MHC gene cluster–deficient animal

Abstract

The present invention provides a production method for cells that have, among chromosomes of a single non-human mammal (non-human mammal 1), a chromosome that includes the major histocompatibility complex (MHC) gene cluster (MHC gene cluster 2) of a mammal (mammal 2) that is not the non-human mammal 1 and a chromosome from which all or a portion of the MHC gene cluster (MHC gene cluster 1) from the non-human mammal 1 has been destroyed or deleted. The production method includes a step for destroying or deleting all or a portion of the MHC gene cluster 1 in cells that are heterozygous for a chromosome obtained by replacing all or a portion of the MHC gene cluster of one wild-type chromosome of a pair of chromosomes of the non-human mammal 1 with the MHC gene cluster of the mammal 2.

Description

MHC gene cluster deficient animals

[0001] The present invention relates to a chromosome in which the MHC gene cluster has been disrupted or deleted, a cell having said chromosome, and the like.

[0002] The major histocompatibility complex (MHC) is a membrane protein that functions to present intracellular antigens to T cells. MHC genes include MHC class I, which presents intracellular antigens to CD8-positive T cells; MHC class II, which presents extracellular antigens to CD4-positive T cells; and non-classical MHC, which includes molecules with physiological functions other than antigen presentation, such as regulating antigen peptide presentation and NK cell activity. In mice, these MHC genes are located in the MHC region on chromosome 17, and although the number varies depending on the strain, it is believed that there are 39 types in the C57BL / 6 strain (Non-Patent Document 1).

[0003] When using mice as model animals for drug development using T cell receptors (TCRs), it is necessary to express human MHC in individuals that do not express mouse MHC in order to reproduce human immune responses. However, because there are many mouse MHC gene clusters, creating mice with all MHC genes deleted is technically extremely difficult, even using genome editing technology, and has not yet been produced. For this reason, mice with deleted classical MHC genes, which play a major role in antigen presentation, or mice with knockout of the β2 macroglobulin (B2M) gene, which forms a complex with MHC class I, are currently used as alternatives to MHC class I-deficient mice. However, in the former, non-classical MHC remains expressed, and in the latter, the mouse MHC genes are not disrupted, so human MHC must be expressed as a fusion protein with B2M, making them not ideal model systems.

[0004] Shiina, T. , Blancher, A. , Inoko, H. , and Kulski, J. K. (2017) Comparative genomics of the human, maque and mouse major histocompatibility complex Immunology 150, 127-138 10.1111 / imm. 12624

[0005] For these reasons, there has been a desire to create animals lacking MHC genes. The creation of MHC gene-deficient mice could solve these problems. Furthermore, the use of MHC gene-deficient mice would also enable specific analysis of the functions of individual MHC proteins.

[0006] As a result of intensive research to solve the above-mentioned problems, the present inventors have succeeded in producing cells having alleles in which the mouse MHC gene cluster has been disrupted or deleted, thereby completing the present invention. Specifically, the present invention is as follows: [1] A chromosome of a non-human mammal in which all or part of the major histocompatibility complex (MHC) gene cluster has been deleted or disrupted. [2] The chromosome according to [1], wherein the non-human mammal is a rodent. [3] The chromosome according to [2], wherein the rodent is a mouse. [4] A cell containing the chromosome according to [1], in a homozygous or heterozygous form. [5] The cell according to [4], which is an embryonic stem cell. [6] A non-human mammal having the chromosome according to [1]. [7] A method for producing a cell having a chromosome from a non-human mammal (non-human mammal 1) in which all or part of the major histocompatibility complex (MHC) gene cluster of one wild-type chromosome of a pair of chromosomes from the non-human mammal has been replaced with the MHC gene cluster of a mammal (mammal 2) other than the non-human mammal 1, the method comprising a step of disrupting or deleting all or part of the MHC gene cluster derived from the non-human mammal 1 (MHC gene cluster 1). [8] The method described in [7], wherein the cell is an embryonic stem cell. [9] The method described in [7], wherein the step of disrupting or deleting MHC gene cluster 1 is performed by genome editing using CRISPR / Cas9.

[10] The method described in [7], wherein the non-human mammal 1 is a rodent.

[11] The method according to claim

[10] , wherein the rodent is a mouse.

