Method for conditionally inactivating gene, and cell therefor

Abstract

The present disclosure provides: a gene disruption method for, by inactivating a cassette including at least one exogenous gene complementing at least one disrupted endogenous gene, inactivating the function of said at least one exogenous gene included in the cassette; and cells used in said method. The present disclosure also provides: a method for inactivating, when a period in which cells require at least one exogenous gene included in a cassette has passed, the cassette that has become unnecessary; and cells used in said method.

Description

Method for conditionally inactivating genes and cells therefor

[0001] The present disclosure relates to a method for conditionally inactivating a gene and a cell for the same. Specifically, the present disclosure relates to a gene disruption method, in which a cassette containing one or more exogenous genes that complement one or more disrupted endogenous genes is inactivated by inactivating the cassette, and to a cell for use in the method. The present disclosure also relates to a method in which a cell inactivates a cassette that is no longer needed when the time when the exogenous genes contained in the cassette are no longer needed has passed, and to a cell for use in the method.

[0002] Various gene disruption techniques have been developed. Systems in which gene disruption is induced only under specific conditions (i.e., conditional knockout systems) have also been established. For example, as disclosed in Patent Document 1, conditional knockout systems typically involve placing two site-specific recombination sequences (e.g., loxP sequences) to sandwich the endogenous gene, and then using a site-specific recombinase (e.g., Cre recombinase) to remove the endogenous gene from the genome. By allowing the site-specific recombinase to act on the genome at the desired time, endogenous genes can be conditionally disrupted. Furthermore, by tissue-specifically expressing the site-specific recombinase, endogenous genes can be disrupted in a tissue-specific manner. Non-Patent Document 1 discloses episomal vectors. Non-Patent Documents 2 and 3 disclose methods for removing episomal vectors such as plasmids. Non-Patent Documents 4, 5, and 6 disclose human artificial chromosomes and yeast artificial chromosomes. Patent Document 2 discloses a plasmid having a temperature-sensitive replication origin and a technique for terminating replication of the plasmid by increasing the temperature. Non-Patent Document 7 discloses a technique for removing artificial chromosomes. Non-Patent Document 7 discloses a technique for removing artificial chromosomes using a CRISPR / Cas system.

[0003] US4959317A Publication US5616480A Publication

[0004] K Van Craenenbroeck et al., Eur J Biochem. 2000. 267(18):5665-78Trevors, JT, FEMS Microbiology Reviews. 1986, 32, 149–157Lee, M. et al., Exp Mol Med. 2019 Jul; 51(7): 82M, Ayabe et al., Biochemical and Biophysical Research Communications., 2004, 321(2):280-90Harrington JJ et al. 1983 Sep;305(5931):189-93Z Ida, Y. et al., DNA Res. 2010, 17(5):293-301Zuo, E. et al., Genome Biol. 2017, 18(1):2

[0005] The present disclosure provides a method for conditionally deleting a gene and cells therefor. Specifically, the present disclosure provides cells in which one or more endogenous genes are disrupted but which have a cassette containing one or more exogenous genes that complement the disrupted one or more endogenous genes. Because the exogenous genes complement the effects of the disrupted endogenous genes, these cells do not exhibit the phenotype caused by the disruption. By inactivating (e.g., removing) the cassette containing the exogenous genes from the cells, the one or more exogenous genes contained in the cassette are inactivated (e.g., removed) simultaneously at the time of inactivation (e.g., removal) of the cassette, thereby allowing the effects of the disrupted endogenous genes to be exerted. In this way, the present disclosure provides a new conditional knockout technique that conditionally inactivates (e.g., removes) an exogenous gene that complements the disrupted endogenous gene. The present disclosure also provides a method for examining the function of a disrupted endogenous gene at a desired time using the conditional knockout technique, and cells for use in the technique and method. The present disclosure further provides a method for maintaining a cassette on DNA when one or more exogenous genes contained in the cassette are needed in a cell, but inactivating (e.g., removing) the cassette when the one or more exogenous genes contained in the cassette are no longer needed, and a cell for use in the method. The present disclosure also provides a method with or without disruption of an endogenous gene. The present disclosure can provide a cell having a cassette containing one or more exogenous genes (e.g., knockdown nucleic acids), and by inactivating (e.g., removing) the cassette, one or more exogenous genes can be removed or inactivated collectively from the cell.

[0006] According to the present disclosure, for example, the following inventions are provided: (1) A genetically modified cell having a cassette containing n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, the cell being capable of expressing each of the exogenous genes, and the cassette being configured to be conditionally inactivated (e.g., removed) {for example, the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and inactivation may be achieved by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on each of the two target sequences, or, if the cassette is present on extrachromosomal DNA, by disrupting the replication apparatus of the extrachromosomal DNA (e.g., a centromere or replication origin)}, preferably in which endogenous genes corresponding to some or all of the n or more exogenous genes have been disrupted {where n is a natural number greater than or equal to 1}. (2) The cell according to (1) above, wherein n is 2 or greater. (3) The cell according to (1) or (2) above, wherein the disrupted endogenous gene comprises a gene associated with or involved in cell survival or proliferation, or a candidate gene therefor. (4) The cell according to (1) or (2) above, wherein the disrupted endogenous gene comprises a gene associated with or involved in cell differentiation control, or a candidate gene therefor. (5) The cell according to (1) or (2) above, wherein the disrupted endogenous gene comprises a gene associated with or involved in cellular physiological function, or a candidate gene therefor. (6) The cell according to any of (1) to (5) above, wherein the cassette is located between two target sequences of a sequence-specific DNA cleavage enzyme or two recognition sequences of a site-specific recombinase, and inactivation is achieved by acting the sequence-specific DNA cleavage enzyme or the site-specific recombinase on each of the two target sequences, and the control sequence is located outside the region between (including) the two target sequences or the two recognition sequences. (7) The cell according to any of (1) to (6) above, wherein the cassette further comprises a gene encoding a knockdown nucleic acid.(8) A composition for use in producing the cell described in any one of (1) to (7) above, comprising cells having an exogenous target region (also referred to as a "landing pad") for integrating the cassette (the landing pad may be present on the genome or extrachromosomal DNA), wherein the exogenous target region comprises a target sequence for a sequence-specific DNA cleaving enzyme. (9) The composition described in (8) above, wherein the exogenous target region comprises two alleles, each having a distinguishably different positive selection marker, and each allele further having a negative selection marker (however, if the positive selection marker also serves as a negative selection marker, an additional negative marker may not be required). (10) A method comprising: (A) culturing the cell described in any one of (1) to (7) above under appropriate conditions; (B) inactivating the cassette after culturing to obtain cells in which the cassette has been inactivated; and (C) further culturing the cells in which the cassette has been inactivated under appropriate conditions. (11) The method according to (10) above, wherein the disrupted gene is a gene associated with or involved in cell survival, a gene associated with or involved in cell proliferation, or a candidate gene thereof, and wherein step (B) is carried out after the cells have been propagated in step (A). (12) The method according to (10) above, wherein the disrupted gene is a gene associated with or involved in cell differentiation control, or a candidate gene thereof, and wherein step (B) is carried out after the cells have been differentiated in step (A). (13) The method according to (10) above, wherein the disrupted gene is a gene associated with or involved in cell differentiation control, or a candidate gene thereof, and wherein step (B) is carried out, followed by subjecting the cells to a differentiation treatment in the culture of step (C). (14) The method according to (10) above, wherein the disrupted gene is a gene associated with or involved in the physiological function of a cell, or a candidate gene thereof, and wherein step (B) is carried out after the cells have been propagated in step (A). (15) A cell according to any one of the above, wherein the cell has extrachromosomal DNA and the cassette is contained in the extrachromosomal DNA {wherein it is preferable that endogenous genes corresponding to some or all of the n or more exogenous genes are disrupted}.(16) The cell according to (15), wherein the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and inactivation is carried out by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on each of the two target sequences. (17) The cell according to (15), wherein the extrachromosomal DNA is an artificial chromosome having alpha satellite DNA (or type I alphoid DNA) in a centromeric region, the alpha satellite DNA having a tetracycline operator sequence (tet-O), and configured such that binding of a transcriptional silencer (e.g., tTS) induces heterochromatinization in the artificial chromosome, causing the extrachromosomal DNA to be excreted from the cell. (18) The cell according to (15), wherein the extrachromosomal DNA is an episomal vector that is configured to be conditionally inactivated (for example, inactivated by cleavage; has a replication origin that is configured to be cleaved or eliminated from the episomal vector (for example, the replication origin is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and is eliminated by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on the two target sequences, respectively); or contains a drug resistance gene that is configured to be eliminated in the absence of the corresponding drug. (19) Any of the above inventions, wherein the cassette is contained in the genome of the cell.

[0007] (101) The cell according to any one of (1) to (7), (15), and (16) above, wherein the cassette contains, in addition to the n exogenous genes, a gene encoding a negative selection marker. (102) The cell according to any one of (1) to (7), (15), and (16) above, wherein the cassette contains, in addition to the n exogenous genes, a gene encoding a selection marker (i.e., a positive selection marker). (103) The cell according to any one of (1) to (7), (15), and (16) above, wherein the cassette contains, in addition to the n exogenous genes, genes encoding a positive selection marker and a negative selection marker. (104) The cell according to any one of (1) to (7), (15), and (16) above, wherein the cassette contains, in addition to the n exogenous genes, a gene encoding a marker that can be used for both positive and negative selection (e.g., a visualization marker).

[0008] (201) A composition comprising a cell having an exogenous target region (also referred to as a "landing pad") {the landing pad may be present on the genome or extrachromosomal DNA}, the exogenous target region having a DNA fragment introduction site {for example, comprising two target sequences of a sequence-specific DNA cleaving enzyme for cassette removal or two recognition sequences of a site-specific recombinase, with the DNA fragment introduction site being between the two target sequences or the two recognition sequences}, the DNA fragment introduction site comprising a target sequence of the sequence-specific DNA cleaving enzyme for DNA fragment introduction, and the two target sequences of the sequence-specific DNA cleaving enzyme for cassette removal are not cleaved by the sequence-specific DNA cleaving enzyme for DNA fragment introduction {wherein the DNA fragment comprises n or more exogenous genes, n is a natural number of 1 or more, preferably 2 or more}. (202) The cell according to (201) above, further comprising a regulatory sequence. (203) The cell according to (201) or (202) above, further comprising a regulatory sequence between the two target sequences or the two recognition sequences. (204) The cell according to (201) or (202) above, further having a regulatory sequence outside the region between the two target sequences or the two recognition sequences. (205) The cell according to any of (201) to (204) above, having insulator sequences at both ends of a series of regions including a regulatory sequence and a target sequence of a sequence-specific DNA cleaving enzyme for cassette introduction. (206) The cell according to any of (201) to (205) above, further having a negative selection marker in the region between the two target sequences of a sequence-specific DNA cleaving enzyme for cassette removal or the two recognition sequences of a site-specific recombinase. (207) The cell according to any of (201) to (205) above, not having a negative selection marker in the region between the two target sequences of a sequence-specific DNA cleaving enzyme for cassette removal or the two recognition sequences of a site-specific recombinase. (208) The cell according to any one of (201) to (205) above, wherein the exogenous target region has two or more alleles, each allele having a distinguishably different positive selection marker. (209) The cell according to (206) above, wherein the exogenous target region has two or more alleles, each allele having a distinguishably different positive selection marker.(210) The cell according to (207) above, wherein the exogenous target region has two or more alleles, each allele having a distinguishably different positive selection marker. (211) The cell according to any of (201) to (210) above, wherein the cell has extrachromosomal DNA, and the exogenous target region is contained in the extrachromosomal DNA. (212) The cell according to (211) above, wherein the cassette is located between two target sequences of a sequence-specific DNA cleavage enzyme or two recognition sequences of a site-specific recombinase, and inactivation is achieved by acting the sequence-specific DNA cleavage enzyme or the site-specific recombinase on the two target sequences, respectively. (213) The cell according to (211), wherein the extrachromosomal DNA is an artificial chromosome and has alpha satellite DNA (or type I alphoid DNA) in the centromere region, the alpha satellite DNA has a tetracycline operator sequence (tet-O), and is configured such that binding of a transcriptional silencer (e.g., tTS) induces heterochromatinization in the artificial chromosome, causing the extrachromosomal DNA to be dropped from the cell. (214) The cell according to (211), wherein the extrachromosomal DNA is an episomal vector, and the episomal vector is configured to be conditionally inactivated {for example, by cleavage; having a replication origin, and configured to be cleaved or eliminated from the plasmid {for example, the replication origin is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and elimination is achieved by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on the two target sequences, respectively}; or comprising a drug resistance gene, and configured to be eliminated in the absence of the corresponding drug}. (215) Any of the above-mentioned inventions, wherein the cassette is contained in the genome of the cell.

[0009] (301) A method for introducing n or more exogenous genes into a cell according to any one of (201) to (210) above, by cleaving a target sequence of a sequence-specific DNA cleaving enzyme for introducing the DNA fragment in the presence of a DNA fragment containing n or more exogenous genes, thereby introducing n or more exogenous genes into the cleavage site.

