification template was present in the sample, or its amount, by counting the presence or absence of amplification for each fraction. PCR primers can be designed to amplify when there is no deletion in the DNA region and not amplify when there is a deletion, or to not amplify when there is no deletion in the DNA region and amplify when there is a deletion. The digital counting technology for nucleic acids using molecular barcodes is a technique that assigns a unique molecular barcode (specifically, a unique base sequence) to each nucleic acid fragment, and then absolutely quantifies the number of nucleic acid molecules by sequencing that sequence. In this digital counting technology for nucleic acids using molecular barcodes, the number of different molecular barcodes corresponds to the number of nucleic acid molecules.

[0051] If the DNA region contains genes that positively regulate cell survival and / or proliferation (in particular, genes essential for cell survival and / or proliferation), the cells will die during culture. Therefore, the effect of the deletion of the DNA region on cell proliferation or survival can be evaluated, for example, by determining the proportion of cells with the deletion in the cell population before and after the death (or by determining that the proportion of colonies or cells with the deletion has decreased below the efficiency of deletion introduction).

[0052] The effect of the deletion of the DNA region on cell proliferation or survival can be determined by comparing it with a control. For example, the effect of the deletion of the DNA region on cell proliferation or survival can be determined by comparing it with the proliferation or survival of control cells. Control cells may be, for example, cells having part or all of the DNA region.

[0053] In one aspect of the present invention, step (c) above (the same applies hereafter to the step of determining the effect of DNA region deletion) includes culturing the cells obtained in step (b). This culturing can be carried out, for example, under predetermined conditions. These predetermined conditions include, in addition to normal cell culture conditions, the presence of stress, growth stimuli, differentiation-inducing stimuli, hypoxic conditions, growth factors, growth inhibitors, differentiation-inducing factors, differentiation-inhibiting factors, and drugs. By culturing the cells obtained in step (b) above under these predetermined conditions, the effect (role) of the deleted DNA region on the behavior of the cells under these predetermined conditions can be determined. It is also possible to investigate the effect of culture conditions (predetermined conditions) on the behavior of cells having a specific DNA region deletion. For example, by investigating the effect of drug addition on the behavior of cells having a specific DNA region deletion, it is possible to perform drug characterization or drug screening.

[0054] The method of the present invention is the same as (a) to (c) above, (d) Compared to a control cell population, determine whether the deleted DNA region contains genes that control cell survival and / or proliferation, based on the presence or magnitude of the effect on survival and / or proliferation in (c) above. It may include and.

[0055] In (d) above, the control or negative control may be unmodified or pre-modified cells or cell populations. In (d) above, the control (or negative control) may be cells with a smaller DNA region deletion or a cell population containing such cells. In (d) above, the control (or positive control) may be cells with a larger DNA region deletion or a cell population containing such cells. In (d) above, the control may be cells with a different DNA region deletion or a cell population containing such cells. The cell populations may be obtained by (b) above.

[0056] In (d) above, by using unmodified or pre-modification cells or cell populations as a control, the effect of genes contained in the deleted DNA region on cell survival and / or proliferation can be evaluated. In (d) above, by using cells with smaller DNA region deletions or cell populations containing such cells as controls, the impact of genes contained in the controls that are deleted due to the DNA deletion being evaluated on cell survival and / or proliferation can be assessed. In (d) above, by using cells with larger DNA region deletions or a cell population containing such cells as a control, the effect of genes not included in the control but included in the cells with the DNA deletion being evaluated on cell survival and / or proliferation can be assessed. In (d) above, by using cells with deletions in different DNA regions or a cell population containing such cells as a control, it is possible to evaluate the effects on cell survival and / or proliferation of (d-1) genes contained in the control that are deleted due to the deletion of the DNA being evaluated and / or (d-2) genes not contained in the control that are contained in cells with the deletion of the DNA being evaluated.

[0057] In one embodiment of the present invention, the above (a) to (c) and (e) further comprising determining the proportion of the cells at at least two different time points, and determining whether the deleted DNA region contains a gene that controls the survival and / or proliferation of at least one cell, based on whether or not it has an effect on the survival and / or proliferation of the cells in (c) over time. It may include the following.

[0058] As described in (b) above, a cell population containing cells with the deletion of the DNA region is obtained. The proportion of cells containing the deletion in the cell population depends on the efficiency of the genome modification. The number of cells containing the deletion in the cell population can be increased, decreased, or maintained by culturing. Even after genome modification, cells may exhibit the same behavior as before the modification for a while due to the persistence of transcripts (e.g., mRNA) from genomic DNA in the cytoplasm and the persistence of translation products (e.g., proteins) from those transcripts. However, after culturing for a while, the amount of the persistence of the transcripts and proteins decreases, and the genotype resulting from the DNA deletion becomes expressed as a phenotype. Subsequently, the phenotype mediated by genes that control cell survival and / or proliferation becomes apparent.

[0059] In (e) above, with respect to the two time points (the first time point and the second time point), the first time point may be before the genotype resulting from the DNA deletion is expressed as a phenotype, and the second time point may be after the genotype resulting from the DNA deletion is expressed as a phenotype. For example, the first time point may be within 3 days of genome modification. Alternatively, the second time point may be 3 days or later of genome modification. The interval between the first time point and the second time point may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 1 week or more. A person skilled in the art can determine the first and second time points appropriately in consideration of the state of the cells, etc., and implement the present invention.