[12] The step of disrupting or deleting all or a part of MHC gene group 1 may be carried out by disrupting or deleting a d1 gene group consisting of H2-M10.2, H2-M10.1, H2-M10.3, H2-M10.4, H2-M11, H2-M9, H2-M1, H2-M10.5, and H2-M10.6 genes; a d2 gene group consisting of H2-D1, H2-Q1, H2-Q2, H2-Q4, H2-Q5, H2-Q6, H2-Q7, and H2-Q10 genes; a d3 gene group consisting of H2-T24, H2-T23, H2-T22, GM11127, H2-Bl, H2-T10, GM7030, GM8909, and H2-T3 genes; The method according to

[11] , comprising a step of disrupting or deleting all or a part of the d1 gene cluster, d2 gene cluster, d3 gene cluster, d4 gene cluster, d5 gene cluster, and d6 gene cluster from a cell having a mouse MHC gene cluster consisting of a d4 gene cluster consisting of H2-Ob, H2-Ab1, H2-Aa, H2-Eb1, and H2-Eb2 genes, a d5 gene cluster consisting of H2-DMa, H2-DMb2, and H2-DMb1 genes, and a d6 gene cluster consisting of H2-K1, H2-Oa, H2-M5, H2-M3, and H2-M2 genes, by genome editing technology using CRISPR / Cas9.

[13] The method according to

[12] , wherein the genome editing vector used in the genome editing technology using CRISPR / Cas9 is a combination of vectors consisting of gRNAs having the nucleotide sequences set forth in SEQ ID NOs: 53 to 67.

[14] A method for producing a non-human mammal 1 having a chromosome in which all or part of the MHC gene group 1 has been disrupted or deleted, the method comprising the steps of: using cells obtained by the method described in [7] to produce a non-human mammal 1 having, of a pair of chromosomes of the non-human mammal 1, a chromosome carrying MHC gene group 2 of the mammal 2 and a chromosome in which all or part of the MHC gene group 1 has been disrupted or deleted, and mating the non-human mammal 1.

[15] A method for producing the cells, the method comprising the step of recovering, from the non-human mammal 1 produced by the method described in

[14] , cells having a chromosome in which all or part of the MHC gene group 1 has been disrupted or deleted.

[16] A method for producing the chromosome, comprising a step of recovering a chromosome in which MHC gene group 1 is disrupted or deleted from a non-human mammal 1 produced by the method of

[14] or a cell produced by the method of claim 15.

[17] A genome editing vector used in the genome editing technique using CRISPR / Cas9 according to

[13] , comprising a combination of vectors consisting of gRNAs having the nucleotide sequences set forth in SEQ ID NOs: 53 to 67.

[0007] The present invention provides MHC gene cluster deficient animals.