[0010] (401) A composition comprising a cell having an exogenous target region (also referred to as a "landing pad"), wherein the exogenous target region has a DNA fragment introduction site {e.g., comprising two target sequences of a sequence-specific DNA cleaving enzyme for cassette removal or two recognition sequences of a site-specific recombinase, with the DNA fragment introduction site being located between the two target sequences or the two recognition sequences}, the DNA fragment introduction site comprising n or more exogenous genes, and the two target sequences of the sequence-specific DNA cleaving enzyme for cassette removal are not cleaved by the sequence-specific DNA cleaving enzyme for DNA fragment introduction {wherein the DNA fragments comprise n or more exogenous genes}. (402) The cell according to (401) above, further comprising a regulatory sequence. (403) The cell according to (401) or (402) above, further comprising a regulatory sequence between the two target sequences or the two recognition sequences. (404) The cell according to (401) or (402) above, further comprising a regulatory sequence outside the region between the two target sequences or the two recognition sequences. (405) The cell according to any of (402) to (405) above, which has insulator sequences at both ends of a series of regions including a control sequence and a target sequence of a sequence-specific DNA cleaving enzyme for cassette introduction. (406) The cell according to any of (401) to (405) above, which has a negative selection marker in a region between two target sequences of a sequence-specific DNA cleaving enzyme for cassette removal or two recognition sequences of a site-specific recombinase. (407) The cell according to any of (401) to (405) above, which does not have a negative selection marker in a region between two target sequences of a sequence-specific DNA cleaving enzyme for cassette removal or two recognition sequences of a site-specific recombinase. (408) The cell according to any of (401) to (405) above, wherein the foreign target region has two or more alleles, and each allele has a distinguishably different positive selection marker. (409) The cell according to (406) above, wherein the exogenous target region has two or more alleles, each allele having a distinguishably different positive selection marker. (410) The cell according to (407) above, wherein the exogenous target region has two or more alleles, each allele having a distinguishably different positive selection marker.(411) The cell according to any one of (401) to (410), wherein the cell has extrachromosomal DNA, and the exogenous target region is contained in the extrachromosomal DNA. (412) The cell according to (411), wherein the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and inactivation is performed by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on each of the two target sequences. (413) The cell according to (411), wherein the extrachromosomal DNA is an artificial chromosome, and has alpha satellite DNA (or type I alphoid DNA) in a centromere region, the alpha satellite DNA has a tetracycline operator sequence (tet-O), and is configured such that binding of a transcriptional silencer (e.g., tTS) induces heterochromatinization in the artificial chromosome, causing the extrachromosomal DNA to be excreted from the cell. (414) The cell according to (411), wherein the extrachromosomal DNA is an episomal vector, and the episomal vector is configured to be conditionally inactivated {for example, by cleavage; having a replication origin, and configured to be cleaved or eliminated from the episomal vector {for example, the replication origin is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and elimination is achieved by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on the two target sequences, respectively}; or comprising a drug resistance gene, and configured to be eliminated in the absence of the corresponding drug}. (415) Any of the above inventions, wherein the cassette is contained in the genome of the cell.

[0011] (1001) A genetically modified cell, comprising a cassette containing n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, the cell being capable of expressing each of the exogenous genes, the cassette being configured to be conditionally inactivated (e.g., removed) {for example, the cassette is located between two target sequences of a sequence-specific DNA cleavage enzyme or two recognition sequences of a site-specific recombinase, and inactivation may be achieved by acting the sequence-specific DNA cleavage enzyme or the site-specific recombinase on each of the two target sequences, or, if the cassette is present on extrachromosomal DNA, by disrupting the extrachromosomal DNA or its replication apparatus (e.g., a centromere or replication origin)}, preferably in which endogenous genes corresponding to some or all of the n or more exogenous genes have been disrupted {wherein n is a natural number greater than or equal to 1.}. (1002) The cell according to (1001), wherein n is 2 or greater. (1003) The cell according to (1001) or (1002) above, wherein the n or more exogenous genes include genes encoding m or more knockdown nucleic acids {wherein m is a natural number of 1 or more or 2 or more and n or less.}. (1004) The cell according to any one of (1001) to (1003) above, wherein the m or more knockdown nucleic acids include one or more knockdown nucleic acids selected from the group consisting of siRNA, shRNA, miRNA, and antisense oligonucleotides.(1005) The cell according to any one of (1001) to (1004) above, wherein the n or more exogenous genes comprise p or more genes having physiological functions (for example, genes associated with or involved in cell survival, genes associated with or involved in cell proliferation, genes associated with or involved in regulating cell differentiation, and genes associated with or involved in the physiological functions of cells), {wherein p is a natural number of 1 or more or 2 or more and is a natural number of n or less, the n or more exogenous genes may or may not contain knockdown nucleic acids, and when the n or more exogenous genes contain m or more knockdown nucleic acids, m is a natural number of 1 or more or 2 or more, and the sum of p and m is n or less.} (1006) The cell according to any one of (1001) to (1005) above, wherein the control sequence is located outside the region between the two target sequences or the two recognition sequences (including the target sequences or the recognition sequences). (1007) A composition for use in producing the cell according to any one of (1001) to (1006) above, comprising a cell having an exogenous target region (also referred to as a "landing pad") for integrating the cassette, wherein the exogenous target region comprises a target sequence for a sequence-specific DNA cleaving enzyme. (1008) The composition according to claim 7, wherein the exogenous target region comprises two alleles, each of which has a distinguishably different positive selection marker, and each of which further has a negative selection marker {however, if the positive selection marker also serves as a negative selection marker, then the composition does not need to have an additional negative marker.} (1009) A method comprising: (A) culturing the cell according to any one of (1001) to (1006) above under appropriate conditions; (B) inactivating (for example, removing) the cassette after the culture to obtain cells in which the cassette has been inactivated (for example, removed); and (C) further culturing the cells in which the cassette has been inactivated (for example, removed) under appropriate conditions. (1010) The method according to (1009) above, wherein the disrupted gene is a gene associated with or involved in cell survival, a gene associated with or involved in cell proliferation, or a candidate gene thereof, and step (B) is carried out after the cells have been proliferated in step (A).(1011) The method according to (1009) above, wherein the disrupted gene is a gene related to or involved in cell differentiation control or a candidate gene thereof, and wherein step (B) is carried out after the cells are differentiated in step (A). (1012) The method according to (1009) above, wherein the disrupted gene is a gene related to or involved in cell differentiation control or a candidate gene thereof, and wherein step (B) is carried out, and then the cells are subjected to a differentiation treatment in the culture of step (C). (1013) The method according to (1009) above, wherein the disrupted gene is a gene related to or involved in the physiological function of a cell (e.g., a gene related to or involved in cellular metabolism, e.g., a gene encoding a cellular metabolic enzyme) or a candidate gene thereof, and wherein step (B) is carried out after the cells are grown in step (A). (1101) Any of the above-mentioned inventions, wherein the cells are human cells. (1102) Any of the above-mentioned inventions, wherein the cells are non-human cells. (1103) Any of the above-mentioned inventions, wherein the cells are microbial cells. (1104) The cell according to any one of the above, wherein the cell has extrachromosomal DNA and the cassette is contained in the extrachromosomal DNA {wherein, preferably, endogenous genes corresponding to some or all of the n or more exogenous genes are disrupted}. (1105) The cell according to (1104), wherein the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and inactivation is performed by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on each of the two target sequences. (1106) The cell according to (1104), wherein the extrachromosomal DNA is an artificial chromosome and has alpha satellite DNA (or type I alphoid DNA) in the centromere region, the alpha satellite DNA has a tetracycline operator sequence (tet-O), and is configured such that binding of a transcriptional silencer (e.g., tTS) induces heterochromatinization in the artificial chromosome, causing the extrachromosomal DNA to be shed from the cell.(1107) The cell according to (1104) above, wherein the extrachromosomal DNA is an episomal vector, and the episomal vector is configured to be conditionally inactivated {for example, by cleavage; having a replication origin, and configured to be cleaved or eliminated from the episomal vector {for example, the replication origin is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and elimination is achieved by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on the two target sequences, respectively}; or comprising a drug resistance gene, and configured to be eliminated in the absence of the corresponding drug}. (1108) Any of the above inventions, wherein the cassette is contained in the genome of the cell.

[0012] (1201) A genetically modified cell, comprising extrachromosomal DNA, wherein the extrachromosomal DNA comprises n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, and the cell is capable of expressing each of the exogenous genes; and wherein the cassette is configured to be conditionally inactivated (e.g., removed) {for example, the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and inactivation may be achieved by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on each of the two target sequences, or, when the cassette is present on the extrachromosomal DNA, by disrupting the extrachromosomal DNA or its replication apparatus (e.g., a centromere or a replication origin)}, preferably wherein endogenous genes corresponding to some or all of the n or more exogenous genes have been disrupted {wherein n is a natural number greater than or equal to 1}. (1202) The cell according to (1201) above, wherein n is 2 or more. (1203) The cell according to (1201) or (1202) above, wherein the disrupted endogenous gene comprises a gene associated with or involved in cell survival, a gene associated with or involved in cell proliferation, or a candidate gene thereof. (1204) The cell according to (1201) or (1202) above, wherein the disrupted endogenous gene comprises a gene associated with or involved in cell differentiation control, or a candidate gene thereof. (1205) The cell according to (1201) or (1202) above, wherein the disrupted endogenous gene comprises a gene associated with or involved in cell physiological function, or a candidate gene thereof. (1206) The cell according to any of (1201) to (1205) above, wherein the regulatory sequence is located outside the region between (including) the two target sequences or the two recognition sequences. (1207) A cell described in any of (1201) to (1206) above, wherein the cassette further comprises a gene encoding a knockdown nucleic acid.(1208) A composition for use in producing the cell according to any one of (1201) to (1207) above, comprising a cell having an exogenous target region (also referred to as a "landing pad") for integrating the cassette, wherein the exogenous target region comprises a target sequence for a sequence-specific DNA cleaving enzyme. (1209) The composition according to (1208) above, wherein the exogenous target region comprises two alleles, each of which has a distinguishably different positive selection marker, and each of which further has a negative selection marker {however, if the positive selection marker also serves as a negative selection marker, the composition does not need to have an additional negative marker.} (1210) A method comprising: (A) culturing under appropriate conditions the cell according to any of (1201) to (1207) above; (B) inactivating (e.g., removing) the cassette after culturing to obtain cells in which the cassette has been inactivated (e.g., removed); and (C) further culturing under appropriate conditions the cells in which the cassette has been inactivated (e.g., removed). (1211) The method according to (1210) above, wherein the disrupted gene is a gene associated with or involved in cell survival, a gene associated with or involved in cell proliferation, or a candidate gene thereof, and wherein step (B) is carried out after the cells have been propagated in step (A). (1212) The method according to (1210) above, wherein the disrupted gene is a gene associated with or involved in control of cell differentiation, or a candidate gene thereof, and wherein step (B) is carried out after the cells have been differentiated in step (A). (1213) The method according to (1210) above, wherein the disrupted gene is a gene related to or involved in the control of cell differentiation or a candidate gene thereof, and the method comprises, after carrying out step (B), subjecting the cells to a differentiation treatment in the culture of step (C). (1214) The method according to (1210) above, wherein the disrupted gene is a gene related to or involved in the physiological function of the cell or a candidate gene thereof, and the method comprises, after growing the cells in step (A), carrying out step (B).(1215) The cell according to (1214), wherein the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and inactivation is carried out by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on each of the two target sequences. (1216) The cell according to (1214), wherein the extrachromosomal DNA is an artificial chromosome and has alpha satellite DNA (or type I alphoid DNA) in the centromere region, the alpha satellite DNA has a tetracycline operator sequence (tet-O), and is configured such that binding of a transcriptional silencer (e.g., tTS) induces heterochromatinization in the artificial chromosome, causing the extrachromosomal DNA to be excreted from the cell. (1217) The cell described in (1214) above, wherein the extrachromosomal DNA is an episomal vector, and the episomal vector is configured to be conditionally inactivated {e.g., inactivated by cleavage; has a replication origin, and is configured to be cleaved or removed from the episomal vector {e.g., the replication origin is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and is removed by acting the sequence-specific DNA cleaving enzyme or site-specific recombinase on each of the two target sequences}; or contains a drug resistance gene, and is configured to be removed in the absence of the corresponding drug}.

[0013] (1301) A genetically modified cell having extrachromosomal DNA, wherein the extrachromosomal DNA comprises n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, the cell being capable of expressing each of the exogenous genes, and the extrachromosomal DNA being configured to be conditionally inactivated {wherein n is a natural number of 1 or more.}. (1302) The cell according to (1301) above, wherein n is 2 or more. (1303) The cell according to (1301) or (1302) above, wherein the n or more exogenous genes comprise genes encoding m or more knockdown nucleic acids {wherein m is a natural number of 1 or more or 2 or more and n or less.}. (1304) The cell according to any one of (1301) to (1303) above, wherein the m or more knockdown nucleic acids comprise one or more knockdown nucleic acids selected from the group consisting of siRNA, shRNA, miRNA, and antisense oligos. (1305) The cell according to any one of (1301) to (1304) above, wherein the n or more exogenous genes comprise p or more genes having physiological functions (for example, genes associated with or involved in cell survival, genes associated with or involved in cell proliferation, genes associated with or involved in regulating cell differentiation, and genes associated with or involved in the physiological functions of cells). {wherein p is a natural number of 1 or more or 2 or more and is a natural number equal to or less than n, the n or more exogenous genes may or may not contain knockdown nucleic acids, and when the n or more exogenous genes comprise m or more knockdown nucleic acids, m is a natural number equal to or greater than 1 or 2, and the sum of p and m is equal to or less than n.} (1306) The cell according to any one of (1301) to (1305) above, wherein the control sequence is located outside the region between the two target sequences or the two recognition sequences (including the target sequences or the recognition sequences). (1307) A composition for use in producing the cell according to any one of (1301) to (1306) above, comprising an exogenous target region (also referred to as a "landing pad") for integrating the cassette.(1308) The composition according to claim 7, wherein the exogenous target region comprises two alleles, each of which has a distinguishably different positive selection marker, and each allele further has a negative selection marker {however, if the positive selection marker also serves as a negative selection marker, an additional negative marker may not be required.} (1309) A method comprising: (A) culturing the cells according to any of (1301) to (1306) above under appropriate conditions; (B) inactivating (e.g., removing) the cassette after culturing to obtain cells in which the cassette has been inactivated (e.g., removed); and (C) further culturing the cells in which the cassette has been inactivated (e.g., removed) under appropriate conditions. (1310) The method according to (1309) above, wherein the disrupted gene is a gene associated with or involved in cell survival, a gene associated with or involved in cell proliferation, or a candidate gene thereof, and wherein step (B) is carried out after the cells are proliferated in step (A). (1311) The method according to (1309) above, wherein the disrupted gene is a gene associated with or involved in cell differentiation control, or a candidate gene thereof, and wherein step (B) is carried out after the cells are differentiated in step (A). (1312) The method according to (1309) above, wherein the disrupted gene is a gene associated with or involved in cell differentiation control, or a candidate gene thereof, and wherein step (B) is carried out, followed by subjecting the cells to a differentiation treatment in the culture of step (C). (1313) The method according to (1309) above, wherein the disrupted gene is a gene associated with or involved in the physiological function of a cell (for example, a gene associated with cellular metabolism, for example, a gene encoding a cellular metabolic enzyme), or a candidate gene thereof, and wherein step (B) is carried out after the cells are proliferated in step (A). (1310) The cell described in (1310) above, wherein the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and inactivation is achieved by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on each of the two target sequences.(1311) The cell described in (1310) above, wherein the extrachromosomal DNA is an artificial chromosome and has alpha satellite DNA (or type I alphoid DNA) in the centromere region, the alpha satellite DNA has a tetracycline operator sequence (tet-O), and is configured such that binding of a transcriptional silencer (e.g., tTS) induces heterochromatinization in the artificial chromosome, causing the extrachromosomal DNA to be shed from the cell. (1312) The cell described in (1310) above, wherein the extrachromosomal DNA is an episomal vector, and the episomal vector is configured to be conditionally inactivated {e.g., inactivated by cleavage; has a replication origin, and is configured to be cleaved or removed from the episomal vector {e.g., the replication origin is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and is removed by acting the sequence-specific DNA cleaving enzyme or site-specific recombinase on each of the two target sequences}; or contains a drug resistance gene, and is configured to be removed in the absence of the corresponding drug}.