[0060] The above (e) further includes determining the proportion of the cells at at least two different time points and assessing whether or not there is an effect over time on the survival and / or proliferation of the cells in (c). In (e) above, if an effect on cell survival and / or proliferation in (c) over time is observed, it is suggested that the deletion of the DNA region causing the effect may contain a gene that controls cell survival and / or proliferation. Furthermore, if an effect on cell survival and / or proliferation in (c) over time is observed, the magnitude of the effect can be evaluated to assess the influence of the gene that controls cell survival and / or proliferation contained in the deletion of the DNA region causing the effect on cell survival and / or proliferation. The magnitude of the effect can be evaluated by comparing it with a control. Examples of controls include positive controls such as genes encoding cell growth factors and / or genes encoding cell growth inhibitors. Negative controls such as genes encoding factors known not to affect cell survival and / or proliferation may also be used as controls.

[0061] If the deleted DNA region contains multiple putative genes (or genes), (f) it may be determined which of these genes controls cell survival and / or proliferation. Determining which of these genes controls cell survival and / or proliferation can be done using various methods. For example, specific gene disruption, fragmentation of the deleted region by gene deletion according to the present invention, gene cloning and functional analysis of that gene can be used to determine which of these genes controls cell survival and / or proliferation.

[0062] If the deleted DNA region contains a gene that controls cell survival and / or proliferation, that gene can be ectopically introduced to another location in the genomic DNA. Therefore, the present invention is (g) Ectopically introducing at least one gene that controls cell survival and / or proliferation (in particular a gene that positively controls cell survival and / or proliferation), which is operablely linked to a regulatory sequence, into genomic DNA having a deletion in the DNA region. It may also include Here, ectopic introduction means introducing something to a location different from the endogenous location.

[0063] By (b) and (g) above, it is possible to induce a large DNA region deletion in the genomic DNA while reducing the impact on cell survival and / or proliferation by ectopically introducing genes that control cell survival and / or proliferation (especially genes that positively control cell survival and / or proliferation, especially genes essential for cell survival) into the genomic DNA, even while causing a deletion of a DNA region in the genomic DNA. In this way, the genomic DNA of a cell can be minimized to a state in which it contains a set of genes essential for survival.

[0064] According to the present invention, (α) Prepare a cell population including cells, where the cells contain a negative selection marker gene in the region to be deleted. (β) The genomic DNA of the cells in the cell population is subjected to sequence-specific nucleic acid cleavage molecules capable of sequence-specifically cleaving two target sequences on the genomic DNA, thereby causing cleavage at the two target sequences on the genomic DNA, and thereby causing deletion of a DNA region in the region between the two cleavage sites in at least some of the cells in the cell population, where the two locations are designed to sandwich the negative selection marker gene. (γ) Selecting cells that do not have a negative selection marker gene, Methods including The present invention provides a method that can be an in vitro method, and the cells may be isolated cells. In one embodiment, the present invention is an in vitro method, and the cells are isolated cells.

[0065] Since (α) above is identical to (a) above except that the cell contains a negative selection marker gene in the region to be deleted, the explanation of the identical parts will be omitted. Here, in a cell population in which cells expressing and not expressing the selection marker are mixed, when selecting cells that do not express the selection marker, the selection marker is referred to as a "negative selection marker" or "selection marker for negative selection."

[0066] Negative selection markers are not particularly limited as long as they can select cells that do not express them. Examples of negative selection marker genes include suicide genes (thymidine kinase (TK)), fluorescent protein genes, luminescent enzyme genes, and chromogenic enzyme genes. If a negative selection marker gene is a gene that negatively affects cell survival (e.g., a suicide gene), it can be functionally linked to an inductive promoter. By functionally linking to an inductive promoter, the negative selection marker gene can be expressed only when it is desired to remove cells that possess the negative selection marker gene. If the negative selection marker gene is an optically detectable marker gene (visible marker gene) such as a fluorescence, luminescence, or chromogenic gene, and has little negative impact on cell survival, it may be constitutively expressed.

[0067] In (α) above, the cell contains a negative selection marker gene (a marker gene that can be used for negative selection) in the DNA region to be deleted. The DNA region to be deleted can be a region that intrinsically contains the negative selection marker gene, or it can be created by exogenously introducing the negative selection marker gene into the DNA region to be deleted. A region that intrinsically contains the negative selection marker gene may be, for example, the region where the HPRT1 gene on the X chromosome (particularly the human HPRT1 gene on the human X chromosome) is located (particularly the DNA region q25-26 on the human X chromosome). Exogenous introduction of the negative selection marker gene into the region to be deleted can be carried out, for example, using gene modification technology (e.g., HDR, genome editing system). For example, if the negative selection marker gene is a visualization marker gene, then, for example, the visualization marker gene can be expressed after being inserted into a specific DNA region of genomic DNA, and it can be determined from its luminescence intensity whether the visualization marker gene has been introduced into one allele or into multiple alleles. Cells containing a negative selection marker gene in the region to be deleted can be obtained by cloning cells into which a visualization marker gene has been inserted in a specific DNA region. Alternatively, cells containing negative selection marker genes in multiple alleles can be obtained by inserting unique, mutually distinguishable selection marker genes and negative selection marker genes into multiple alleles, and selecting cells based on the expression of the unique selection markers incorporated into each allele. Insertion of a gene into a specific DNA region can be achieved by using a genome modification system to cleave the target sequence in that DNA region, and by inducing HDR with donor DNA containing the gene to be inserted between the upstream homology arm, which is capable of homologous recombination upstream of the cleavage site, and the downstream homology arm, which is capable of homologous recombination downstream of the cleavage site.If the negative selection marker gene is a gene that negatively affects cell survival, such as a suicide gene, then after cloning, the suicide gene is expressed in each clone, and the cells that commit suicide are those in which the negative selection marker gene has been introduced into at least one allele. If the negative selection marker gene is to be introduced into other alleles, it is sufficient to functionally (operably) link it to another inductive promoter, introduce the negative selection marker gene into the other alleles, and confirm that the cells do not die even when the promoter is activated. Diploid cells, preferably haploid cells, can be used. From the viewpoint of simplicity of the assay system, HAP1 cells can be used as haploid cells, for example.