[0008] [Figure 1] A schematic diagram showing recombination for replacing non-human MHC with human MHC in the present invention. The diagram is a schematic diagram when the non-human animal is a mouse. [Figure 2] A schematic diagram showing the MHC region in a human chromosome. [Figure 3] A diagram showing the region targeted when using a genome editing targeting vector. [Figure 4] A schematic diagram of a method for producing an MHC region-humanized mouse. [Figure 5] A diagram showing the structure of the targeting vector. (A) Structure of TV3R-dHLA. (B) Structure of TV4-dH2. (C) Structure of TV-Tl1 / 2-BS. The genomic positions of the homology arms used in the targeting vector are as shown in the diagram. [Figure 6] A diagram showing the isolation of an A9-MRC5 fusion cell clone carrying human chromosome 6. (A) Genomic DNA was prepared from isolated A9-MRC5 fusion cell clones (hChA9#1, 5, 13, 17) and analyzed by genomic PCR for the presence of sequences on human chromosome 6. (B) FISH analysis confirmed the presence of human chromosome 6 in hChA9#5. Red: human chromosome 6 centromere-specific probe. [Figure 7] TV3R targeting. (A) TV3R-dHLA targeting was performed on A9-MRC5 fusion cell clones, and hygromycin-resistant clones were isolated and analyzed by genomic PCR for recombination. * indicates the TV3RhChA9#5-45(128) clone. (B) TV3RhChA9#5-45(128) was recloned and analyzed by genomic PCR. (C) TV3RhChA9#5-45a was analyzed by FISH. Red: human chromosome 6 centromere-specific probe. [Figure 8] TV4 targeting diagram. (A) TV4-dH2 targeting analyzed by genomic PCR. * indicates the TV4RENKA#17 clone. (B) DAPI staining confirmed that TV4RENKA#17 had 40 chromosomes. [Figure 9] TV3-4RENKA clone isolation diagram. FISH analysis was performed on TV3-4RENKA#17 / 5-45a-3 cells introduced with modified human chromosome 6. The arrowhead indicates the introduced modified human chromosome 6. The chromosome number was confirmed to be 41.Red: Mouse Cot-1 (probe for mouse genomic sequence), Purple: RP11-54H13 (probe within the human MHC region). [Figure 10] Isolation of the TV3-4RENKATl3 / 4 clone. (A) Cre / loxP-mediated recombination translocation. (B) Confirmation of the translocation by genomic PCR. * denotes TV3-4RENKATl3 / 4#17-1. (C) TV3-4RENKATl3 / 4#17-1 was subjected to FISH analysis. The numbers in the FISH image correspond to the translocated chromosome numbers in the schematic diagram. Red: Human Cot-1 (probe for human genomic sequence), Green: Mouse Cot-1 (probe for mouse genomic sequence). (D) TV3-4RENKATl3 / 4#17-1 was subjected to FISH analysis. The numbers in the FISH image correspond to the translocated chromosome numbers in the schematic diagram. Red: RP11-54H13 (probe within the human MHC region), green: RP23-147G23 (probe on the centromeric side of the MHC region on mouse chromosome 17), purple: RP23-119G24 (probe on the telomeric side of the MHC region on mouse chromosome 17). [Figure 11] Isolation of the TV3-4RENKAT1b clone. (A) Chromosomal translocation analyzed by genomic PCR. * indicates the TV3-4RENKAT1b#38 clone. (B) TV3-4RENKAT1b#38 was analyzed by FISH. The numbers in the FISH image correspond to the numbers of the translocated chromosomes in the schematic diagram. Red: RP23-147G23 (probe located closer to the centromere than the MHC region of mouse chromosome 17), green: human Cot-1 (probe for the human genome sequence), purple: RP23-306H20 (probe located closer to the telomere than the MHC region of mouse chromosome 17). [Figure 12] Isolation of the TV3-4RENKATlbdh6 clone. (A) Sorting of TV3-4RENKATlbdh6. (B) FISH analysis of TV3-4RENKATlb#38dh6#3. The numbers in the FISH image correspond to the numbers of the translocated chromosomes in the schematic diagram. The number of chromosomes was confirmed to be 40. Red: RP23-147G23 (probe located on the centromeric side of the MHC region on mouse chromosome 17), green: human Cot-1 (probe for the human genome sequence), purple: RP23-361C19 (probe located on the telomeric side of the MHC region on mouse chromosome 17). (C) Results of whole-genome sequencing analysis of the HLA haplotype of TV3-4RENKATlbdh6#3.[Figure 13] Figure showing MHC-humanized chimeric mice. (A) Appearance of a chimeric mouse derived from TV3-4RENKATlbdh6#3. (B) Structure of MHC-humanized mouse chromosome 17. An EGFP expression cassette has been inserted into the distal side of the MHC region. (C) Flow cytometry analysis of EGFP expression in testicular cells of a chimeric mouse derived from TV3-4RENKATlbdh6#3. [Figure 14] Figure showing the generation of MHC-humanized (heterozygous) mice. (A) MHC-humanized (heterozygous) cells have MHC-humanized mouse chromosome 17 with an EGFP expression cassette inserted into the distal side of the MHC region. (B) Individual born by insemination using testicular cells of a chimeric mouse derived from TV3-4RENKATlbdh6#3. Left: Bright-field image. Right: EGFP fluorescence detected. (C) Genomes were prepared from offspring obtained by ICSI and analyzed by PCR for the presence of sequences within the human MHC region. Individual numbers 7 and 12 were EGFP-negative offspring. [Figure 15] Schematic diagram showing the generation of MHC-humanized (heterozygous) mice from XO ES cells. (A) Chimeric mice generated from the TV3-4XOTlbdh6#11-1-4-57-3 clone. Arrowheads indicate 100% chimeric mice. (B) Offspring of an XO chimeric mouse (female) and a C57BL / 6 (male). Black individuals are not born unless XO ES cells are transmitted through the germline. (C) Genome typing method targeting the DAXX locus within the MHC region. Three primers, 1 to 3, are used to determine whether the MHC region is human or mouse-derived. The red arrows indicate the positions of primers designed to match the sequences between human and mouse. Black indicates human- or mouse-specific primers. (D) Example of genotyping of offspring obtained by mating an XO chimeric mouse (female) with a C57BL / 6 (male). W: wild-type mouse; H: MHC humanized (heterozygous) mouse. [Figure 16] Results of analysis of human MHC protein expression in MHC humanized (heterozygous) mice. (A) Expression of human MHC protein in splenocytes was analyzed by Western blotting. Wt, splenocytes derived from wild-type mice; Het, splenocytes derived from MHC humanized (heterozygous) mice. (B) Surface expression of human MHC protein in splenocytes was analyzed by flow cytometry.Wt, wild-type mouse-derived spleen cells; Het, MHC-humanized (heterozygous) mouse-derived spleen cells. [Figure 17] Schematic diagram of the generation of mouse MHC gene-deficient alleles using MHC-humanized (heterozygous) ES cells. [Figure 18] Diagram showing the structure of the MHC region of MHC-humanized (heterozygous) ES cells and the distribution of mouse MHC genes. The upper row shows MHC-humanized alleles, and the lower row shows wild-type alleles. Mouse MHC genes were classified into six gene groups, d1 to d6. [Figure 19] Diagram showing the procedure for generating mouse MHC-deficient alleles. [Figure 20] Diagram showing the isolation of d1 clones using CRISPR / Cas9. (A) Schematic diagram of the d1 cluster. CR indicates the CRISPR / Cas9 target site. Arrows indicate the positions of primers used to confirm deletion. (B) Isolated clones were analyzed by genomic PCR. Arrowheads indicate the positions of expected PCR amplification products. (C) FISH analysis of clones that were positive for genomic PCR. Red indicates mouse chromosome 17, purple indicates the human MHC region, and green indicates the staining image using a probe for the d1 cluster. [Figure 21] Diagram showing the isolation of d2 clones using CRISPR / Cas9. (A) Schematic diagram of the d2 cluster. CR indicates the target site of CRISPR / Cas9. Arrows indicate the positions of primers used to confirm deletion. (B) The isolated clones were analyzed by genomic PCR. Arrowheads indicate the expected positions of PCR amplification products. (C) FISH analysis of clones that were positive for genomic PCR. Red indicates mouse chromosome 17, purple indicates the human MHC region, and green indicates the staining image using a probe for the d2 cluster. [Figure 22] Diagram showing the isolation of d3 clones using CRISPR / Cas9. (A) Schematic diagram of the d3 cluster. CR indicates the target site of CRISPR / Cas9. The arrows indicate the positions of the primers used to confirm the deletion. (B) The isolated clones were analyzed by genomic PCR. The arrowheads indicate the expected positions of the PCR amplification products. (C) FISH analysis of clones that were positive for genomic PCR. Red indicates mouse chromosome 17, purple indicates the human MHC region, and green indicates a stained image using a probe for the d3 cluster. [Figure 23] Diagram showing the isolation of d4 clones using CRISPR / Cas9. (A) Schematic diagram of the d4 cluster. CR indicates the target site of CRISPR / Cas9.Arrows indicate the positions of primers used to confirm the deletion. (B) Isolated clones were analyzed by genomic PCR. Arrowheads indicate the expected positions of PCR amplification products. [Figure 24] Diagram showing the isolation of d5 clones using CRISPR / Cas9. (A) Schematic diagram of the d5 cluster. CR indicates the target site of CRISPR / Cas9. Arrows indicate the positions of primers used to confirm the deletion. (B) Isolated clones were analyzed by genomic PCR. Arrowheads indicate the expected positions of PCR amplification products. (C) Gene disruption of H2-M5 in genomic PCR-positive clones was analyzed by sequence analysis. [Figure 25] Diagram showing the isolation of d6 clones using CRISPR / Cas9. (A) Schematic diagram of the d6 gene cluster. (B) Sequence analysis results of isolated d6 clone #198 are shown. [Figure 26] Diagram showing SNP analysis near H2-M2. (A) The position of SNPs near H2-M2 is indicated by black circles. (B) Analysis of SNPs 1 and 2 (indicated by numbers with circles) located upstream or downstream of H2-M2 was performed on d6 clone #198. (C) Analysis of the chromosome number of d6 clone #198. [Figure 27] T cell differentiation in d5 homozygous mice. (A) Spleen cells or thymocytes were isolated from wild-type or d5 homozygous mice, and the expression of CD4 or CD8 in CD3-positive cells was analyzed. (B, C) Wild-type or d5 homozygous mice were immunized with SARS CoV-2 S protein, and anti-S1 IgG1 (B) or IgG2 (C) antibodies in the serum were analyzed by ELISA. [Figure 28] Generation of mouse MHC gene cluster-deficient mice. (A) Genomic PCR analysis of offspring obtained by mating chimeric mice generated from d6 ES clones with wild-type mice. (B) Germline-transmitted d6 heterozygous mouse. (C) Genomic PCR analysis of offspring obtained by mating a d6 heterozygous male with a d6 heterozygous female. (D) Individual number 3, a d6 homozygous mouse obtained by mating.