[0014] (1400) The cell according to any one of the above, wherein the extrachromosomal DNA is an artificial chromosome. (1401) The cell according to any one of the above, wherein the extrachromosomal DNA is a human artificial chromosome. (1402) The cell according to any one of the above, wherein the extrachromosomal DNA is a yeast artificial chromosome. (1403) The cell according to any one of the above, wherein the extrachromosomal DNA is a bacterial artificial chromosome. (1404) The cell according to any one of the above, wherein the extrachromosomal DNA is a P1 bacteriophage artificial chromosome. (1405) The cell according to any one of the above, wherein the extrachromosomal DNA is an episomal vector. (1406) The cell according to any one of the above, wherein the extrachromosomal DNA is a BK virus-derived episomal vector. (1407) The cell according to any one of the above, wherein the extrachromosomal DNA is a bovine papillomavirus 1-derived episomal vector. (1408) The cell according to any one of the above, wherein the extrachromosomal DNA is an Epstein-Barr virus-derived episomal vector. (1409) The cell according to any one of the above, wherein the extrachromosomal DNA is a Sendai virus vector. (1410) The cell according to any one of the above, wherein the extrachromosomal DNA is a plasmid DNA.

[0015] (1501) The cell according to any one of (1400) to (1409) above, wherein the extrachromosomal DNA has a suicide gene operably linked to an inducible promoter. (1502) The cell according to any one of (1400) to (1409) above, wherein the extrachromosomal DNA has a drug resistance gene operably linked to a promoter. (1503) The cell according to any one of the above, wherein the extrachromosomal DNA has recognition sequences for two or more site-specific recombinases and target sequences for one or more sequence-specific DNA recombinases, and wherein the extrachromosomal DNA is destroyed by allowing the site-specific recombinase or the sequence-specific DNA recombinase to act on the extrachromosomal DNA. (1504) The cell according to any one of the above, wherein the extrachromosomal DNA is temperature-sensitive and is configured to be removed from the cell by raising the temperature. (1505) The cell according to any one of the above, wherein the extrachromosomal DNA is a temperature-sensitive Sendai virus vector and is removed from outside the cell in a temperature-sensitive manner. (1506) The cell according to any of the above, wherein the cell has extrachromosomal DNA, the cassette is contained in the extrachromosomal DNA, and one or more, preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1507) The cell according to (1400) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1508) The cell according to (1401) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1509) The cell according to (1402) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1510) The cell described in (1403) above, wherein the cassette is contained in the extrachromosomal DNA and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted.(1511) The cell according to (1404) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1512) The cell according to (1405) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1513) The cell according to (1406) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1514) The cell according to (1407) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1515) The cell according to (1408) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette have been disrupted. (1516) The cell according to (1409) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette have been disrupted. (1517) The cell according to (1410) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette have been disrupted. (1518) The cell according to (1501) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette have been disrupted. (1519) The cell described in (1502) above, wherein the cassette is contained in the extrachromosomal DNA and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted.(1520) The cell according to (1503) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1521) The cell according to (1504) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted. (1522) The cell according to (1505) above, wherein the cassette is contained in the extrachromosomal DNA, and one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are disrupted.

[0016] This technique involves ectopically introducing genes A, B, C, and D into DNA containing a landing pad, then disrupting endogenous genes A, B, C, and D to congregate genes A to D in one location, and then conditionally knocking out the congregated genes A to D. Figure 1 shows an example of a conditional knockout technique in which a cassette containing genes A to D is flanked by loxP, a recognition sequence for a site-specific recombinase. Cre recombinase is induced and allowed to act on the DNA to remove the cassette from the DNA. An example of the configuration of a landing pad is shown. In the figure, the triangle represents, for example, the recognition sequence for the site-specific recombinase. A cassette is the region between two recognition sequences for the site-specific recombinase; in this example, the control sequence is located outside the cassette. A target sequence for a sequence-specific DNA cleavage enzyme is present within the cassette, and a fragment containing n or more foreign genes is inserted at the target sequence. In the figure, the triangle represents the recognition sequence for the site-specific recombinase. Note that an insulator does not have to be present. An example of the configuration of a landing pad is shown. In the figure, triangles represent recognition sequences for site-specific recombinases. A cassette is a region between the recognition sequences for two site-specific recombinases; in this example, a control sequence is located inside the cassette. Target sequence 1 for a sequence-specific DNA cleavage enzyme is present inside the cassette, and a fragment containing n or more foreign genes is inserted at the location of target sequence 1. Target sequence 2 for a sequence-specific DNA cleavage enzyme is present inside the cassette, and after cassette removal, target sequence 2 is cleaved, destroying the cassette. Note that an insulator does not have to be present. An example in which a control sequence is present outside the cassette is shown. In this example, after cassette removal, the removed cassette is dissociated from the control sequence. An example in which a control sequence is present inside the cassette is shown. In this example, after cassette removal, the removed cassette is accompanied by a control sequence. An example in which a control sequence is present inside the cassette is shown. The cassette contains a target sequence for a sequence-specific DNA cleavage enzyme inside. The diagram shows a scheme for destroying the cassette by applying a sequence-specific DNA cleavage enzyme to the target sequence of the removed cassette. The cleaved DNA is then removed by the intracellular mechanism.

[0017] <Definition of Terms> As used herein, singular terms do not exclude plurals.

[0018] As used herein, "comprise" means that an unspecified third component may be contained. As used herein, "consist of" means that an unspecified third component is substantially not contained. Substantially not containing does not exclude the inclusion of a third component to an extent that cannot be removed in the manufacturing process or below the detection limit. As used herein, "consist only of" means that an unspecified third component is not contained.

[0019] As used herein, a "cell" is the basic structural and functional unit of all living organisms. A cell has a closed space surrounded by a cell membrane and contains genomic DNA. A cell can be a cell of a prokaryotic organism (prokaryotic cell) or a cell of a eukaryotic organism (eukaryotic cell). A cell can be a cell of a unicellular organism or a cell of a multicellular organism. A cell can be a single-celled cell. A cell can be an isolated cell. In some aspects, a cell can be a cell selected from the group consisting of a pluripotent cell and a pluripotent stem cell (such as an embryonic stem cell and an induced pluripotent stem cell). In some aspects, the pluripotent cell and the pluripotent stem cell are naive. In some aspects, the pluripotent cell and the pluripotent stem cell can be primed. In some aspects, a cell can be an epiblast stem cell or is not an epiblast stem cell. In some aspects, a cell can be a tissue stem cell. In some aspects, a cell can be a somatic cell. In some aspects, the cell may be a germline cell (e.g., a germ cell, a gamete, e.g., an oocyte, egg, spermatogonia, etc.). In some aspects, the cell may be a cell line. In some aspects, the cell may be an immortalized cell. In some aspects, the cell may be a cancer cell. In some aspects, the cell may be a non-cancer cell. In some aspects, the cell may be a cell from a patient with a disease. In some aspects, the cell may be a cell from a healthy individual. In some aspects, the cell may be an animal cell (e.g., a human cell), such as a cell selected from the group consisting of 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, and derivatives thereof. In some aspects, the cell may be a cell from a non-human organism.In some aspects, the non-human organism is a yeast (e.g., a fission yeast or budding yeast, e.g., a species of the genus Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces fragilis, Saccharomyces rouxii, Candida utilis, Candida tropicalis, etc.). The non-human organism may be an organism selected from the group consisting of Candida species such as Candida tropicalis, Pichia, Kluyveromyces, Yarrowia, Hansenula, and Endomyces. In some embodiments, the non-human organism may be a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium, and Penicillium species). In some embodiments, the non-human organism is Bacillus subtilis. In some embodiments, the non-human organism is a hydrogen bacterium. Hydrogen bacteria are bacteria that have the ability to oxidize hydrogen and use the energy generated by the oxidation reaction to perform carbon dioxide assimilation. (Also referred to as "hydrogen-oxidizing bacteria"). Hydrogen bacteria (e.g., aerobic hydrogen bacteria, Knallgas bacteria) can use hydrogen as an electron donor and oxygen as an electron acceptor, and have the ability to reductively assimilate carbon dioxide to synthesize organic matter. Examples of hydrogen bacteria include, but are not limited to, bacteria of the genus Caminibacter, bacteria of the genus Aquifex, bacteria of the genus Ralstonia, and bacteria of the genus Paracoccus. Examples of hydrogen bacteria also include bacteria of the genus Alcaligenes, bacteria of the genus Pseudomonas, bacteria of the genus Bacillus, and bacteria of the genus Hydrogenobadcter. Hydrogen bacteria can grow well in the presence of sufficient nutrients, hydrogen, oxygen, and carbon dioxide.Hydrogen bacteria produce substances such as proteins from carbon dioxide, which not only reduces carbon dioxide emissions but also produces organic substances such as proteins (e.g., animal proteins), foods, plastics, chemicals, and biofuels, making them highly anticipated for use. Hydrogen bacteria preferably have group 1h / 5 hydrogenase. In a preferred embodiment, the hydrogen bacteria may be Ralstonia eutropha.

[0020] As used herein, the term "control sequence" refers to a sequence that promotes transcription from DNA to RNA (particularly mRNA). Examples of control sequences include promoters. As used herein, the term "promoter" refers to a moiety that is located near (particularly upstream of) a DNA region to be expressed and has the function of promoting transcription from the DNA to RNA (particularly mRNA). The promoter is not particularly limited, and various Pol II promoters can be used. Examples of Pol II promoters include, but are not limited to, the CMV promoter, the EF1 promoter (EF1α promoter), the SV40 promoter, the MSCV promoter, the hTERT promoter, the β-actin promoter, the CAG promoter, and the CBh promoter. The promoter may be an inducible promoter. An inducible promoter is a promoter that can induce expression of a polynucleotide operably linked to the promoter only in the presence of an inducer that drives the promoter. Examples of inducible promoters include promoters that induce gene expression by heating, such as heat shock promoters. Examples of inducible promoters include promoters in which the inducer that drives the promoter is a drug. Examples of such drug-inducible promoters include Cumate operator sequences, λ operator sequences (e.g., 12×λOp), and tetracycline-inducible promoters. Examples of tetracycline-inducible promoters include promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline), or reverse tetracycline-controlled transactivator (rtTA). Examples of tetracycline-inducible promoters include the TRE3G promoter.

[0021] As used herein, "operably linked" means linking a gene (DNA) to a regulatory sequence (e.g., a promoter) so that its expression is driven. By operably linking a gene to a regulatory sequence, the expression of the gene can be induced by the action of the regulatory sequence.

[0022] As used herein, "gene" refers to a polynucleotide that contains at least one open reading frame that encodes a specific protein or that contains a frame that encodes a non-coding RNA. A gene preferably encodes a protein and may contain both exons and introns. A gene preferably encodes a protein and may have only exons. A gene preferably has a complementary DNA (cDNA) sequence of the RNA (e.g., mRNA) that is transcribed from the gene.

[0023] The term "allele" refers to a set of sequences present at the same locus on a chromosomal genome. In some embodiments, a diploid cell has two alleles at the same locus, and a triploid cell has three alleles at the same locus. In some embodiments, additional alleles may be formed by an abnormal copy of the chromosome or an abnormal additional copy of the locus.

[0024] The terms "genome modification" and "genome editing" are used interchangeably and refer to the induction of mutations at desired locations (target regions) on a genome. Genome modification may involve the use of sequence-specific nucleic acid cleaving molecules designed to cleave target region DNA. In a preferred embodiment, genome modification may involve the use of nucleases engineered to cleave target region DNA. In a preferred embodiment, genome modification may involve the use of nucleases (e.g., TALENs or ZFNs) engineered to cleave target sequences having specific base sequences in the target region. In a preferred embodiment, genome modification may involve the use of a restriction enzyme with only one cleavage site in the genome, such as a meganuclease (e.g., a restriction enzyme with 16-base sequence specificity (theoretically, 4 bases)), to cleave target sequences having specific base sequences in the target region. １６ A restriction enzyme with 17 base sequence specificity (theoretically, 4 １７ and restriction enzymes with 18-base sequence specificity (theoretically, 4 １８In some cases, sequence-specific endonucleases such as site-specific nucleases (which occur at a ratio of one per base) can also be used. Typically, a double-strand break (DSB) is induced in the DNA of the target region by the use of a site-specific nuclease, and the genome is then 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 a double-strand break 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 is also capable of introducing desired mutations into the target region. A preferred example of a genome modification technique is the CRISPR / Cas system.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-CrepsbIIIIP, 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 restriction enzymes 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 derivatives thereof, preferably a meganuclease that is a restriction enzyme having sequence specificity of 18 bases or more and its cleavage site (or recognition site), particularly a meganuclease that does not cleave the genome of a cell at one or more sites and its cleavage site, can be used.Genome modification can also be used to modify extrachromosomal DNA.