[0068] In (β) above, two target sequences are determined so as to flank the DNA region in which the negative selection marker gene was inserted in (α) above, and a sequence-specific nucleic acid cleavage molecule can be designed to cleave these sequences in a sequence-specific manner. When the sequence-specific nucleic acid cleavage molecule is applied to genomic DNA, the DNA region between the two target sequences is deleted from the genomic DNA. Since the DNA region between the two target sequences contains the negative selection marker gene, the negative selection marker gene is also deleted as a result of the above deletion.

[0069] In the above (γ), cells having a deletion in the DNA region between the two target sequences obtained in the above (β) are selected. Cells having a deletion in the DNA region between the two target sequences do not have the negative selection marker gene as described above. Therefore, even when the cells are maintained under conditions in which the negative selection marker gene is induced, the negative selection marker gene will not be expressed. Therefore, under conditions in which the negative selection marker gene is induced, the cells can be maintained, and cells having a deletion in the DNA region between the two target sequences can be selected using the fact that the negative selection marker gene is not expressed as an indicator. When the negative selection marker gene is a gene that kills cells (suicide gene), these genes may be operably linked to an inducible promoter, and an inducer is allowed to act on the inducible promoter to induce the expression of the negative selection marker gene, thereby removing cells that do not have a deletion in the DNA region. When the negative selection marker gene is a visualization marker gene, cells that do not have a deletion in the DNA region can be removed using the fact that the visualization marker gene is expressed as an indicator. When the negative selection marker gene is a visualization marker gene, cells having a deletion in the DNA region can also be selected using the fact that the visualization marker gene is not expressed as an indicator.

[0070] <Cells with DNA region deletions> According to the present invention, by the method of the present invention described above, cells having a deletion in a target DNA region can be obtained. Therefore, according to the present invention, cells having a deletion in a target DNA region are provided. According to the present invention, the target DNA region can contain a gene essential for cell survival. Therefore, according to the present invention, cells having a deletion in a target DNA region, wherein the target DNA region contains a gene essential for cell survival are provided. According to the present invention, cells having a deletion in a target DNA region, wherein the target DNA region contains a gene essential for cell survival may have genomic DNA into which a gene essential for cell survival has been ectopically inserted. Therefore, according to the present invention, cells having a deletion in a target DNA region, wherein the target DNA region contains a gene essential for cell survival and having genomic DNA into which a gene essential for cell survival of the cell has been ectopically inserted are provided. The ectopic insertion site is not particularly limited and can be a region where no other genes are present. In the above (a) and (α), cells having a deletion in a target DNA region, wherein the target DNA region contains a gene essential for cell survival and having genomic DNA into which a gene essential for cell survival of the cell has been ectopically inserted may be used as the cells. Thereby, according to the above (b) or (β), the DNA region to be further deleted can be expanded, and the deletion can be extended to a gene that controls the survival and / or proliferation of the next cell (for example, a gene that positively controls the survival and / or proliferation of the cell, for example, a gene essential for the survival and / or proliferation of the cell). Cells having a deletion in a target DNA region obtained by the method of the present invention described above may have a growth rate or survival rate of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more compared to the cells before the deletion. In one aspect, the cells having a deletion in the target DNA region have deleted 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the original genome.

[0071] <Method for producing cells having a deletion in a DNA region> Cells having a deletion in the target DNA region can be produced by the method of the present invention described above. Therefore, according to the present invention, there is provided a method for producing a cell having a deletion in a target DNA region, the method comprising performing the method of the present invention. The cells having a deletion in the DNA region may be selected by not having a negative selection marker gene.

Example

[0072] Materials and methods cell culture HAP1 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% (v / v) fetal bovine serum and 100 U / mL penicillin / streptomycin at 37 °C in an atmosphere containing 5% CO2. HCT116 cells were cultured under the same conditions except that McCoy's 5A medium was used. HAP1 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% (v / v) fetal bovine serum and 100 U / mL penicillin / streptomycin at 37 °C in an atmosphere containing 5% CO2. HCT116 cells were cultured under the same conditions except that McCoy's 5A medium was used.

[0073] Estimation of essential genes based on past large-scale experiments Essential genes of HAP1 cells were defined by the following two criteria: (1) being annotated as essential in at least one of three reports (OGEE database 1 , CRISPR screening 2 , Gene-trap screening 3 ), and (2) being transcribed in HAP1 cells [Transcript per million > 0; data is available from Human Protein Atlas (www.proteinatlas.org / humancell)].