[0009] The present invention relates to a chromosome of a non-human mammal in which major histocompatibility complex (MHC) genes are deleted or disrupted, to cells that have the chromosome in a homozygous or heterozygous state, and to non-human mammals and the like that are produced from the cells and have a chromosome in which the MHC genes are deleted or disrupted.

[0010] The present invention further provides a method for producing cells from a cell heterozygous for a chromosome in which the MHC gene cluster (also referred to as "MHC gene cluster 1") of the wild-type chromosome of one of a pair of chromosomes of a non-human mammal (referred to as "non-human mammal 1") has been replaced with the MHC gene cluster (also referred to as "MHC gene cluster 2") of a mammal other than the non-human mammal 1, the cell comprising a chromosome from the non-human mammal 1 that has MHC gene cluster 2 of the mammal 2 and a chromosome in which all or part of the MHC gene cluster 1 of the non-human mammal 1 has been disrupted or deleted. The production method comprises the step of disrupting or deleting MHC gene cluster 1 in the other wild-type chromosome of the non-human mammal 1.

[0011] 1. Overview Because autosomal sequences exist in pairs, gene disruption or deletion of genome sequences using genome editing technology occurs randomly in one or both alleles. However, to generate an MHC-deficient non-human mammal, all MHC genes must be disrupted or deleted in one allele. Furthermore, if the MHC region of a non-human mammal is present in only one allele, it is possible to generate an MHC-deficient allele. Therefore, the present inventors used mice as an example to generate MHC-humanized (heterozygous) ES cells in which the mouse MHC region is present in only one allele. Using these ES cells, they devised a method to disrupt or delete mouse MHC genes on the same allele to generate a mouse MHC-deficient allele, and established ES cells carrying a mouse MHC-deficient allele.

[0012] The following describes the preparation of a cell heterozygous for a chromosome in which the MHC gene group 1 of one of a pair of chromosomes from a wild-type non-human mammal has been replaced with the MHC gene group 2 of a mammal (referred to as "mammal 2") other than the non-human mammal 1. Next, a method for disrupting or deleting all or part of the MHC gene group 1 in the other wild-type chromosome of the non-human mammal 1 from the chromosome of the cell heterozygous for the chromosome will be described.

[0013] Non-human mammal 1 is an animal whose own MHC region is replaced with the MHC region of another mammal. The type of non-human mammal 1 is not particularly limited, and examples include rodents such as mice, rats, guinea pigs, rabbits, and hamsters; livestock such as cows, horses, pigs, and sheep; pets such as dogs and cats; and primates such as Japanese macaques and common marmosets. Non-human mammal 1 is not limited to the above animals. Mammal 2 is an animal whose MHC region is replaced with that of non-human mammal 1. The type of mammal 2 is not particularly limited, and includes humans as well as the above non-human mammal 1.

[0014] 2. Production of cells in which the MHC of non-human mammal 1 has been replaced with the MHC of mammal 2, and non-human mammals (1) Method for replacing the MHC region of non-human mammal 1 with the MHC region of mammal 2 An overview of the replacement of the MHC region of non-human mammal 1 with the MHC region of mammal 2 is shown in Figure 1. For ease of explanation, Figure 1 will be described using a human as an example of mammal 2.

[0015] In Figure 1, (a) is a schematic diagram of chromosome 6 of mammal 2 (human) and the chromosome of non-human mammal 1 (non-human) before replacement, and (c) is a schematic diagram of the chromosome after replacement of the MHC region. Replacement of the MHC region utilizes two-step recombination, namely, translocation recombination from (a) to (b1) and translocation recombination from (b1) to (c) (this is referred to as "two-step recombination 1"), or two-step recombination, namely, translocation recombination from (a) to (b2) and translocation recombination from (b2) to (c) (this is referred to as "two-step recombination 2"). In the present invention, the order of recombination is arbitrary, and either two-step recombination 1 or two-step recombination 2 may be used.

[0016] In Figure 1 , the telomere side of the MHC region that is the target of translocation recombination (also referred to herein as "replacement" or simply "recombination") is referred to as the distal side, and the centromere side is referred to as the proximal side. An arbitrary position distal to the human MHC region, i.e., between the human MHC region and the distal end, that serves as the boundary of recombination, is designated as P1, and an arbitrary position distal to the non-human MHC region, i.e., between the non-human MHC region and the distal end, that serves as the boundary of recombination and corresponds to P1, is designated as p1. Also in Figure 1 , an arbitrary position proximal to the human MHC region that is the target of recombination, i.e., between the human MHC region and the centromere, that serves as the boundary of recombination, is designated as P2, and an arbitrary position proximal to the non-human MHC region, i.e., between the non-human MHC region and the centromere, that serves as the boundary of recombination and corresponds to P2, is designated as p2.

[0017] In two-step recombination 1, the recombination from (a) to (b1) is the first recombination, which involves recombination between the region from P1 to the distal end (telomere) and the region from p1 to the distal end (telomere). The recombination from (b1) to (c) is the second recombination, which involves recombination between the region from P2 to the distal end (telomere) and the region from p2 to the distal end (telomere).

[0018] On the other hand, in two-step recombination 2, the "first recombination" occurs between the region from the centromeric side (P2) to the distal end of the human MHC region, which is the target of recombination in Figure 1, and the region from the centromeric side (p2) to the distal end of the non-human MHC region (Figure 1(b2)). At this stage, recombination has not occurred between the region from P1 to the distal end of the human chromosome and the region from p1 to the distal end of the non-human chromosome. Thereafter, recombination occurs between the region from P1 to the distal end of the human chromosome and the region from p1 to the distal end of the non-human chromosome, which is the "second recombination."