[0025] The term "target region" refers to a region of DNA that is targeted by a sequence-specific DNA cleaving enzyme, which typically cleaves DNA within or near the target region.

[0026] The term "corresponding" in the context of a gene means an identical gene or a functional counterpart. An identical gene is a gene present at the same locus in the same animal species, and is capable of performing the same physiological function, although it does not exclude differences in sequence. Thus, an endogenous gene corresponding to a foreign gene means an endogenous gene that is recognized as the same gene as the foreign gene, although it does not exclude differences in sequence. A functional counterpart means a gene that has an equivalent function.

[0027] The term "donor DNA" refers to DNA used to repair a double-strand break in DNA, capable of homologous recombination with DNA surrounding the target region. The donor DNA contains, as homology arms, nucleotide sequences upstream and downstream of the target region (e.g., nucleotide sequences adjacent to the target region). Herein, a homology arm consisting of a nucleotide sequence upstream of the target region (e.g., a nucleotide sequence adjacent to the upstream side) may be referred to as an "upstream homology arm," and a homology arm consisting of a nucleotide sequence downstream of the target region (e.g., a nucleotide sequence adjacent to the downstream side) may be referred to as a "downstream homology arm." The donor DNA can contain a desired nucleotide sequence between the upstream homology arm and the downstream homology arm. The length of each homology arm is preferably 300 bp or more, typically about 500 to 3,000 bp. The lengths of the upstream homology arm and the downstream homology arm may be the same or different. If the target region successfully induces homologous recombination with the donor DNA after sequence-dependent cleavage, the sequence between the upstream and downstream base sequences of the target region will be replaced with the sequence of the donor DNA.

[0028] "Upstream" of a target region refers to a DNA region located 5' of a reference nucleotide strand in the double-stranded DNA of the target region. "Downstream" of a target region refers to a DNA region located 3' of a reference nucleotide strand. When a target region includes a protein-coding sequence, the reference nucleotide strand is usually the sense strand. Generally, a promoter is located upstream of a protein-coding sequence. A terminator is located downstream of a protein-coding sequence.

[0029] The term "sequence-specific nucleic acid cleaving molecule" refers to a molecule that can recognize a specific nucleic acid sequence and cleave a nucleic acid at the specific nucleic acid sequence. A sequence-specific nucleic acid cleaving molecule is a molecule that has the activity of cleaving nucleic acids in a sequence-specific manner (sequence-specific nucleic acid cleaving activity).

[0030] The term "target sequence" refers to a DNA sequence in DNA that is the target of cleavage by a sequence-specific nucleic acid cleaving molecule. When the sequence-specific nucleic acid cleaving molecule is a Cas protein, the target sequence refers to a DNA sequence that is the target of cleavage by the Cas protein. When a Cas9 protein is used as the Cas protein, the target sequence must be a sequence adjacent to the 5' side of a protospacer adjacent motif (PAM). The target sequence is typically selected as a sequence of 17 to 30 bases (preferably 18 to 25 bases, more preferably 19 to 22 bases, and even more preferably 20 bases) immediately adjacent to the 5' side of the PAM. Known design tools such as CRISPR DESIGN (crispr.mit.edu / ) can be used to design the target sequence. From the perspective of reducing off-target cleavage, the target sequence is preferably a unique sequence on DNA (particularly the genome).

[0031] As used herein, "under suitable conditions" refers to conditions under which a purpose can be achieved. For example, conditions suitable for culture refer to conditions suitable for maintaining and / or growing cells. Conditions suitable for differentiation may be conditions under which, in addition to maintaining cells, differentiation factors necessary for the cells to differentiate into specific cells are sufficiently supplied.

[0032] As used herein, the term "cassette" refers to a unit of nucleic acid that can be inactivated or removed from other parts. The elements contained within a cassette can be inactivated (e.g., removed) together in a single operation.

[0033] As used herein, "conditional" means activated by a specific condition. Conditional knockout means knockout that is activated by a specific condition. Conditional inactivation means inactivation that is activated by a specific condition. The specific condition can be met by the application of a specific stimulus to a cell. Inactivation of a cassette means inactivating the function of the exogenous gene contained in the cassette, for example, by disrupting (e.g., cleaving), inhibiting, or removing the cassette; disrupting (e.g., cleaving), inhibiting, or removing the promoter; or disrupting (e.g., cleaving), inhibiting, or removing the functional linkage between the gene and the promoter; or, as is possible when the cassette is contained in extrachromosomal DNA, eliminating the entire extrachromosomal DNA containing the inactivated cassette. In a preferred embodiment, inactivation can be removal of the cassette or extrachromosomal DNA.

[0034] Conditional inactivation can be achieved by conditional knockout. Conditional knockout can be caused, for example, by the presence, expression, or addition of a site-specific recombinase or a sequence-specific DNA cleavage enzyme in cells. The presence, expression, or addition of a site-specific recombinase or a sequence-specific DNA cleavage enzyme in cells can be achieved by operably linking these enzymes to an inducible control sequence and allowing them to be present in the cells, thereby driving the inducible control sequence. Driving the inducible control sequence can be achieved by contacting cells with a factor that drives the inducible control sequence. Examples of site-specific recombinases include Cre recombinase, Flp recombinase, Dre recombinase, phage integrase, and various site-specific recombinases derived therefrom. Examples of site-specific recombinases also include transposases contained in DNA transposons. Cre recombinase recognizes loxP sequences and removes the region flanked by two loxP sequences from DNA. Flp recombinase recognizes FRT sequences and removes the region flanked by the two FRT sequences from DNA. Dre recombinase removes the region flanked by the two Dre sequences from DNA. Phage integrase removes the region flanked by attL and attR from DNA with the assistance of an excision factor (Xis). Transposase recognizes the inverted terminal repeats (ITRs) that transposons have at each end of their genome and removes the region flanked by the two ITRs from DNA. Methods for introducing a desired cassette (or gene) into DNA or removing a cassette from DNA using a piggyBac vector (or piggyBac transposon vector) are well known to those skilled in the art. Phage integrases include tyrosine-type integrases such as λ phage integrase and HK022 bacteriophage integrase, and serine-tap integrases such as phiC31 integrase and TG1 integrase.Sequences derived from the loxP sequence include lox511, lox2272, and loxFAS, which are recognized by the Cre recombinase, but these sequences do not recombine with loxP. Those skilled in the art can appropriately select a site-specific recombinase and its recognition sequence to use for cassette removal. The recognition sequence for the site-specific recombinase can be selected from the group consisting of loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attL, attR, and Dre sites. Recombinases that induce recombination at these recognition sequences are well known. The two recognition sequences are oriented and positioned on the DNA so that the flanking region is removed. Those skilled in the art can determine appropriate conditions for activating the above-mentioned recombinase, and under these appropriate conditions, the expression of the function of the foreign gene in the cassette can be inhibited or stopped, for example by removing the cassette and / or regulatory sequence between the recognition sequences.

[0035] Conditional inactivation can also be achieved by transcription suppression technology such as CRISPRi. In CRISPRi, a transcription suppression domain is linked to a dCas9 protein that has lost endonuclease activity (a mutant in which an amino acid mutation is introduced into the active center of DNA cleavage, inhibiting nuclease activity, such as a mutant of Streptococcus pyogenes Cas9 with D10A and H840A (Streptococcus pyogenes Cas9 has, for example, UniProt accession number Q99ZW2 Cas9)), and the transcription suppression domain is recruited to DNA in a target sequence-specific manner, thereby suppressing transcription from the target regulatory sequence. The transcription suppression ability is strong, and for example, expression can be reduced to several hundred times. Examples of the transcription suppression domain include a KRAB domain (Kruppel-associated box domain) and a MeCP2 transcription suppression (TRD) domain, as well as fusion proteins thereof. Examples of transcriptional repression domains include peptides or transcriptional repression domains thereof selected from the group consisting of KRAB, DMNT1, SET1, HDAC1, DMNT3A, SETD8, EXH2, SUV39H1, PHF19, SALI, NUE, SUR4, KYP, DIM5, HDAC8, SIRT3, SIRT6, MESOLO4, SET8, HST2, COBB, NCOR, MeCP2, SIN3A, HDT1, MBD2B, NIPP1, and HP1A, or fusion proteins of two peptides or transcriptional repression (TRD) domains thereof (see Nat Methods. 2018 Aug; 15(8): 611-616). CRISPRi can inactivate cassettes, for example, by targeting regulatory sequences or exogenous genes. Such CRISPRi can be achieved using a guide RNA (e.g., a single-stranded guide RNA or crRNA) having a regulatory sequence or a sequence that targets an exogenous gene as the guide RNA.

[0036] To perform conditional inactivation, an inactivation protein such as a site-specific recombinase, a sequence-specific DNA cleavage enzyme, or dCAS9 can be linked to a tissue-specific promoter or an inducible promoter. Tissue-specific promoters and inducible promoters are widely used in the field of conditional knockout, and those skilled in the art can select an appropriate tissue-specific promoter or inducible promoter to achieve conditional inactivation of a cassette. The inducible promoter may be as described above.

[0037] <Cells of the Present Disclosure> The present disclosure provides genetically modified cells. The cells have a cassette containing n or more exogenous genes, the exogenous genes are operably linked to one or more regulatory sequences, and the cells are capable of expressing each of the exogenous genes (where n is a natural number of 1 or greater, or 2 or greater).

[0038] According to certain aspects of the present disclosure, the cell or cassette is configured such that the cassette can be conditionally inactivated, which can be achieved by disruption (e.g., cleavage), inhibition, or removal of the cassette.

[0039] According to one aspect (first aspect) of the present disclosure, in the cell, the n or more exogenous genes include p or more genes encoding physiologically functional products {where p is a natural number greater than or equal to 1 or 2, and is n or a natural number less than n}. By introducing n or more exogenous genes into a cell, a phenotype can be induced or observed, and then the cassette can be inactivated (e.g., removed). Furthermore, one aspect (first aspect) of the present disclosure can be used to produce the cell of the second aspect described below. The cell of the first aspect survives and preferably proliferates in culture under appropriate conditions.

[0040] According to one aspect (second aspect) of the present disclosure, in the cell, the n or more exogenous genes include genes encoding p or more physiologically functional products {wherein p is a natural number greater than or equal to 1 or 2, and is n or a natural number less than n.}, and endogenous genes corresponding to some (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, or 20 or more) or all of the n or more exogenous genes have been disrupted. Alternatively, in the cell, the n or more exogenous genes include genes encoding p or more physiologically functional products {wherein p is a natural number greater than or equal to 1 or 2, and is n or a natural number less than n.}, and endogenous genes corresponding to some or all of the genes encoding the p or more physiologically functional products have been disrupted. In this aspect, the exogenous genes encoding physiologically functional products can complement (e.g., the function of) the disrupted endogenous genes. Of the disrupted endogenous genes, the number of genes whose functions are complemented by the exogenous genes is q (q is a natural number equal to or greater than 1, preferably equal to or greater than 2, and is a natural number equal to or less than p). It is expected that the effects of disrupting the q endogenous genes will appear in the cell by inactivating (e.g., removing) the cassette, as described below. The cells of the second aspect survive and preferably proliferate when cultured under appropriate conditions, at least before inactivating (e.g., removing) the cassette. After inactivating (e.g., removing) the cassette, the cells of the second aspect, for example, (i) survive and do not proliferate, (ii) survive and proliferate, or (iii) do not survive.

[0041] In some embodiments, n and p are equal. In some embodiments, n and p are different. Also, in some embodiments, p and q are equal. In some embodiments, p and q are different. In some embodiments, n and p are equal and p and q are equal. In some embodiments, n and p are equal and p and q are different. In some embodiments, n and p are different and p and q are equal. In some embodiments, n and p are different and p and q are different.

[0042] In a second aspect, the exogenous gene introduced into the cassette comprises the endogenous gene to be disrupted or a gene capable of at least complementing its physiological function, preferably a gene identical to the endogenous gene. This allows the exogenous gene introduced into the cassette to complement the endogenous gene and ensure the necessary cellular functions even after the endogenous gene is disrupted. It is not necessary for all disrupted endogenous genes to be complemented by exogenous genes. This is because, as long as the disrupted gene retains its necessary function, it is not necessary to exogenously supply the same gene to the cell. Furthermore, the cassette may contain a gene different from the endogenous gene to be disrupted. A gene different from the endogenous gene to be disrupted in the cassette can confer a new function to the cell. An identical gene is a gene present at a corresponding locus (i.e., the same locus) in the same biological species. Since the endogenous gene and the exogenous gene are the same gene, the function of the disrupted endogenous gene is complemented by the exogenous gene. Inactivation (eg, removal) of the cassette is expected to have the effect of disrupting q endogenous genes in the cell.

[0043] According to a third aspect of the present disclosure, in the cell, the n or more exogenous genes include m or more knockdown nucleic acids (where m is a natural number of 1 or more or 2 or more). The knockdown nucleic acid is a nucleic acid that inhibits the expression of one or more genes (preferably a single gene) in nucleic acids (e.g., a genome, etc.) present in the cell. As the knockdown nucleic acid, a knockout nucleic acid composed of RNA is advantageously used, and may be selected from the group consisting of, for example, siRNA, shRNA, miRNA, and antisense oligonucleotides. In the third aspect, the endogenous gene may or may not be disrupted, but the important point is that by inactivating (e.g., removing) the cassette, the gene encoding the knockdown nucleic acid is inactivated (e.g., removed), thereby reducing or eliminating the expression of the knockdown nucleic acid. When the cassette contains multiple knockdown nucleic acids, the expression of multiple knockdown nucleic acids can be reduced or eliminated collectively. This means that the knockdown effect can be reversed by conditionally inactivating (e.g., removing) the cassette. When a cassette contains multiple knockdown nucleic acids, the knockdown effects of the multiple knockdown nucleic acids can be reversed by inactivating (e.g., removing) the cassettes at the desired timing in a single operation. In one embodiment, n and m are equal. In another embodiment, n and m are different. The cassette can be inactivated (e.g., removed) at the timing of expression of a sequence-specific DNA cleavage enzyme or site-specific recombinase for inactivating (e.g., removing) the cassette. The enzyme can be expressed by driving an inducible promoter operably linked to a gene encoding the enzyme. Alternatively, the cassette can be inactivated (e.g., removed) by introducing a sequence-specific DNA cleavage enzyme or site-specific recombinase into the cell. In this way, the cassette can be inactivated (e.g., removed) by acting on the sequence-specific DNA cleavage enzyme or site-specific recombinase when necessary. The cell of the third embodiment survives and preferably proliferates in culture under appropriate conditions, at least before inactivation (e.g., removal) of the cassette.The cells of the third aspect, after inactivation (eg, removal) of the cassette, (i) survive and do not proliferate, (ii) survive and proliferate, or (iii) do not survive.