[0074] Plasmid construction For the construction of 24 gRNA / Cas9 expression plasmids (gpHY001-pJS067), gRNA sequences were designed using CRISPRdirect software 4 . gRNA oligonucleotides (Table 1) were annealed and ligated to linearized pX330 (Addgene) using standard protocols 5 .

[0075] [Table 1]

[0076] The ligated product was transformed into DH5 alpha E. coli cells, and the plasmid was purified using the EndoFree Plasmid mini Kit (Qiagen) according to the manufacturer's protocol. pHY262 is a pWZ267 vector. 6 It was constructed by inserting a sequence called the sgRNA(sg-A) site, which is targeted by pJS050, into the 5'UTR of the IRES-puro cassette. pHY262 was created using TransformMax TM EPI300 TM It was transformed into E. coli (Epicentre). pHY263 was constructed by inserting an IRES-GFP-2A-Puro cassette into pGEM(trademark)-T Easy Vector (Promega). pHY271 (thymidine kinase negative selection marker) was constructed by conjugating the amplification products shown in Tables 2-1 and 2-2 using Gibson assembly. The Gibson assembly products were used to transform DH5 alpha E. coli cells. The plasmids were purified using the EndoFree Plasmid Midi Kit (Qiagen) according to the manufacturer's protocol.

[0077] [Table 2-1] [Table 2-2]

[0078] pHY269 (RBMX2 locus cloning) and pHY270 (MMGT1 locus cloning) were constructed. pHY262 (~100 ng) was linearized using BamHI and EcoRI (New England Biolabs) and co-transformed into yeast with ~100 ng of amplified genome (Supplementary Table 6) covering the RBMX2 or MMGT1 locus, including a 3 kb region upstream of the transcription start site and a 3 kb region downstream of the polyadenylation site. Each fragment has ~300 bp overlap. Yeast transformation was performed according to the protocol described previously. 7 The plasmid was extracted from the yeast colony and processed according to the protocol described above using TransforMax. TM EPI300 TM E. coli (Epicentre) recovered 8 Copy Control of E. coli TM The plasmids were cultured in LB using Induction Solution (Epicentre) and purified using the EndoFree Plasmid Midi Kit (Qiagen) according to the manufacturer's protocol. To confirm the constructed plasmids, the purified plasmids were digested with appropriate restriction enzymes and separated by agarose gel electrophoresis.

[0079] Transfection HAP1 cells were placed in a 12-well plate in a 6x10⁶ arrangement. 5 Cells were seeded at a density of cells / well and incubated overnight. 50 μL of 2.5 M CaCl2 containing 1.5 μg of DNA was prepared and mixed with 50 μL of 2 × BBS buffer (400 mM borate, 300 mM NaCl, 5 mM EDTA). This solution was incubated at 25°C for 5 minutes, then mixed with 1 mL of IMDM and added to the cell culture, where it was incubated for 4–8 hours. The medium was then aspirated and the cells were washed twice with D-PBS (Nacalai Tesque, #14249-24). Fresh IMDM medium was then added and incubated at 37°C and 5% CO2. HCT116 cells were placed in 12-well plates in a 3 × 10⁶ layer. 5Cells were seeded at the cell / well density and incubated overnight. FuGENE® HD (Promega) transfection was performed in a 3:1 ratio according to the manufacturer's instructions (3 μL of FuGENE® HD transfection reagent and 900 ng of DNA per well).

[0080] Digital Junction PCR For each target region, 48 reactions were performed using nested PCR. In the first PCR, 100 genomes (3.3 pg of genomic DNA in the case of haploid human cells) were used per reaction. For the second PCR, 2× Quick Taq HS DyeMix (Toyobo) and the primers listed in Tables 3-1 and 3-2 were used, with a 1 / 100 dilution of the first PCR mixture as the template. The deletion efficiency (λ) was calculated using Poisson statistics: λ = -ln(1-p) {where λ is the average number of genomes with deletion junctions among 100 genomes, and p is the percentage of positive reactions in the 48 PCRs}. 9 .

[0081] [Table 3-1] [Table 3-2]

[0082] Isolation of surviving colonies after 6-TG selection After transfection of HAP1 cells with a gRNA / Cas9 expression vector, approximately 2,000 cells were trypsin-treated and re-seeded in 10 cm dishes, then cultured in 5 μM 6-thioguanine (6-TG) (Sigma, A4882-100MG) for 12 days. Independent colonies were then picked from the 10 cm dishes and spread separately into each well of a 24-well plate. A portion of the cells were then spread in a 12-well plate for genomic DNA isolation, while the remainder were frozen as stocks for further analysis of individual clones.

[0083] Colony formation assay after 6-TG selection After transfecting HAP1 or HCT116 with a gRNA / Cas9 expression vector, ~5 × 10⁻⁶ 4 Individual cells were trypsin-treated and re-seeded in a 6 cm dish, then cultured in 5 μM 6-TG (Sigma, A4882-100MG) for 9 days. Afterward, the culture medium was removed from the 6 cm dish, and EtBr solution (10 mg / mL ethidium bromide (Nacalai Tesque) 0.5% in 50% ethanol) was added. The mixture was incubated at room temperature for 30 seconds. The EtBr solution was removed, and the colonies were visualized using a UV illuminator. 10 The number and size of stained colonies were quantified using the "find maxima" and "analysis particle" functions in ImageJ software, respectively.