[0019] After the recombination is complete, cells with humanized MHC, such as ES cells, can be produced and transplanted into early embryos of the non-human mammal 1 to produce chimeric non-human animals. After the recombination is complete, it is preferable to remove any human chromosomes remaining in the cells. The human chromosomes can be removed by being dropped off by subculturing the cells in the absence of a selective agent. Furthermore, although chromosomes in non-human mammals are diploid, the chromosomes of the non-human mammal after the recombination is complete in the present invention, in which the MHC region has been replaced with the MHC region of a human chromosome, may be homozygous (both MHC regions of the two chromosomes are humanized) or heterozygous (the MHC region of one chromosome is humanized). However, heterozygous is preferred in order to delete or disrupt the MHC region in the non-human chromosome, as described below.

[0020] Therefore, MHC-humanized heterozygous animals can be produced by natural mating, intracytoplasmic sperm injection, etc. Furthermore, MHC-humanized homozygous animals can be produced by further natural mating, intracytoplasmic sperm injection, etc.

[0021] Here, human MHC is the same molecule as HLA, and the genes that determine this molecule are arranged in series on chromosome 6. The MHC region of non-human mammal 1 varies depending on the animal, and is located, for example, on chromosome 17 in mice, chromosome 20 in rats, chromosome 12 in rabbits, and chromosome 7 in pigs. Non-human mammal 1 is not limited to the above animals, and other examples include chromosome 20 in horses, chromosome 23 in cattle, chromosome 12 in dogs, and chromosome 4 in common marmosets.

[0022] When describing a gene encoding MHC in a gene region, it may be expressed as an "MHC region." Furthermore, since the MHC region of a mouse, for example, among non-human mammals 1, is present on the long arm, when a mouse chromosome is shown as a non-human chromosome in Figure 1, for example, the orientation of the long and short arms of the mouse is opposite to the orientation of the long and short arms of a human.

[0023] The MHC region to be recombined may be the entire MHC region or a part of it. In class I, it may be a classical class I molecule or a non-classical class I molecule. In humans, there are three types of classical class I molecules: HLA-A, HLA-B, and HLA-C, while the non-classical class I molecules include HLA-E, HLA-F, HLA-G, MICA, and MICB.

[0024] Human class II genes are located in the human MHC class II region, and the DR, DQ, and DP loci encode the major products of this region. Mouse MHC also contains mouse H2-M2 and H2-M3, but these MHCs do not need to be included in the target region for replacement in the present invention. The class III region contains various genes other than MHC, and in humans, it is a region distal to HLA-DRA and proximal to MICB.

[0025] For example, Figure 2 is a schematic diagram showing human MHC regions. The MHC region to be replaced may be only the class I region, only the class II region, or only the class III region, or a combination of these regions (including all combinations). Furthermore, the class I region may be a region containing classical class I MHC genes, a region containing non-classical class I MHC genes, or both. Therefore, in the present invention, the region constituting each class may be, for example, any one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, MICA, or MICB, or a combination of two or more of these (including all combinations). For class II, the region may be any one of HLA-DP, HLA-DQ, HLA-DR, HLA-DO, or HLA-DM, or a combination of two or more of these (including all combinations). The class III region contains various genes other than MHC, and in humans is a region located distal to HLA-DRA and proximal to MICB.

[0026] Here, the MHC region to be recombined may be the entire region or a part of the region, as described above. Therefore, depending on the selection of the target MHC region, the P1, p1, P2, and p2 may be located in an MHC region that has not been selected.

[0027] In the present invention, it is preferable that the human MHC class I region includes the entire region from MICB to HLA-F (the region designated by L2 in FIG. 2), and the class II region includes the entire region from HLA-DP to HLA-DR (the region designated by L1 in FIG. 2). The class III region is a region distal to HLA-DRA and proximal to MICB (the region designated by L3 in FIG. 2). In one embodiment of the present invention, it is even more preferable to replace the entire MHC region shown in FIG. 2 (the region designated by L4 in FIG. 2).

[0028] (2) Targeting Vector (2-1) First Recombination in Two-Step Recombination 1 and Second Recombination in Two-Step Recombination 2 In the present invention, the vector used to cause recombination is called a targeting vector. The targeting vector used here is not limited as long as it causes the above-mentioned recombination, and may be a vector that uses a recombinase recognition sequence and a recombinase, or a vector that uses a genome editing tool. If necessary, a genome editing tool can also be used as a tool to cut at the P1 and p1 positions. Using a genome editing tool makes it possible to cut the chromosome at the desired position, making it easier for subsequent recombination to occur.

[0029] <Recombination Using Recombinase> Recombination using a recombinase utilizes, for example, a recombinase recognition sequence called loxP and a recombinase called Cre. The Cre / loxP specific recombination system is well known. The Cre enzyme is a protein consisting of 343 amino acids derived from P1 phage, and it recognizes a specific 34-bp base sequence called a loxP site, enabling site-specific recombination. The loxP site can be divided into three parts: 13 bases, 8 bases, and 13 bases. The 13-base sequence has a complementary inverted repeat structure. Meanwhile, the 8-base sequence determines the orientation of the loxP site. Many mutant loxPs, called Lox511 and lox2272, are also known, and their recombinase recognition sequences can also be used.