[0044] siRNA, also known as small interfering RNA, is a molecule capable of silencing the expression of RNA (target RNA) with a homologous sequence. While siRNA is not particularly limited as long as it can silence the target RNA, it is typically a double-stranded RNA comprising a 20-25-mer RNA (guide strand) with a sequence complementary to the target RNA and a 20-25-mer complementary strand (passenger strand). In siRNA, the guide strand and passenger strand hybridize to form double-stranded RNA. The siRNA may have a non-double-stranded sequence (overhang) at the 5' or 3' end. The overhang may be, for example, approximately 1-3 mer. shRNA, also known as small hairpin RNA, is typically a single-stranded RNA in which two RNA strands in the siRNA are linked by a hairpin. Under physiological conditions, the guide strand and passenger strand contained in the shRNA can hybridize. shRNA is advantageous for intracellular expression. This is because shRNAs are single-stranded RNAs that can be transcribed from a single gene encoding the shRNA, and the transcribed shRNA can form a functional form with a hairpin structure within cells. miRNAs are mature microRNAs, approximately 21-25 single-stranded RNAs, molecules that can suppress the expression of RNAs with complementary sequences. siRNAs, shRNAs, and miRNAs are thought to silence target RNAs by being incorporated into a protein complex called the RNA-induced silencing complex (RISC). Antisense oligos are single-stranded nucleic acids with a sequence complementary to the target RNA. Antisense oligos can be single-stranded RNAs, and the optimal length is generally considered to be 12-mer to 28-mer. Antisense oligos shorter than 12-mer increase the risk of nonspecific silencing, while antisense oligos longer than 25-mer are said to have reduced cellular uptake efficiency. For example, antisense oligos can be 18-mer to 30-mer or 20-mer to 25-mer in length.In the present disclosure, antisense oligos can be generated by intracellular transcription from DNA, and therefore longer antisense oligos can also be used. Those skilled in the art can appropriately determine the sequence and length of these knockdown nucleic acids, taking into account their target specificity, expression efficiency, etc. The knockdown nucleic acid is typically operably linked to a regulatory sequence for RNA polymerase III (e.g., U6 promoter, T7 promoter, etc.).

[0045] The first to third aspects of the cell described above are not necessarily exclusive and may be compatible with each other. In a fourth aspect, in the first to third aspects described above, of the n or more exogenous genes, p genes encode physiologically functional products, and m genes may be knockout nucleic acids {wherein p and m are independently a natural number of 1 or more, preferably 2 or more, and the sum of p and m is a natural number of n or less}. In the fourth aspect, endogenous genes corresponding to some or all of the genes encoding physiologically functional products may be disrupted. In one aspect, p and q are equal. In another aspect, p and q are different. In a preferred aspect, the sum of p and m may be a natural number of 2 or more, preferably 3 or more, and more preferably 4 or more. The cell of the fourth aspect survives and preferably grows in culture under appropriate conditions, at least before inactivation (e.g., removal) of the cassette. The cells of the fourth aspect, after inactivation (eg, removal) of the cassette, (i) survive and do not proliferate, (ii) survive and proliferate, or (iii) do not survive.

[0046] A cassette contains n or more exogenous genes. Here, n can be a natural number of 1 or more, preferably 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, but can be, for example, a natural number of 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, or 10 or less. n can be, for example, a natural number from 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10, 2 to 50, 2 to 40, 2 to 30, 2 to 20, or 2 to 10. A cell may contain two or more cassettes. The two cassettes contain a total of n or more exogenous genes. Here, n can be a natural number of 1 or more, preferably 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, but can be, for example, a natural number of 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, or 10 or less. n can be a natural number, for example, from 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10, from 2 to 50, 2 to 40, 2 to 30, 2 to 20, or 2 to 10. Two or more cassettes can be removed independently, consecutively, sequentially, or simultaneously or together.

[0047] In the second aspect, the effects of the endogenous gene disruption can be mitigated by disrupting an endogenous gene and providing an exogenous gene identical to the disrupted gene in a cassette. However, a single operation of inactivating (e.g., removing) the cassette allows the exogenous genes to be inactivated (e.g., knocked out) en masse, thereby eliminating the effect of mitigating the effects of the endogenous gene disruption. In order to inactivate (e.g., disrupt) multiple genes that are originally present separately in the nucleic acid (e.g., genome) of a cell, they must be inactivated (e.g., disrupted) individually, and it is not easy to inactivate (e.g., disrupt) multiple genes from the nucleic acid (e.g., genome) in a single operation. However, according to the present disclosure, the cell or cassette is configured so that the entire cassette can be inactivated. According to the present disclosure, for example, the cassette can be removed from the nucleic acid (e.g., cell, genome, or extrachromosomal DNA) possessed by the cell. Furthermore, by removing the cassette from the nucleic acid or cell, the n or more exogenous genes can be inactivated (e.g., knocked out) en masse. In cells in which the cassette has been inactivated (e.g., removed), the endogenous gene is disrupted, and after cassette inactivation (e.g., removal) (particularly, triggered by cassette inactivation (e.g., removal)), the cell will exhibit a phenotype due to the disruption. This is also true in the fourth aspect.

[0048] In the third aspect, the functions of the knockdown nucleic acids contained in the cassette can be stopped collectively by inactivating (e.g., removing) the cassette, and the knockdown effect on the cell can be canceled at the timing of inactivating (e.g., removing) the cassette. This is also true for the fourth aspect.

[0049] The cassette carries the exogenous gene to be conditionally inactivated (e.g., deleted). In addition to the exogenous gene, the cassette may contain additional natural or artificial sequences. Examples of additional natural or artificial sequences include tool genes such as marker genes and sequences for promoting or suppressing the expression of the exogenous gene in the cassette. Here, a "tool gene" can be used as a tool to enhance the convenience of gene modification techniques, such as detecting the success or failure of genetic modification or obtaining only successfully modified cells. Sequences for promoting or suppressing the expression of the exogenous gene in the cassette include regulatory sequences, polyadenylation sequences, and insulators. A marker gene can be used to confirm the introduction of a cassette into DNA, or the removal of a cassette from DNA. The cassette may further contain a gene encoding a site-specific recombinase. A cassette may contain r tool genes (r is a natural number greater than or equal to 1 and does not exceed n when added to the number of exogenous genes). Such a tool gene may also be included in the exogenous gene. r may be a natural number of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and may be a natural number from 1 to 20 or from 1 to 10.

[0050] The cassette may be present in the genome of the cell or in extragenomic (or extra-host chromosomal) DNA. Therefore, inactivation (e.g., removal) of the cassette can be achieved by removal from the genome, removal from the host extrachromosomal DNA, and removal of the host extrachromosomal DNA. The cassette does not necessarily need to be removed from the cell as long as expression is inhibited; however, in some embodiments, removal of the cassette is removal from the cell. When the cassette is present in the genome, it may be located or integrated into, for example, a transcriptionally active gene locus or a safe harbor region on the genome. Here, "host" refers to a cell into which the cassette is introduced or a cell derived from the cell. The insertion site of the cassette may be, for example, the ROSA26 locus. The insertion site of the cassette may be, for example, the CH25h locus. The insertion site of the cassette may be, for example, the AAVS1 locus. Even if the cassette is not a safe harbor region, the cassette may be integrated anywhere as long as it does not cause unacceptable and significant impairment of cellular function due to, for example, disruption of existing genes, or does not significantly inhibit expression of the cassette. When the cassette is present outside the host chromosome, one or more, or preferably two or more, endogenous genes corresponding to part or all of the exogenous genes contained in the cassette are genetically engineered (preferably, disrupted). In a preferred embodiment, the cassette is present outside the host chromosome, and two or more endogenous genes corresponding to part or all of the exogenous genes are disrupted.

[0051] The cassette may be inserted into extragenomic (or extra-host chromosomal) DNA, and may be contained on extrachromosomal DNA such as, but not limited to, artificial chromosomes (e.g., artificial chromosomes for eukaryotic cells such as human artificial chromosomes (HACs) and yeast artificial chromosomes (YACs), as well as bacterial artificial chromosomes such as bacterial artificial chromosomes (BACs) and P1 bacteriophage artificial chromosomes (PACs)) and episomal vectors (e.g., circular DNA such as plasmids and viral vectors). HACs are DNAs created by deleting regions unnecessary for chromosomal maintenance, including telomere and centromere regions. HACs include, but are not limited to, mini-chromosomes created by shortening chromosomes by inserting telomere sequences into native chromosomes, and those in which centromeres, telomeres, and various genes are incorporated into linear YACs. YACs contain centromeres, telomeres, genes encoding selectable markers, and autonomously replicating sequences (ARSs). By having a centromere region, artificial chromosomes can be replicated intracellularly and maintained in dividing cells. BACs have an F factor replicon and can be maintained in a single copy in host cells (e.g., E. coli). PACs are plasmids that utilize the replication system of the P1 bacteriophage and can be maintained in host cells (e.g., E. coli) at a low copy number. These bacterial artificial chromosomes can be removed from host cells using techniques similar to those used with plasmids. These artificial chromosomes preferably each have a cloning site. The cloning site has, for example, a restriction enzyme cleavage site, allowing the insertion of foreign DNA. Artificial chromosomes are replicated and maintained independently without being inserted into a host chromosome, and when a gene is loaded, they do not interfere with the induction of gene expression. Artificial chromosomes may further contain a selectable marker gene. Episomal vectors are vectors that can replicate outside of the host chromosome. Episomal vectors include DNA vectors and RNA vectors. A typical example of a DNA vector is a circular DNA such as a plasmid, and a typical example of an RNA vector is a mononegavirus such as a Sendai virus vector.A plasmid is a circular DNA containing a replication origin necessary for replication in a host cell and a selection marker gene. Plasmids usually have a drug resistance gene and a replication origin and can be maintained intracellularly in the presence of a drug. The viral vector is not particularly limited as long as it is not inserted into the genome and is maintained episomally, but examples include Sendai virus vectors. Sendai virus is a virus classified in the Paramyxoviridae family that has a single-stranded, non-segmented, negative-strand RNA genome. Sendai virus lacking the F protein necessary for infection is used as a vector. During production, F protein is supplied from packaging cells, but after cell infection, infectious virus is not produced due to the lack of F protein.

[0052] Inactivation (e.g., removal) of the cassette is also possible by removing the entire extrachromosomal DNA. For example, extrachromosomal DNA can be removed from host cells by disrupting the replication origin or centromere of the extrachromosomal DNA, by culturing in the absence of a drug corresponding to the drug resistance gene carried, or by cleaving the chromosomal DNA. Destruction of the replication origin or centromere can be achieved by DNA cleavage using a site-specific recombinase or sequence-specific DNA cleavage enzyme. In episomal vectors, the replication origin is essential for replication, and the episomal vector can be removed by disrupting the replication origin. Destruction of the replication origin can be achieved, for example, by removing or cleaving the replication origin. In artificial chromosomes, the centromere is essential for replication, and the artificial chromosome can be removed by destroying or removing the centromere. Destruction of the centromere can be achieved, for example, by removing the centromere or promoting heterochromatinization of the centromere. In some embodiments, the extrachromosomal DNA contains, for example, a recognition sequence for a sequence-specific DNA cleaving enzyme, and is cleaved by the sequence-specific DNA cleaving enzyme, resulting in the destruction and disappearance of the extrachromosomal DNA. In some embodiments, the extrachromosomal DNA contains, for example, two site-specific recombinase recognition sequences or target sequences for a sequence-specific DNA cleaving enzyme, flanking the replication origin or centromere, allowing the site-specific recombinase or sequence-specific DNA cleaving enzyme to remove the region containing the replication origin or centromere from the extrachromosomal DNA. In some embodiments, the extrachromosomal DNA contains one or more target sequences for sequence-specific DNA cleaving enzymes within the replication origin or centromere, and is cleaved by the sequence-specific DNA cleaving enzyme, resulting in the destruction and disappearance of the extrachromosomal DNA. The sequence-specific DNA cleaving enzyme may be a CRISPR / Cas system or a meganuclease. Bacterial artificial chromosomes can be removed from host cells using plasmid incompatibility.Bacterial artificial chromosomes can also be removed by one or more chemicals (e.g., plasmid removal agents) selected from the group consisting of acriflavine, acridine orange, ethidium bromide, quinacrine, coumermycin, novobiocin, mitomycin C, rifampicin, and sodium dodecyl sulfate (SDS) (Trevors, JT, FEMS Microbiology Reviews. 1986, 32, 149-157). Episomal vectors include, for example, BK virus-derived episomal vectors, bovine papillomavirus 1-derived episomal vectors, and Epstein-Barr virus-derived episomal vectors (K Van Craenenbroeck, P Vanhoenacker, G Haegeman, Eur J Biochem. 2000 Sep;267(18):5665-78). BK virus-derived episomal vectors typically contain a BKV early promoter (BKVE), BKori, BKV small T antigen (BKt), and BKV large T antigen (BKT), and may also contain a gene encoding a drug selection marker in E. coli, an origin of replication, and a gene encoding a drug selection marker in eukaryotic cells. Bovine papillomavirus 1-derived episomal vectors (BPV-1-derived vectors) contain a BPV-1ori and E1 to E8, and may also contain a gene encoding a drug selection marker in E. coli, an origin of replication, and a gene encoding a drug selection marker in eukaryotic cells. Epstein-Barr virus (EBV)-derived episomal vectors contain EBNA1 and OriP, and may also contain a gene encoding a drug selection marker in E. coli, an origin of replication, and a gene encoding a drug selection marker in eukaryotic cells.Techniques for removing extrachromosomal DNA include, for example, a method of incorporating a suicide gene, selecting, and selecting only cells that have lost extrachromosomal DNA during the division process (Lee, M. et al., Exp Mol Med. 2019 Jul; 51(7): 82), a method of inhibiting the replication of artificial chromosomes by inhibiting the centromere of artificial chromosomes for eukaryotic cells, and obtaining cells that have lost the artificial chromosome (Iida, Y. et al., DNA Res. 2010 Oct; 17(5): 293-301), and a method of removing extrachromosomal DNA from cells by cleaving it at one or more locations, preferably multiple locations, using a CRISPR / Cas system or meganuclease (Zuo, E. et al., Genome Biol. 2017 Nov 24; 18(1): 224) by applying such techniques to extrachromosomal DNA. Temperature-sensitive plasmid removal technology (US Pat. No. 5,616,480A) can also be applied to extrachromosomal DNA. Those skilled in the art would be able to appropriately remove extrachromosomal DNA from cells. Temperature-sensitive Sendai virus vectors have temperature-sensitive mutations and can be removed from cells by increasing the temperature (WO 2023 / 127871A). Various temperature-sensitive mutations are known, and those skilled in the art can select and use an appropriate one. Sendai virus vectors have a component protein (e.g., P protein) linked to a degron and can be removed from cells by the action of a proteolysis promoter (WO 2023 / 127871A).