[0084] Genome DNA extraction Cells cultured in a 12-well plate were transferred to a 1.5 mL tube, resuspended in 118 μL of breaking buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.04% (w / v) SDS, 0.2 mg / mL proteinase K (Wako), 2.5 mg / mL RNAsA (Nippon Gene)), and incubated at 37°C for approximately 18 hours. One volume of phenol / chloroform / isoamyl alcohol (25:24:1) was added, and the mixture was rotated at 60 rpm for 30 minutes. After centrifugation at 12,000 rpm for 10 minutes, the upper aqueous layer was transferred to a fresh tube, and one volume of absolute isopropanol was added. After centrifugation at 13,000 rpm for 5 minutes, the pellet was washed with 300 μL of 70% ethanol and air-dried. Genomic DNA was dissolved in 100 μL of TE buffer and stored at -20°C.

[0085] Detection and sequencing of deleted junctions Using 2× Quick Taq HS DyeMix (Toyobo) and the PCR primers listed in Supplementary Table 5, deletion junctions were amplified from extracted genomic DNA using nested PCR. The base sequences of all PCR products were determined using an ABI 3100 DNA sequencer with the ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems) (Figure 1d).

[0086] Detection of gene deletions in deletion clones Gene deletions were detected by amplifying the extracted genomic DNA using 2x Quick Taq HS DyeMix (Toyobo) and the PCR primers listed in Tables 4-1 and 4-2. Nested PCR was performed to confirm that the gene to be deleted was missing from the genome. The obtained PCR products were analyzed by 1% agarose gel electrophoresis (Figure 3a).

[0087] [Table 4-1] [Table 4-2]

[0088] Analysis of growth rate Each HAP1 deletion clone or WT clone was mixed with hHY224 (IRES-GFP-2A-Puro cassette incorporated into the 3'UTR of the GAPDH locus in HAP1 cells) and cultured in IMDM medium. After 2 days of culture, cells were collected as the "day_0" sample, and the remaining cells were cultured for a further 16 days and collected as the "day_16" sample. GFP-positive and GFP-negative cells were measured by flow cytometry. The relative population was calculated by dividing the ratio of GFP-negative cells in the deletion clone by the ratio of GFP-negative cells in the WT clone. Flow cytometry analysis was performed using an EC800 flow cytometry analyzer (Sony Biotechnology).

[0089] Microarray analysis Total RNA was extracted from HAP1 cells using the RNeasy Mini RNA isolation kit (Qiagen). Microarray analysis was performed using a SurePrint G3 Human GE 8x60K Microarray (Agilent). Data analysis was performed using Agilent Feature Extraction Software (Agilent). For data plotting, only genes with a signal evaluation score of 2 in all wild-type or deletion clones were selected. A p-value < 0.05 and a 2x change > 1 were considered statistically significant.

[0090] Transplantation of essential genomic loci or the IRES-GFP-2A-Puro cassette to the GAPDH locus pHY269 (RBMX2 locus cloning), pHY270 (MMGT1 locus cloning), or pHY263 (IRES-GFP-2A-Puro cassette plasmid for creating hHY224 cells) were co-transfected into HAP1 cells using pJS039 (gRNA expression vector targeting the 3'UTR of the GAPDH region) and pJS050 (gRNA expression vector targeting the sgRNA (sg-A) site of pHY262). Two days after transfection, the cells were cultured for approximately 10 days in IMDM medium supplemented with 1 ng / ml of puromycin. Since some populations of HAP1 cells become diploid, only the haploid population was collected using SH800Z (Sony Bio). Haploid cells were cultured in large cultures for 7 days before cryopreservation for long-term storage. The cell lines were thawed and cultured in normal medium for 5 days, after which MEGES was applied.

[0091] Integration of TK markers into the HCT116 genome pHY271 was co-transfected into HCT116 using pJS067 (a gRNA expression vector targeting the downstream OCRL region) and pJS050 (a gRNA expression vector targeting the sg-A site of pHY271). After 2 days of transfection, cells were cultured in McCoy's 5A medium (10% FBS, 1% penicillin / streptomycin, 1 ng / μL puromycin) for ~10 days. Single colonies were enlarged and cultured for another 7 days before cryopreservation for long-term storage. Marker integration was tested by PCR using HY2223 and HY2224 primers (Supplementary Table 1). Cell stocks were thawed and cultured in standard medium for 7 days before MEGES was applied.

[0092] Analysis of DNA content 2 x 10 6 Cells were suspended in 1 mL of 4% PFA / PBS and incubated at 4°C for 10 minutes. The cells were then spun down at 500 g for 3 minutes. The cell pellet was resuspended in 70% EtOH and stored at -20°C. For PI staining, cells were spun down at 500 g for 3 minutes, resuspended in 500 μL of PI solution (D-PBS (Nacalai Tesque), 50 μg / mL propidium iodide (Wako), 0.25 mg / mL RNase A (Nippon Gene)), and incubated at 37°C for 10 minutes. Flow cytometry analysis was performed using an EC800 flow cytometry analyzer (Sony Bio).