[0030] In another aspect of the present invention, other examples of site-specific recombination systems using site-specific recombinases, in addition to the Cre / loxP system, include the Flp / FRT system derived from yeast plasmid 2μ, the PhiC31 integrase system derived from bacteriophage PhiC31, and the Bxb1 integrase system derived from bacteriophage Bxb1.

[0031] In Figure 1, the targeting vector for recombination from (a) to (b1) or from (b2) to (c) is a combination of the following targeting vector 1 and targeting vector 2, or the following targeting vector 3. The combination of targeting vector 1 and targeting vector 2 is a vector that causes recombination using a recombinase recognition sequence and a recombinase, and targeting vector 3 is a vector that causes recombination using a genome editing tool.

[0032] (i) Targeting Vector 1 Targeting Vector 1 is integrated into human chromosome 6 at an arbitrary position P1 distal to the MHC (human MHC) region present on the short arm of the chromosome and targeted for recombination. Targeting Vector 1 induces translocation recombination between the region from P1 to the distal end and a region in a non-human mammalian chromosome distal to the non-human MHC region targeted for recombination and corresponding to P1, from position p1 to the distal end. Targeting Vector 1 also includes a gene cassette 1 containing a recombinase recognition sequence, a partial sequence of drug resistance gene 1 for drug selection that is reconstituted during translocation recombination, and drug resistance gene 2 for drug selection after targeting.

[0033] The construct contained in gene cassette 1 can have, for example, a recombinase recognition sequence of loxP, a drug resistance gene 1 of neomycin resistance gene, and a drug resistance gene 2 of hygromycin resistance gene.

[0034] Here, "corresponding" refers to the positional relationship of the site where recombination takes place. That is, the above P1, p1, P2, and p2 are markers indicating the positional relationship of the distal or proximal side from the MHC region to be recombined. Therefore, it is not required that the genes adjacent to P1 and p1, or P2 and p2, be identical. Depending on the type of mammal, the MHC region may be inverted compared to the orientation of the MHC region in humans. In such cases, when the loci on both sides of the site where the targeting vector is integrated (P1) are between the MOG locus and the GABBR1 locus in humans (see the Examples and Figure 5), p1 of an animal with an inverted MHC region can be, for example, between the Kifc1 locus and the Daxx locus.

[0035] 5 is a schematic diagram showing the structure of one embodiment of the targeting vector of the present invention. For convenience of explanation, a targeting vector for replacing a mouse MHC region with a human MHC region will be described as an example.

[0036] In Figure 5A, "TV3R" represents the gene cassette 1. Recombinase recognition sequences are indicated by horizontal "▲" in the figure and are, for example, loxP sequences. In the present invention, "3'neo" is used as one partial sequence of drug resistance gene 1 for drug selection reconstituted during translocation recombination (a predetermined partial sequence of drug resistance gene 1 used in targeting vector 1). In this case, the drug resistance gene is a neomycin resistance gene. Drug resistance gene 2 for drug selection after targeting is indicated as "Hyg," and the drug resistance gene in this case is a hygromycin resistance gene.

[0037] The gene cassette 1 prepared in the examples of the present invention contains the sequence shown in the following formula (1): BGHpA-3'Neo-SA-loxP-EF1-Hyg-P2A-mRuby2-RGpA (1)

[0038] In formula (1), BGHpA represents the bovine growth hormone-derived poly A addition signal sequence, 3'Neo represents a partial sequence on the 3' side of the neomycin resistance gene, SA represents a splicing acceptor sequence, loxP represents a recombinase recognition sequence, EF1 represents the human elongation factor 1α-derived promoter sequence, Hyg represents the hygromycin resistance gene, P2A represents the porcine teschovirus-derived 2A sequence, mRuby2 represents the red fluorescent gene, and RGpA represents the rabbit β-globin-derived poly A addition signal sequence. However, in the present invention, a Flp recombinase recognition sequence (FRT) can also be added to cassette 1 shown in (1) above.

[0039] "TV3R-dHLA" is targeting vector 1, in which TV3R is linked on both sides with homologous sequences to the regions on both sides of the position (P1) where TV3R is integrated.

[0040] In FIG. 5A, "PX458a-dHLACR1" is a genome editing vector that artificially cuts an arbitrary position P1 of a chromosome to facilitate recombination of the targeting vector 1. Any known genome editing tool can be used for the genome editing vector. Examples include tools that use zinc finger nucleases (ZFNs), tools that use tail nucleases (TALENs), and tools that use CRISPR-Cas9 (CRISPR-Cas9). PX458a-dHLACR1 is a vector that uses CRISPR-Cas9, and is composed of, for example, a guide RNA sequence, a Cas9 sequence, and a gene sequence for the fluorescent protein ametrine.