[0053] Centromere heterochromatinization can be achieved by incorporating a transcriptional silencer target sequence into the centromere. For example, centromere heterochromatinization can be achieved by introducing a tetracycline operator sequence (tet-O) into the centromere and contacting it with a transcriptional silencer. A fusion protein (tTS) of the Tet repressor protein (TetR) and the KRAB silencing domain (SDKid-1) can be used as the transcriptional silencer. Upon binding to tet-O, ​​the transcriptional repressor promotes centromere heterochromatinization, thereby allowing the artificial chromosome to be removed from the cell. In some embodiments, the centromere can be, for example, α satellite DNA (or type I alphoid DNA, e.g., type I alphoid DNA of chromosome 21 (α21-I)). Conditional inactivation of artificial chromosomes can be achieved by producing a transcriptional repressor from a gene operably linked to an inducible promoter.

[0054] The disruption or removal can be achieved by site-specific recombinase or sequence-specific DNA cleavage enzyme. By operably linking the site-specific recombinase or sequence-specific DNA cleavage enzyme to an inducible promoter, the extrachromosomal DNA can be conditionally inactivated (e.g., removed from cells). Furthermore, by operably linking the site-specific recombinase or sequence-specific DNA cleavage enzyme to a tissue-specific promoter, the extrachromosomal DNA can be inactivated (e.g., removed from cells) only in specific tissues. Extrachromosomal DNA can also be inactivated (e.g., removed from cells) by removing from the culture medium a drug corresponding to a selectable marker gene (particularly a drug resistance gene), but this method has the disadvantage that inactivation (e.g., removal from cells) may be incomplete.

[0055] When it is not necessary to preserve the extrachromosomal DNA within the cell, the extrachromosomal DNA itself can be removed from the cell, thereby inactivating the cassette, but when it is necessary to preserve the extrachromosomal DNA within the cell, it is preferable to remove only the cassette from the extrachromosomal DNA without removing the remaining parts. In the extrachromosomal DNA preserved within the cell, other genes that were not removed can still function within the cell even after the cassette is removed.

[0056] There are known techniques for removing Sendai virus from within cells based on temperature sensitivity, for removing it from within cells by adding a tag containing a degron to a component, and for removing it from within cells by incorporating a target sequence for a microRNA, and such techniques can also be used in the method of the present disclosure.

[0057] The cell contains at least one such cassette. It is sufficient that one cassette is inserted into the DNA; it is not necessary that two or more cassettes, for example, be inserted into each allele of the same locus. However, in some embodiments, the cassette may be inserted into each allele of the same locus on the genome. The cell may contain, but is not particularly limited to, two or more different cassettes that are integrated so that each can be inactivated (e.g., removed from the cell). The two or more different cassettes can be inactivated (e.g., removed from the cell) independently (e.g., at different times).

[0058] The size of the cassette is not particularly limited. For example, when the cassette is removed using a site-specific recombinase, the size may be 500 bp to 1 Mbp, for example, 1 kbp to 1 Mbp, 1 kbp to 900 kbp, 1 kbp to 800 kbp, 1 kbp to 700 kbp, 1 kbp to 600 kbp, 1 kbp to 500 kbp, 1 kbp to 400 kbp, 1 kbp to 300 kbp, 1 kbp to 500 kbp, 1 kbp to 600 kbp, 1 kbp to 500 kbp, 1 kbp to 400 kbp, 1 kbp to 300 kbp, 1 kbp to 500 kbp, 1 kbp to 600 kbp, 1 kbp to 500 kbp, 1 kbp to 500 kbp, 1 kbp to 600 kbp, 1 kbp to 700 kbp, 1 kbp to 800 kbp, 1 kbp to 900 kbp, 1 kbp to 900 kbp, 1 kbp to 10 ... kbp to 200 kbp, 1 kbp to 100 kbp, 1 kbp to 50 kbp, 1 kbp to 40 kbp, 1 kbp to 30 kbp, 1 kbp to 20 kbp, 1 kbp to 10 kbp, 1 kbp to 9 kbp, 1 kbp to 8 kbp, 1 kbp to 7 kbp, 1 kbp to 6 kbp, 5 kbp, 1 kbp to 4 kbp, 1 kbp to 3 kbp, or 1 kbp to 2 kbp. The size of the cassette is not particularly limited, but when removing the cassette using a sequence-specific DNA cleavage enzyme, the size may be 500 bp to 100 Mbp, 500 bp to 90 Mbp, 500 bp to 80 Mbp, 500 bp to 70 Mbp, 500 bp to 60 Mbp, 500 bp to 50 Mbp, 500 bp to 40 Mbp, 500 bp to 30 Mbp, 500 bp to 20 Mbp, 500 bp to 10 Mbp, 500 bp to 9 Mbp, 500 bp to 8 Mbp, 500 bp to 7 Mbp, 500 bp to 6 Mbp, 500 bp to 5 Mbp, 500 bp to 4 Mbp, 500 bp to 3 Mbp, 500 bp to 2 Mbp , 500bp to 1Mbp, 1kbp to 900kbp, 1kbp to 800kbp, 1kbp to 700kbp, 1kbp to 600kbp, 1kbp ~500kbp, 1kbp~400kbp, 1kbp~300kbp, 1kbp~200kbp, 1kbp~100kbp, 1kbp~50kbp p, 1 kbp to 40 kbp, 1 kbp to 30 kbp, 1 kbp to 20 kbp, 1 kbp to 10 kbp, 1 kbp to 9 kbp, 1 kbp to 8 kbp, 1 kbp to 7 kbp, 1 kbp to 6 kbp, 5 kbp, 1 kbp to 4 kbp, 1 kbp to 3 kbp, or 1 kbp to 2 kbp. When both ends of the cassette are cleaved with sequence-specific DNA cleavage enzymes, the cassette can be removed regardless of its size.Although the use of a sequence-specific DNA cleavage enzyme is generally more suitable for removing larger regions than the use of a site-specific recombinase, those skilled in the art can appropriately select a site-specific recombinase and a sequence-specific DNA cleavage enzyme depending on the size of the cassette to be removed. Furthermore, those skilled in the art can appropriately determine the size of the cassette depending on the site-specific recombinase and sequence-specific DNA cleavage enzyme used. The removed cassette is expected to disappear from the cell over time, but the removed cassette may be further cleaved or degraded to be inactivated.

[0059] In a second embodiment, inactivation (e.g., removal) of the cassette is useful for evaluating the function of a disrupted endogenous gene that has been complemented by an exogenous gene. Inactivation (e.g., removal) of the cassette can also be performed conditionally, and by inactivating (e.g., removing) the cassette at the required time, it is possible to induce a phenotype resulting from the disruption of the endogenous gene at the timing of inactivation (e.g., removal) of the cassette. As with general conditional knockout, tissue- or organ-specific inactivation (e.g., removal) can also be useful.

[0060] Tissue- or organ-specific inactivation (e.g., removal) of the cassette can be achieved by linking the genes encoding the site-specific recombinase and sequence-specific DNA cleavage enzyme to tissue- or organ-specific regulatory sequences.

[0061] The endogenous gene to be disrupted is not particularly limited and may be any gene. This is because even if the gene is essential for cell survival, as long as it is complemented by the exogenous gene in the cassette, cell survival will be maintained by the exogenous gene. The endogenous gene to be disrupted may be a gene of unknown function. This is because the system of the present disclosure can also be used for analyzing gene function.

[0062] The endogenous gene to be disrupted is not particularly limited, but may be, for example, a gene having a physiological function, and may be selected from the group consisting of genes associated with or involved in cell survival, genes associated with or involved in cell proliferation, genes associated with or involved in the control of cell differentiation, and genes associated with or involved in the physiological function of cells. Here, the control of cell differentiation may be the maintenance of the undifferentiated state of cells (e.g., stem cell state or pluripotency) or the promotion of cell differentiation. Endogenous genes to be disrupted include, but are not limited to, genes related to, involved in, or essential for cell survival, or candidate genes thereof; genes related to, involved in, or essential for cell proliferation, or candidate genes thereof; genes related to, involved in, or essential for cell differentiation, or candidate genes thereof; genes related to, involved in, or essential for maintaining cell stemness, or candidate genes thereof; genes related to, involved in, or essential for cell reprogramming, or candidate genes thereof; genes related to, involved in, or essential for cell metabolism, or candidate genes thereof; genes encoding enzymes in cellular metabolic pathways, or candidate genes thereof; genes related to, involved in, or involved in cell anabolism, or candidate genes thereof; genes encoding enzymes in cellular anabolic pathways, or candidate genes thereof; genes related to, involved in, or involved in cell motility, or candidate genes thereof; genes encoding cellular skeletons, or candidate genes thereof; genes encoding transcription factors, or candidate genes thereof; genes encoding transcription repressors, or candidate genes thereof; genes encoding non-coding RNA (e.g., miRNA), or candidate genes thereof. Disruption of an endogenous gene can be performed in all alleles in which the gene is present. In some embodiments, it is sufficient to disrupt only some of the endogenous genes, rather than all alleles. For example, it is useful when testing whether or not the endogenous gene is haploinsufficient, and when the endogenous gene is haploinsufficient.

[0063] In some embodiments, the endogenous gene in the cell is not disrupted, disrupted but not completely disrupted, or completely disrupted. Completely disrupted means that all gene functions are disrupted, such as the complete deletion of a specific gene. Disrupted but not completely disrupted means that at least one specific gene function is disrupted, but not all gene functions are lost. The introduction of an exogenous gene can complement part or more of the endogenous gene disruption, allowing the removal of the cassette containing the exogenous gene to observe or benefit from the effects of changing the cell from a complemented state to a non-complemented state.

[0064] The exogenous gene that complements the disruption of an endogenous gene is usually the same gene as the endogenous gene. This configuration allows the cassette containing the exogenous gene to be conditionally inactivated (e.g., removed) to create a situation in which the endogenous gene is disrupted, thereby enabling conditional knockout of n or more genes. However, as long as the exogenous gene can functionally complement the disruption of the endogenous gene, the exogenous gene usually does not need to be the same as the endogenous gene.

[0065] In one aspect, there is provided a genetically modified cell, the cell having on its genome a cassette containing n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, and the cell being capable of expressing each of the exogenous genes, endogenous genes corresponding to some or all of the n or more exogenous genes being disrupted, and some or preferably all of the disrupted endogenous genes being complemented by the exogenous genes, the cassette being located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, such that the cassette can be removed by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on the two target sequences, respectively {where n is a natural number greater than or equal to 1.} In a preferred aspect, the endogenous genes and the exogenous genes are the same gene.

[0066] In one aspect, there is provided a genetically modified cell, wherein the cell has on its genome a cassette containing n or more exogenous genes encoding physiologically functional products, the exogenous genes are operably linked to one or more regulatory sequences, and the cell is capable of expressing each of the exogenous genes, endogenous genes corresponding to some or all of the n or more exogenous genes are completely disrupted (preferably deleted), and some or preferably all of the disrupted endogenous genes are complemented by the exogenous genes, and the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, and the cell is configured so that the cassette can be removed by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on the two target sequences, respectively {where n is a natural number greater than or equal to 1.} In a preferred aspect, the endogenous genes and the exogenous genes are the same gene.

[0067] In one aspect, there is provided a genetically modified cell comprising: extrachromosomal DNA; and a cassette on the extrachromosomal DNA comprising n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, such that the cell is capable of expressing each of the exogenous genes; endogenous genes corresponding to some or all of the n or more exogenous genes are disrupted, and some or preferably all of the disrupted endogenous genes are complemented by the exogenous genes; the cassette is located between two target sequences of a sequence-specific DNA cleaving enzyme or two recognition sequences of a site-specific recombinase, such that the cassette can be removed by acting the sequence-specific DNA cleaving enzyme or the site-specific recombinase on the two target sequences, respectively {where n is a natural number greater than or equal to 1.} In a preferred aspect, the endogenous genes and the exogenous genes are the same gene.

[0068] In one aspect, there is provided a genetically modified cell comprising: a cassette on extrachromosomal DNA containing n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, and the cell being capable of expressing each of the exogenous genes; endogenous genes corresponding to some or all of the n or more exogenous genes being completely disrupted (preferably deleted), and some or preferably all of the disrupted endogenous genes being complemented by the exogenous genes; the cassette is located between two target sequences of a sequence-specific DNA cleavage enzyme or two recognition sequences of a site-specific recombinase, and the cell is configured so that the cassette can be removed by acting the sequence-specific DNA cleavage enzyme or the site-specific recombinase on the two target sequences, respectively {where n is a natural number greater than or equal to 1.} In a preferred aspect, the endogenous genes and the exogenous genes are the same gene.