[0093] Quantitative RT-qPCR Total RNA was extracted from HAP1 cells using the RNeasy Mini RNA isolation kit (Qiagen). 400 ng of RNA was reverse transcribed using the PrimeScript RT Reagent Kit with gDNA eraser (Takara). Quantitative PCR was performed using TB Green™ Premix Ex Taq. TMThe experiment was conducted using (Takara). Each data point is the mean of three independent experiments performed in overlapping fashion. ACTB mRNA was used as an internal control to normalize the variability in gene expression levels. Primer sequences are shown in Table 5.

[0094] [Table 5]

[0095] [result] Development of methodologies for large-scale genome deletion To demonstrate the concept of this experimental platform, we attempted to delete the genomic region on chromosome X containing the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene in the human cell line HAP1 (Figure 1b). HPRT1 has been identified as an essential gene in certain human cells based on the Online Gene Essentiality (OGEE) database [REF], but it is established as a negative selection marker for 6-TG (Nature Reviews Genetics, 6, 507-512 (2005)). If the same applies to HAP1 cells, then HPRT1 cannot be used as a negative selection marker. (Blomen's gene trapping) 3 or Mair's CRISPR screening 2While HAP1 cell dysfunction screening had shown that HPRT1 is not necessary for HAP1 cell proliferation, deletion using the paired gRNAs we developed (e.g., gRNA L and gRNA R shown in Figure 1a) is a different method from gene knockout and may yield different results. Therefore, we used a paired gRNA approach to investigate whether the 0.51 Mb region containing HPRT1 is essential. Two days after introducing paired gRNAs (R1 and L1) into HAP1 cells, a portion of the cells were collected and junction PCR was performed. Of the 48 PCR reactions containing ~100 genomes as templates, 14 reactions produced amplification, indicating that ~0.3% of transfected HAP1 cells had a targeted deletion between the R1 and L1 cleavage sites after any proliferation effect had occurred. In the negative control using gRNA R1 alone, no amplification occurred in any of the 48 reactions, which is explained by the fact that the 0.51 Mb region was intact and therefore too long to be amplified by junction PCR. Thus, subsequent 6-TG selection was expected to result in a significant difference in the number of surviving cell colonies (Figure 1c). The above process successfully removed 11 genes, including HPRT1, and non-genetic regions within 0.51 Mb within them. Furthermore, analysis of the genomic DNA sequences extracted from the three clones that survived after paired gRNA cleavage and 6-TG selection confirmed that the target region was clearly deleted from the gene locus (Figure 1d). Our platform using HPRT1-6-TG negative selection was validated as useful for obtaining HAP1 cell clones lacking the non-essential region we targeted.

[0096] Expansion of deletion loci for detecting essential genomic regions Next, we expanded the deleted region from HPRT1 in both the centromere and telomere directions to investigate whether our platform could be used to identify essential gene regions and / or intergenetic regions (Figure 2). Based on the OGEE database, the first essential gene candidate from HPRT1 in the centromere direction is ARHGAP36, with 22 potentially non-essential gene regions and intervening non-coding regions between them (corresponding to the 3.12 Mb region between the gRNA L1 and L2 sites in Figure 2b). The next essential gene candidate is RBMX2. Between ARHGAP36 and RBMX2 (corresponding to the 0.62 Mb region between the gRNA L3 and L4 sites in Figure 2b), there are three non-essential gene candidates, mostly intergenetic regions. Here, we separated a region of approximately 4 Mb between gRNA L1 and L5 into four sections (two essential gene candidate regions and two non-essential gene candidate clusters) and applied our platform to investigate whether any of the essential gene candidates and / or the vast intergeneric region are essential for HAP1 growth (Figure 2b).

[0097] Digital junction PCR on day 2 confirmed that deletion of the target region was achieved with comparable efficiency (0.3-1.1%) using five different gRNA pairs: L1, L2, L3, L4, L5, and R1. Notably, after 6-TG selection, several hundred colonies were formed with four of the five gRNA pairs, but when the region between L4 / L5 was deleted, viable cells were drastically lost. This data reveals that the RBMX2 gene located between the L4 and L5 gRNA target sites is crucial for normal HAP1 proliferation, and that the region between them is deleted. This result also supports Blomen's gene trapping. 3 Mair's CRISPR screening 2This is consistent with the decline-of-function screening, suggesting that the RBMX2 locus contains essential elements, but ARHGAP36 is absent in HAP1 cells. However, while these conventional LOF screenings can only assess the essentiality of a single gene at a time, our platform provides new information on the entire Mb-scale genomic region containing RBMX2, 38 other genes, and the dispersion of large intergene regions. Due to this high level of scalability, our platform was named MEGES.

[0098] The deletion was continued to expand in the telomere direction (Figure 2c). The essential gene candidate supported by the first OGEE was SMIM10, whose essentiality in HAP1 cells had not been verified until now. The next OGEE candidate, MMGT1, was suggested to be essential in HAP1 cells by Mair's CRISPR screening, but was suggested to be non-essential by Biomen's gene trap. It should be noted that there are also 30 non-essential gene candidates and their inter-gene sequences in the 1.1 Mb region between HPRT1 and MMGT1 (between gRNA R1 and R4 sites). MEGES induced deletions using five different gRNA pairs, each of R2, R3, R4, and R5, and L4. It efficiently induced deletions in all target regions, as verified by digital junction PCR on day 2. Although MMGT1 is essential, the entire 5.48 Mb region between the L4 and R4 sites of the gRNA containing SMIM10 was shown to be deletable without significant impact on HAP1 cell growth.