[0069] In one aspect, there is provided a genetically modified cell comprising extrachromosomal DNA and a cassette on the extrachromosomal DNA comprising n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, the cell being capable of expressing each of the exogenous genes, endogenous genes corresponding to some or all of the n or more exogenous genes being disrupted, and some or preferably all of the disrupted endogenous genes being complemented by the exogenous genes, wherein the extrachromosomal DNA can be removed from the cell {wherein n is a natural number greater than or equal to 1.} In a preferred aspect, the endogenous genes and the exogenous genes are the same gene.

[0070] In one aspect, there is provided a genetically modified cell, comprising: a cassette on extrachromosomal DNA containing n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, the cell being capable of expressing each of the exogenous genes; endogenous genes corresponding to some or all of the n or more exogenous genes being completely disrupted (preferably deleted), and some or preferably all of the disrupted endogenous genes being complemented by the exogenous genes; and the extrachromosomal DNA being configured to be removed from the cell {where n is a natural number greater than or equal to 1.} In a preferred aspect, the endogenous genes and the exogenous genes are the same gene.

[0071] Inactivation (e.g., removal) of a cassette from a nucleic acid (e.g., genome) possessed by a cell can be carried out according to conventional methods. For example, a recognition sequence for a site-specific recombinase can be placed at each end of the cassette, and the cassette can be inactivated (e.g., removed) from the nucleic acid by reacting the site-specific recombinase with the recognition sequence. Alternatively, a DNA cleavage enzyme (e.g., sequence-specific DNA cleavage enzyme such as the CRISPR / Cas9 system, TALEN, zinc finger nuclease, meganuclease, etc.; or a site-specific recombinase such as Cre, Flp, Dre, phage integrase, or transposase) that targets each end of the cassette can be used to introduce cleavage at each end of the cassette, thereby inactivating the cassette (e.g., removal from the nucleic acid). By placing a unique sequence recognized by the DNA cleavage enzyme at both ends of the cassette, it is possible to introduce cleavage specifically at both ends of the cassette.

[0072] The cell may have, within the cell or within the DNA, a gene encoding a site-specific recombinase operably linked to a control sequence (preferably an inducible control sequence) and / or a gene encoding a sequence-specific DNA cleavage enzyme.

[0073] The relationship between the regulatory sequence and the exogenous gene in the cassette will be described. The regulatory sequence is operably linked to the exogenous gene so as to drive the exogenous gene in the cassette. In some embodiments, the regulatory sequence is contained within the cassette. In some embodiments, the regulatory sequence is not contained within the cassette but is contained in a region outside the cassette. When the regulatory sequence is contained within the cassette, one regulatory sequence may be operably linked to the exogenous gene so as to drive only one exogenous gene. When the regulatory sequence is contained within the cassette, or when it is not contained within the cassette but is contained in a region outside the cassette, one regulatory sequence may be operably linked to two or more exogenous genes so as to drive these two or more exogenous genes. In some embodiments, two or more exogenous genes are transcribed monocistronically. In some embodiments, two or more exogenous genes may be configured to be transcribed as a single polycistronic RNA. When the exogenous gene encodes a protein, the RNA may have an internal ribosome entry site (IRES) between the genes. When the exogenous genes encode proteins, two or more exogenous genes may be linked by a cleavable peptide. Examples of cleavable peptides include cleavage sites of 2A self-cleaving peptides, furin, etc. Examples of 2A self-cleaving peptides (2A peptides) include, but are not limited to, the T2A sequence, P2A sequence, E2A sequence, and F2A sequence. These sequences are well known to those skilled in the art and can be obtained appropriately by those skilled in the art.

[0074] In one embodiment, the regulatory sequence is not contained within the cassette, but is contained in a region outside the cassette (see, for example, Figure 2). By doing so, the cassette fragment containing the exogenous gene generated after cassette removal is physically separated from the regulatory sequence, which is thought to make it easier to stop gene expression from the exogenous gene.

[0075] In one embodiment, the regulatory sequence is contained within the cassette (see FIG. 4A). Even in this case, the cassette fragment containing the foreign gene resulting from cassette removal does not have a replication origin or the like and is likely to be lost over time. However, if it is desired to destroy the cassette fragment, a target sequence for a sequence-specific DNA cleavage enzyme can be incorporated into the cassette and the target sequence can be cleaved after cassette removal (see FIG. 4B). For example, as shown in FIG. 4B, a target sequence for a sequence-specific DNA cleavage enzyme can be placed between the regulatory sequence and the gene in the cassette, and the regulatory sequence and the gene can be separated by cleavage. Thus, in one embodiment, the regulatory sequence is contained within the cassette, and the cassette can have a target sequence for a sequence-specific DNA cleavage enzyme within it.

[0076] According to the present disclosure, there is provided a composition comprising a cell population comprising the cells of the present disclosure. In some embodiments, the cell population comprising the cells of the present disclosure may be a cell population consisting of the cells of the present disclosure. In some embodiments, the cell population comprising the cells of the present disclosure comprises cloned cells of the present disclosure. In some embodiments, the cell population comprising the cells of the present disclosure comprises isolated cells of the present disclosure.

[0077] According to the present disclosure (e.g., a second aspect), there is provided a gene knockout system comprising the cell of the present disclosure. The gene knockout system of the present disclosure may be a conditional knockout system. According to the present disclosure (e.g., a third aspect), there is provided a silencing release system comprising the cell.

[0078] The gene knockout system of the present disclosure is preferably suitable for the conditional inactivation of two or more genes (e.g., conditional knockout), but is also suitable for the conditional inactivation of a single gene (e.g., conditional knockout). For example, a single gene on the genome can be moved to another location and then disrupted. For example, by introducing a negative selection marker into the cassette, knockout cells can be identified or determined using the deletion of the negative selection marker associated with the deletion of the cassette as an indicator. While a selection marker indicates that cells that select for that selection marker have been successfully treated, the presence of a negative selection marker indicates that the treatment has failed. Examples of negative selection markers include suicide genes (e.g., thymidine kinases such as herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9), fluorescent protein genes (e.g., green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene, etc.), luminescent enzyme genes (e.g., luciferase gene), chromogenic enzyme genes (e.g., β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene, etc.). When the negative selection marker gene is a gene that negatively affects cell viability (e.g., a suicide gene), the negative selection marker gene can be operably linked to an inducible promoter. By operably linking the negative selection marker gene to an inducible promoter, the negative selection marker gene can be expressed only when it is desired to eliminate cells carrying the negative selection marker gene. When the negative selection marker gene has little negative impact on cell viability, such as when it is an optically detectable marker gene (visible marker gene) that emits fluorescence, luminescence, or color, it may be constitutively expressed. The above-mentioned mechanism for conditional knockout of a single gene can be beneficial both in vivo and in vitro.For example, in vivo, cells from which the cassette has been removed in a cell population such as a tissue or organ can be determined by using the loss of a negative selection marker (e.g., a visual marker, particularly a fluorescent dye or a chromogenic dye, can be used as an indicator). In vitro, by using a suicide gene or a toxin as a negative selection marker, cells from which the cassette has not been removed can be killed, and only cells from which the cassette has been removed can be enriched. This method also has the advantage of allowing conditional knockout without being affected by unexpected influences due to the gene's natural genomic location.

[0079] The conditional gene inactivation system (e.g., conditional knockout system) of the present disclosure can also be advantageous in that it can conditionally inactivate (e.g., knockout) multiple genes scattered across a genome all at once, thereby providing a useful inactivation (e.g., knockout) system that can knockout multiple genes all at once when the cassette is removed.

[0080] <Method for producing cells of the present disclosure> The cells of the present disclosure can be produced, for example, as described in the Examples below, without being particularly limited thereto. The cells of the present disclosure can also be produced by the method described in detail below.

[0081] According to the present disclosure, there is provided a method for producing a cell of the present disclosure. The method includes providing a cell (a starting cell). The starting cell can be, for example, a cell that has an endogenous gene to be disrupted.

[0082] The starting cells can be any of the cells described above.

[0083] In some embodiments, the starting cell may not have a disrupted endogenous gene.

[0084] In some embodiments, one or more endogenous genes may be disrupted in the starting cells, provided that disruption does not significantly interfere with the implementation of the present disclosure. Preferably, the cells are capable of surviving or growing in culture under appropriate conditions. These endogenous genes may be disrupted but not completely disrupted, or may be completely disrupted. Completely disrupted means that all gene functions are disrupted, such as by complete deletion of the gene. Disrupted but not completely disrupted means that at least one gene function is disrupted, but not all gene functions are lost. For example, it will be understood that even if the starting cells already have a disrupted endogenous gene, this disruption is acceptable as long as it does not interfere with subsequent cell modification steps. In the subsequent modification steps, this endogenous gene does not need to be complemented with an exogenous gene, but if it is complemented with an exogenous gene, it can be used to test the function of the exogenous gene.

[0085] The method also includes inserting into the cell (e.g., into a transcriptionally active locus of the cell) a cassette containing n or more exogenous genes. The present disclosure provides cells containing such cassettes, and methods for making the cells.

[0086] In the second and fourth aspects, the method further comprises disrupting the endogenous genes. The number of endogenous genes to be disrupted is one or more, preferably two or more. Disruption of an endogenous gene may also have a limited effect on the cell, since its function is complemented by an exogenous gene provided in the cassette. For the function to be complemented by the exogenous gene, the endogenous gene and the exogenous gene are preferably identical, but do not need to have completely identical sequences. According to the present disclosure, a method for simultaneously inactivating (e.g., knocking out) one or more, preferably two or more, genes is provided. The method first comprises providing a cell having the cassette described above.

[0087] In the second, third, and fourth aspects, the method further includes inactivating (e.g., removing) the cassette. This allows n or more exogenous genes contained in the cassette to be inactivated (e.g., removed) from the cell at once, thereby enabling simultaneous inactivation of n or more exogenous genes. Therefore, in the second and fourth aspects, by using this method, the effect of endogenous gene disruption can be significantly achieved by inactivating (e.g., removing) the cassette, rather than by timing the disruption of the endogenous gene. This enables conditional inactivation (e.g., knockout) of endogenous genes (preferably, simultaneous conditional inactivation (e.g., knockout) of two or more genes). In the method of the present disclosure, if the method is designed to allow inactivation (e.g., removal) of the entire cassette, all genes contained in the cassette can be inactivated (e.g., removed) at once, achieving more complete simultaneous conditional inactivation (e.g., knockout) of multiple genes. In addition, in the third and fourth aspects, by removing the cassette, the knockdown nucleic acid provided in the cassette is inactivated (e.g., removed), and its silencing effect can be terminated.

[0088] In producing the cells of the present disclosure, it is possible to produce cells (intermediate cells) into which a landing pad (LP; an exogenous target region, e.g., see FIG. 1(i)) has been inserted, which allows for the introduction of an exogenous gene into a region on the cell's genome or into the extrachromosomal DNA of the host, as described below. The landing pad functions as a platform for integrating a cassette and may contain various factors other than the cassette. This intermediate cell is suitable for producing any of the cells of the first to fourth aspects.

[0089] For example, the landing pad may include a regulatory sequence.

[0090] For example, the landing pad may contain one recognition sequence for a site-specific recombinase or one target sequence for a sequence-specific DNA cleavage enzyme (i.e., two in total), one outside the site where the cassette is inserted.

[0091] For example, the landing pad may contain an insulator sequence near each end. An insulator sequence is a sequence that blocks or alleviates the influence of the adjacent chromosomal environment and ensures or enhances the independence of transcriptional regulation of the DNA flanked by that region. An insulator is defined by its enhancer-blocking effect (the effect of blocking the effect of the enhancer on promoter activity by being inserted between the enhancer and the promoter) and its suppression of position effects (the effect of flanking the transgene with insulators to prevent the expression of the transgene from being affected by the inserted position).

[0092] The relationship between the control sequence and the cassette is as described above.

[0093] In one embodiment, as shown in Figure 1C, the landing pad may contain recognition sequences for two site-specific recombinases or target sequences for sequence-specific DNA cleavage enzymes, with a region for inserting a cassette located between the two recognition sequences and the target sequence. In one embodiment, insulator sequences may be located before and after the two recognition sequences for site-specific recombinases or target sequences for sequence-specific DNA cleavage enzymes, flanking them. In one embodiment, the landing pad may contain one or more regulatory sequences near the region for inserting a cassette so as to operably link the foreign gene in the cassette.

[0094] In one aspect, the region in the landing pad into which the cassette is inserted can contain a target sequence for a sequence-specific DNA cleavage enzyme. This is because the cassette can be inserted into the target sequence by cleaving the target sequence in the presence of donor DNA containing the cassette. Here, the donor DNA has an upstream homology arm capable of homologous recombination with a region upstream of the target sequence in the landing pad and a downstream homology arm capable of homologous recombination with a region downstream of the target sequence, and the cassette is located in a region between the upstream and downstream homology arms. By using such donor DNA, the region between the upstream and downstream homology arms of the donor DNA (i.e., the region containing the cassette) is inserted between the upstream and downstream regions.

[0095] In one embodiment, as shown in Figure 1B, the region in the landing pad into which the cassette is inserted does not need to contain the target sequence of the sequence-specific DNA cleavage enzyme. A region in the landing pad to which insertion is desired (target region) can be envisioned, which has an upstream homology arm capable of homologous recombination with the upstream region of the target sequence in the landing pad and a downstream homology arm capable of homologous recombination with the downstream region of the target sequence, with the cassette located in the region between the upstream and downstream homology arms. By using such donor DNA, the region between the upstream and downstream homology arms of the donor DNA (i.e., the region containing the cassette) will be inserted between the upstream and downstream regions.