[0099] L4-R4 Deletion Clone Characterization Four HAP1 cell clones (hHY131, 145, 148, 149; L4-R4 deletion clones) obtained by single-clonal development after MEGES using R4 and L4 paired gRNAs were analyzed. The obtained clones were confirmed to be haploids using flow cytometry with propidium-iodine staining. 16First, PCR genotyping was performed on individual loci located within the deletion region. It is hypothesized that the genomic fragments cleaved by the paired gRNAs are randomly integrated into different chromosomal locations, and it is possible that essential genes in this region were judged to be non-essential genes. There are 69 annotation genes between the R4 and L4 regions of the gRNA, but many of these genes are either duplicated or located very close together. Therefore, using genomic DNA extracted from the L4-R4 deletion clone, genotyping PCR was performed on 39 loci that could cover all 69 loci. As shown in Figure 3a, compared to two clones of original HAP1 (hHY153, 154) that retained all the tested loci, the L4-R4 deletion clone was found to have all the tested loci deleted from its genome.

[0100] [Table 6]

[0101] The data shown in Table 6 were obtained by analyzing the genotypes of the clones shown in Figure 2. Cell clones with the desired gene deletion were counted as the number of cells with the deletion.

[0102] Furthermore, the effects of 69 gene deletions on the transcriptome were evaluated (Figure 3b). This transcriptome analysis showed that RBMX2 and MMGT1 showed almost no change in transcriptional levels due to deletions of adjacent 5.5 Mb regions [Figure 3c], indicating that distal cis-regulatory elements of these genes are not present in these deletion regions.

[0103] Interchromosomal transfer of essential elements The RBMX2 and MMGT1 genes, lacking distal cis-regulatory elements, could be treated as compact, independent gene units, as many bacterial and yeast genes have been in genome design and synthesis. To verify this, we attempted to transplant both genes to different chromosomal locations. Using a BAC-YAC vector, we cloned the entire gene region ("rescue cassette"), including a ~3 kbp region upstream of the transcription start site and a ~3 kbp region downstream of the polyadenylation (pA) site [Figure 4a]. On the ring-closed pHY262 vector, a puromycin N-acetyl-transferase (PNAT) coding sequence is present between the IRES sequence and the pA signal. Transfecting HAP1 with the resulting rescue vector resulted in co-transfection with two different gRNA / Cas9 vectors; one targeted at the 3'UTR of the GAPDH gene on chromosome 12, and the other targeted just outside the IRES sequence on the rescue locus vector. 15 By puromycin selection, cells with the IRES-Puro-pA+ rescue cassette integrated into the GAPDH locus can be obtained. Real-time RT-PCR demonstrated that the obtained cell clones showed a 1.5-2.0-fold increase in RBMX2 or MMGT1 mRNA levels [Figure 4b]. Subsequently, MEGES, which involved deletion of the RBMX2 or MMGT1 gene from the endogenous locus on the X chromosome, showed that transplanted cells exhibited a greater number of viable cell colonies with comparable gRNA-mediated deletion efficiency [Figure 4c]. These data revealed that both loci can be transplanted to non-endogenous chromosomal regions and play a role as essential genes. Furthermore, it was revealed that the upstream and downstream 3kb regions were sufficient to express both genes at levels adequate for normal cell proliferation. In particular, by transplanting essential genes between chromosomes, MEGES was able to further delete the HPRT1 locus genome and reach the next candidate essential gene [Figure 5].

[0104] MEGES at non-HPRT1 locus To broaden the scope of MEGES, we used a different negative selection marker to delete genomic regions other than the HPRT1 locus in different human cell lines, HCT116. HCT116 cells, established from male colorectal cancer patients, have only one HPRT1 locus because they possess only one X chromosome. The herpes simplex virus thymidine kinase (TK) expression cassette contains a PNAT flanked by a 2A peptide following the TK encoding sequence, which is expressed by the CMV promoter and SV40 pA signaling [Figure 6a]. When the plasmid containing the TK expression cassette was co-transfected with two different gRNA / Cas9 vectors, one gRNA targeted the 3'UTR of the non-essential HCT116 gene OCRL (Nucleic Acid Research, 2020, doi: 10.1093 / nar / gkaa884) on chromosome X, while the other gRNA targeted just upstream of the CMV promoter on the TK expression cassette vector. 15 Cells selected for puromycin resistance were confirmed by junction PCR to have a TK expression cassette at the targeted OCRL locus [Figures 6a and 6b]. These cells were then used in MEGES. All five gRNA pairs (L8, L9, L10, L11, or L12 in pairs with R9) efficiently deleted the genomic region containing the OCRL locus, as indicated by deletion efficiencies of 0.3–1.2 measured by junction digital PCR on day 2 [Figure 6c]. Ganciclovir selection, which induces cell proliferation impairment when TK is present in the genome and expressed intracellularly, produced significantly more colonies in all gRNA pairs except the L12 and R9 pair, suggesting that the region between the L11 and L12 gRNA target sites contains a crucial element for cell proliferation. Thus, the basic strategy of MEGES was demonstrated to be applicable to any chromosomal region by using different selection markers and different human cells.