[0096] Of course, the cells of the present disclosure can be produced without using a landing pad. Donor DNA can be incorporated into DNA, for example, by the method disclosed in International Publication No. 2021 / 206054. The advantage of using a landing pad is that by providing components common to modified cells on the landing pad, the number of components to be included in the donor DNA to be inserted into the modified cell can be reduced. Alternatively, by introducing a unique cleavable sequence (e.g., a meganuclease recognition sequence or a genome editing enzyme target sequence) into the DNA (particularly the genome) onto the landing pad, the risk of off-target genomic cleavage at a different site can be reduced during exogenous gene introduction.

[0097] Cassettes can be prepared by conventional gene recombination techniques or DNA synthesis techniques. For example, a technique is known and widely used in which multiple DNA fragments are ligated to obtain a single DNA fragment by synthesizing DNA encoding the genes carrying the cassette, making the ends of the DNA sticky, and then ligating them to other DNA. By making full use of such techniques, cassettes carrying n or more genes can be easily obtained.

[0098] <Culture Method of Cells of the Present Disclosure> The present disclosure provides a method for culturing cells of the present disclosure. This method includes providing cells of the present disclosure (having a cassette containing n or more exogenous genes). The method also includes culturing the cells of the present disclosure under conditions suitable for culturing, inactivating the cassette (e.g., deleting it from the DNA (e.g., removing it from the genome or extrachromosomal DNA)) by applying a site-specific recombinase to the site-specific recombinase recognition sequences flanking the cassette, and then further culturing the cells. The site-specific recombinase may be expressibly integrated into extrachromosomal DNA, or outside or within the cassette in the genome. This configuration allows the cassette to be inactivated (e.g., removed) by inducing expression of the enzyme. Induction of expression of the enzyme can be achieved by those skilled in the art, for example, using an inducible promoter. Induction of expression of the enzyme can also be achieved by those skilled in the art, for example, using a tissue- or organ-specific promoter.

[0099] <Examples of Use of the Cells of the Present Disclosure> Further examples of use of the cells of the present disclosure are as described in the Examples below.

[0100] Example 1: Construction of a new knockout system We devise a new knockout system that differs from conventional conditional knockout techniques. This new knockout system can efficiently knock out multiple genes scattered across the genome in a single operation.

[0101] To construct this knockout system, cells can be prepared by inserting a landing pad (LP; exogenous target region, e.g., see Figure 1(i)) into a region on the genome of the cell, which allows the introduction of an exogenous gene. It is desirable to insert the landing pad at a location on the genome that does not affect the expression of existing genes. For example, the landing pad can be inserted into a transcriptionally active gene locus or a safe harbor region of the genome. The cells thus obtained, which have a landing pad inserted, can be advantageously used in the subsequent construction of a knockout system. Naturally, cells without a landing pad can also be used in the subsequent construction of a knockout system.

[0102] Next, a fragment (cassette) containing one or more genes to be knocked out is introduced into any target region or landing pad on the genome (see, for example, Figure 1(ii)). The cassette may contain only one gene, but preferably contains two or more genes. Figure 1(ii) shows an example of inserting a cassette containing four genes. The genes contained in this cassette are preferably operably linked to regulatory sequences, so that their expression can be induced. This is because, even if an endogenous gene is disrupted in a later step, the phenotype caused by the disruption can be rescued, or a rescue attempt can be made, by expressing the genes contained in the cassette.

[0103] Furthermore, endogenous genes corresponding to the genes contained in the cassette are each disrupted. Gene disruption can be achieved by known methods or commonly used techniques, such as disrupting the coding region and the regulatory sequence. The number of endogenous genes to be disrupted is one or more, preferably two or more (see, for example, Figure 1 (iii)). Genes corresponding to the endogenous genes to be disrupted are incorporated into the cassette in a manner that allows for inducible expression, and the phenotype resulting from the disruption of the endogenous genes can be rescued.

[0104] The cassette can be removed at the desired timing. For example, FIG. 1 shows that the cassette is located between loxP sequences and can be removed by the action of Cre recombinase. The cassette contains multiple genes, allowing these to be knocked out all at once. Thus, the knockout system of the present disclosure is useful for conditionally knocking out one or more genes.

[0105] Example 2: Application of the Knockout System (1) Gene Functional Analysis The knockout system of the present disclosure can be used for gene functional analysis. More specifically, because it can conditionally knock out one or more genes, preferably multiple genes, the knockout system of the present disclosure can be advantageously used when examining the function of one or more genes, preferably a combination of multiple genes. Furthermore, even if the endogenous gene to be disrupted includes a gene essential for cell survival or proliferation, the cell has a gene corresponding to that gene that can be expressed prior to cassette removal, so the endogenous gene is not affected until the cassette is removed. By removing the cassette at the desired timing, knockout can be performed at the desired timing, and the phenotype resulting from the disruption can be observed, allowing the function of the disrupted gene to be estimated. It is acceptable for the cassette to contain additional genes in addition to the endogenous gene to be disrupted. This is because the function of the additional genes can be utilized, or if the function is unknown, its function can be investigated. Furthermore, the cassette does not necessarily need to contain genes corresponding to all of the endogenous genes to be disrupted. If supplementation by a cassette is not necessary, there is no need to introduce the gene into the cassette to induce gene expression.

[0106] Since genes can be supplemented using a cassette, there are no particular limitations on the endogenous gene to be disrupted, and the endogenous gene to be disrupted may include any existing gene. For example, a gene essential for cell survival or its candidate gene, a gene essential for cell proliferation or its candidate gene, a gene essential for cell differentiation or its candidate gene, a gene essential for maintaining cell stemness or its candidate gene, a gene essential for cell initialization or its candidate gene, a gene encoding an enzyme in a cell's metabolic pathway or its candidate gene, a gene encoding an enzyme in a cell's anabolic pathway or its candidate gene, a gene encoding a cell skeleton or its candidate gene, a gene encoding a transcription factor or its candidate gene, a gene encoding a transcription repressor or its candidate gene, a gene encoding a non-coding RNA or its candidate gene, etc. The gene to be supplemented in the cassette may be a gene with a known function. This is because it is possible to evaluate whether the disruption of the endogenous gene can be rescued by its known function, and it may also be possible to elucidate unknown gene functions.

[0107] (2) Cellular Substance Production Cell-based production systems for useful substances are used industrially to advantage. In some cases, disrupting an enzyme, such as a metabolic enzyme, alters the metabolic pathway, resulting in the production of a desired substance in larger quantities than before disruption. However, such enzyme disruption can sometimes have a negative effect on cell growth. Therefore, during the cell growth phase (e.g., logarithmic growth phase), the cells are cultured in an environment where the enzyme is present, and after cell growth, the cassette is removed and then substance production is initiated. This can be used to increase the yield of the desired substance while preventing any effects on cell growth. During substance production, nutrients, including a sufficient carbon source, can be supplied to the cells under appropriate conditions.

[0108] In substance production using cells, a gene required for cell growth can be disrupted and cells containing a cassette carrying the corresponding gene can be used. During the cell growth phase (e.g., logarithmic growth phase), the cells are cultured in an environment in which the gene functions. After cell growth, the cassette is removed and then substance production is initiated. This can increase the yield of a desired substance while preventing any effects on cell growth. In particular, cells allocate available energy between cell growth and substance production. By reducing the amount of energy and / or carbon source allocated to cell growth, it is expected that the amount of energy and / or carbon source available for substance production can be increased. This can improve the efficiency of substance production by cells. During substance production, nutrients containing a sufficient carbon source can be supplied to the cells under appropriate conditions. In this embodiment, it is desirable for the cells to survive and proliferate before cassette removal, and it is acceptable for the cells to at least survive but not proliferate after cassette removal. For example, in one embodiment, the cells exhibit reduced proliferation capacity after cassette removal compared to before cassette removal. In one aspect, after cassette removal, the cells do not exhibit substantial proliferation capacity, e.g., do not increase cell number by 2-fold or more, 1.5-fold or more, 1.4-fold or more, 1.3-fold or more, 1.2-fold or more, or 1.1-fold or more.

[0109] More specific examples include the following. In this specific example, the cell is a bacterium (preferably a hydrogen bacterium). The bacterium has a cassette containing multiple exogenous genes with physiological functions. The exogenous genes include one or more, particularly two or more, endogenous genes selected from the group consisting of enzymes in the peptidoglycan synthesis pathway (e.g., glmS and glmU), enzymes in the fatty acid synthesis pathway (e.g., fabH and fabD), leucine synthase (e.g., leuA1 and leuA2), enzymes in the pathway from acetyl-CoA to the TCA cycle (e.g., cisY), and hbdH. The exogenous genes can complement the disrupted endogenous genes, and preferably include at least exogenous genes corresponding to all of the disrupted endogenous genes. Such a bacterium can be grown under conditions suitable for culture in the presence of the cassette. Once the bacterium has grown sufficiently, the cassette can be inactivated (e.g., removed). It is believed that further culturing the bacteria after cassette removal will enable the bacteria to efficiently synthesize polyhydroxyalkanes (PHAs), because inactivation (particularly removal) of the enzymes described above is expected to stop production pathways other than PHA, allowing carbon and energy sources to be used efficiently for PHA synthesis.

[0110] (3) Maintenance of Cell Undifferentiated State or Differentiation Induction Maintenance of cells in an undifferentiated state is important from the perspective of quality control of stem cells (particularly pluripotent cells or multipotent stem cells). On the other hand, when maintaining an undifferentiated state is no longer necessary, it may be desirable to turn off gene expression for maintaining the undifferentiated state. In such cases, by disrupting the cell's endogenous gene and supplementing it with the corresponding gene on a cassette, the undifferentiated state can be maintained by the supplemented gene function, and the cassette can be removed when maintaining the undifferentiated state is no longer necessary. An example of a time when maintaining an undifferentiated state is when inducing differentiation of cells. Examples of genes that maintain cells in an undifferentiated state include Oct4. Other examples of genes that maintain cells in an undifferentiated state include klf4, c-mic, sox2, nanog, lin28, etc. Therefore, by introducing one or more of these into a cassette, making them expressible, and then disrupting one or more endogenous genes, cells can be obtained that are beneficial for maintaining an undifferentiated state, but can also be differentiated by inactivating (e.g., removing) the cassette when differentiation is desired to promote differentiation induction. In particular, sox2 plays an important role in maintaining the pluripotency of pluripotent stem cells and in their differentiation into ectoderm. Inactivating (e.g., removing) sox2 is thought to promote the differentiation of cells into mesoderm and endoderm while eliminating pluripotency. This approach is thought to be useful, for example, in differentiating pluripotent stem cells into pancreatic islets or pancreatic beta cells.

[0111] Maintenance of an undifferentiated state may be beneficial not only for pluripotent stem cells, but also for the maintenance of other types of tissue stem and progenitor cells.

[0112] It may also be beneficial in inducing cell differentiation. This is because, although not limited to, a gene required in one step of the cell differentiation induction process may become unnecessary in the subsequent differentiation step. In such cases, although not limited to, the corresponding endogenous gene may be disrupted, and the corresponding gene may be replenished into the cassette. After completing one differentiation step, the cassette may be removed and the next differentiation step may be performed. By removing the cassette and the gene contained therein, it is expected that unexpected cell differentiation induction due to expression of the gene may be prevented. When a cell can differentiate into both cell A and cell B, removing a gene that promotes differentiation into cell B can promote differentiation into cell A. It is also possible to further promote differentiation into cell A by introducing a knockdown nucleic acid for a gene that promotes differentiation into cell A into the cassette and then removing the cassette when promoting differentiation into cell A.

Claims

1. A genetically modified cell, comprising a cassette containing n or more exogenous genes encoding physiologically functional products, the exogenous genes being operably linked to one or more regulatory sequences, the cell being capable of expressing each of the exogenous genes, endogenous genes corresponding to some or all of the n or more exogenous genes being disrupted, and the cassette being configured to be conditionally inactivated {wherein n is a natural number greater than or equal to 1.}.

2. The cell described in claim 1, wherein n is a natural number of 2 or more.

3. The cell of claim 1 or 2, wherein the disrupted endogenous gene comprises a gene associated with or involved in cell survival or proliferation, or a candidate gene thereof.

4. The cell according to claim 1 or 2, wherein the disrupted endogenous gene includes a gene related to or involved in the control of cell differentiation or a candidate gene thereof.

5. The cell of claim 1 or 2, wherein the disrupted endogenous gene comprises a gene associated with or involved in the physiological function of the cell, or a candidate gene thereof.

6. A cell according to any one of claims 1 to 5, wherein the control sequence is located outside the region between (including) the two target sequences or the two recognition sequences.

7. The cell according to any one of claims 1 to 6, wherein the cassette further comprises a gene encoding a knockdown nucleic acid.

8. A composition for use in producing the cell according to any one of claims 1 to 7, comprising a cell having an exogenous target region for integrating the cassette, the exogenous target region comprising a target sequence for a sequence-specific DNA cleaving enzyme.

9. The composition according to claim 8, wherein the exogenous target region comprises two alleles, each of which has a distinguishably different positive selection marker, and each of which further has a negative selection marker (provided that, when the positive selection marker also serves as a negative selection marker, the negative marker may not be present).

10. A method comprising: (A) culturing a cell according to any one of claims 1 to 7 under suitable conditions; (B) inactivating the cassette after culturing to obtain a cell in which the cassette has been inactivated; and (C) further culturing the cell in which the cassette has been inactivated under suitable conditions.

11. The method according to claim 10, wherein the disrupted gene is a gene associated with or involved in cell survival or proliferation, or a candidate gene thereof, and step (B) is carried out after the cells are proliferated in step (A).

12. The method according to claim 10, wherein the disrupted gene is a gene related to or involved in the control of cell differentiation or a candidate gene thereof, and step (B) is carried out after the cells are differentiated in step (A).

13. The method according to claim 10, wherein the disrupted gene is a gene related to or involved in the control of cell differentiation or a candidate gene thereof, and the method comprises, after carrying out step (B), subjecting the cells to a differentiation treatment in the culture of step (C).

14. The method according to claim 10, wherein the disrupted gene is a gene related to or involved in the physiological function of a cell, or a candidate gene thereof, and step (B) is carried out after the cells are grown in step (A).

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