[0105] MEGES: Avoiding negative choices To date, we have demonstrated the usefulness and advantages of MEGES for screening and identifying genomic regions crucial for cell proliferation, and for isolating cell clones with deletions in target genomic regions on a megabase-pair scale, thereby further elucidating the functional significance of target regions. However, screening only essential regions requires the prior integration of negative selection markers, and colony formation assays using negative selection have low throughput. Therefore, instead of negative selection, we devised an improved version of MEGES that includes digital junction PCR at multiple time points. If the deleted region is essential or important for cell proliferation, the proportion of cells with the deleted region decreases as cell culture progresses. This change in proportion can be evaluated by comparing the deletion efficiency determined using digital junction PCR at multiple time points after gRNA / Cas9 transfection. We call this strategy digital junction PCR-based MEGES (dMEGES). To demonstrate this concept, we applied dMEGES to the HPRT1 locus (Figure 7). We performed digital junction PCR on days 2 and 17 after cleavage by two gRNA pairs (R1-L4 and R1-L5) and demonstrated the expected results. The percentage of cells cleaved by R1-L4 remained largely unchanged from day 2 to day 17 (1.8% to 2.0%), while the percentage of cells cleaved by R1-L5 decreased significantly from 1.0% to 0.2%. Here, we tested cleavage by another gRNA pair, R1 and L13, which cleaves inside the RBMX2 locus, and found that cleavage of R1-L5 significantly decreased from 1.0% to 0.2%. This also resulted in a significant decrease in the cell population with deletion (2.1% to 0.6%). These data are consistent with the data in Figure 2b, indicating that the 4.35 Mb region between R1 and L4 could be deleted without affecting cell proliferation, while the R1-L5 region could not be deleted because it is an essential region of RBMX2.

[0106] Furthermore, cell populations with deletions in each of the 22 regions of the X chromosome were created and analyzed similarly (Figure 8). As a result, as shown in Figure 8, deletions in regions 7 and 16 were found to be potentially essential for cell proliferation or survival. In addition, regions 1, 9-15, and 17-19 were found to potentially contain genes that positively influence cell proliferation or survival. While deletions in these 22 regions had previously been considered non-essential for survival in CRISPR screening, the present invention allowed for the highly sensitive determination of essential chromosomal regions.

[0107] In this way, the present invention allows MEGES to be performed without prior introduction of negative selection markers, in which case MEGES can induce large-scale genomic deletions, and furthermore, regions containing genes essential for cell survival can be determined. Once genes essential for survival are determined, they can be integrated onto other chromosomes, further expanding the genomic deletion. dMEGES may be a useful technique for identifying genes essential for cell survival and / or for large-scale genomic deletions.

[0108] TIFF0007872590000010.tif155170

[0109] TIFF0007872590000011.tif78170TIFF0007872590000012.tif126170TIFF0007872590000013.tif127170TIFF0007872590000014.tif85170

Claims

1. This is an in vitro method. (a) Prepare a cell population containing isolated cells, (b) Applying sequence-specific nucleic acid cleavage molecules to the genomic DNA of the cells in the cell population in such a way that they sequence-specifically cleave two target sequences on the genomic DNA, thereby causing cleavage at two target sequences on the genomic DNA, and thereby obtaining a cell population containing multiple cells in which a deletion of the DNA region occurs in the region between the two cleavage sites common to the cells, (c) The process then involves culturing the obtained cell population and determining the effect of the deletion of the DNA region on cell proliferation or survival, In (c) above, the determination is made by determining the deletion efficiency or an estimate of the DNA region, culturing this cell population, and comparing the proportion of cells having the DNA region deletion in the cultured cell population with the determined deletion efficiency or an estimate of the DNA region. In (c) above, the deletion efficiency or its estimate, and the percentage of cells having a DNA region deletion after culture, are determined as the percentage of cells having the deletion relative to the total number of cells in the suspension containing the cell population. A method in which cell sorting is not performed between (b) and (c) above.

2. The method according to claim 1, wherein the above ratio is determined by a counting technique for genomic DNA having and not having the deletion contained in the suspension.

3. A method according to claim 1 or 2, (d) Determining whether the deleted DNA region contains genes that control cell survival and / or proliferation, compared to the control genomic DNA, based on the presence or magnitude of the effect on cell survival and / or proliferation in (c) compared to a control cell population. Methods that further include the above.

4. A method according to claim 3, wherein the control cell population is a cell population comprising cells having deletions of a larger DNA region, a smaller DNA region, or a different DNA region as described in (b).

5. A method according to claim 3 or 4, further comprising identifying at least one gene that controls cell survival and / or proliferation from genes present in the deleted DNA region compared to control genomic DNA.

6. A method according to any one of claims 1 to 5, (f) Identify at least one gene from the genes present in the deleted DNA region that controls cell survival and / or proliferation. Methods that further include the above.

7. (g) Ectopically introducing at least one gene that controls cell survival and / or proliferation, which is operablely linked to a regulatory sequence, into genomic DNA having a deletion in the said DNA region. The method according to claim 5 or 6, further comprising:

8. The method according to any one of claims 1 to 7, wherein the size of the DNA region deleted in (b) is 0.5 Mbp or more.

9. The method according to any one of claims 1 to 8, wherein the size of the DNA region deleted in (b) is 1 Mbp or more.

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