[0041] Figure 1(d) shows an enlarged view of the region from near the MHC region to the distal end. In the examples of the present invention, P1 was designated between the MOG locus and the GABBR1 locus in the human chromosome, and targeting vector 1 was designed to be integrated into this region (Figure 5A).

[0042] (ii) Targeting Vector 2 Targeting Vector 2 is integrated into a chromosome of a non-human mammal in which an MHC region is present, at position p1, which corresponds to P1, and is distal to the non-human MHC region to be the target of recombination.

[0043] In the mouse of the example, targeting vector 2 induces translocation recombination between the region from p1 to the distal end and the region from P1 to the distal end of a human chromosome, and contains gene cassette 2 including a recombinase recognition sequence, another partial sequence of drug resistance gene 1 for drug selection (a partial sequence other than the predetermined partial sequence of drug resistance gene 1 used in targeting vector 1) that is reconstituted during translocation recombination, and drug resistance gene 3 for drug selection after targeting.

[0044] The construct contained in gene cassette 2 can have, for example, a loxP recombinase recognition sequence, a neomycin resistance gene as drug resistance gene 1, and a puromycin resistance gene as drug resistance gene 3. As mentioned above, some animal species have an inverted MHC region. Even in this case, the targeting vector 2 can be integrated into position p1 corresponding to any position P1.

[0045] In Figure 5B, "TV4" represents gene cassette 2. Recombinase recognition sequences are indicated by horizontal "▲" in the figure, and are, for example, loxP sequences. In the present invention, "5'neo" is used as the partial sequence of drug resistance gene 1 for drug selection that is reconstituted during translocation recombination. In this case, the drug resistance gene is a neomycin resistance gene. Drug resistance gene 2 for drug selection after targeting is indicated as "Puro," and in this case, the drug resistance gene is a puromycin resistance gene.

[0046] The gene cassette 2 prepared in the example of the present invention contains the sequence shown in the following formula (2): EF1-EGFP-P2A-5'Neo-SD-loxP-SA-T2A-Puro-RGpA (2)

[0047] In formula (2), EF1 represents the promoter sequence derived from human elongation factor 1α, EGFP represents the green fluorescent gene, P2A represents the porcine teschovirus-derived 2A sequence, 5'Neo represents a partial sequence on the 5' side of the neomycin resistance gene, SD represents a splicing donor sequence, loxP represents a recombinase recognition sequence, SA represents a splicing acceptor sequence, T2A represents the Thosea asigna virus-derived 2A sequence, Puro represents the puromycin resistance gene, and RGpA represents a rabbit β-globin-derived poly(A) addition signal sequence. In the present invention, FRT can also be added to cassette 2 shown in formula (2) above.

[0048] TV4-dH2 is targeting vector 2, and both sides of TV4 are linked to homologous sequences of the regions on both sides of the position (p1) where TV4 is integrated. In Figure 5B, "PX458a-dH2CR1" is a genome editing vector, which artificially cuts an arbitrary position p1 of the chromosome to facilitate recombination of targeting vector 2. The genome editing tools used in genome editing vectors are the same as those described above. PX458a-dH2CR1 is a vector that uses CRISPR-Cas9, and is composed of, for example, a guide RNA sequence, a Cas9 sequence, and the gene sequence of the fluorescent protein ametrine.

[0049] Similar to Figure 1(d), in this example of the present invention, targeting vector 2 was designed to be integrated between the Mog locus and the Gabbr1 locus in the non-human chromosome (Figure 5B).

[0050] <Recombination Using a Genome Editing Vector> (iii) Targeting Vector 3 In another aspect of the present invention, a genome editing tool can be used instead of recombination using a recombinase to perform the first recombination. Targeting Vector 3 causes translocation recombination between a region on human chromosome 6 that is distal to the human MHC region to be recombined and that is targeted for cleavage by the genome editing tool, from an arbitrary position P1 to the distal end, and a region on a chromosome of a non-human mammal that contains an MHC, that is distal to the non-human MHC region and that corresponds to position P1, from the distal end.

[0051] Even in this case, the replacement of the MHC region can utilize translocation recombination from (a) to (b1) and from (b1) to (c) in Figure 1 (two-step recombination 1), or translocation recombination from (a) to (b2) and from (b2) to (c) (two-step recombination 2).

[0052] The targeting vector 3 contains a homologous sequence to the sequence of a portion of the region between the P1 and the human MHC region (this region is referred to as "R1a"), a drug resistance gene 4 for drug selection after targeting, and a homologous sequence to the sequence of a portion of the region distal to the p1 (this region is referred to as "R2b"), or contains a homologous sequence to the sequence of a portion of the region distal to the P1 (this region is referred to as "R1b"), a drug resistance gene 4 for drug selection after targeting, and a homologous sequence to the sequence of a portion of the region between the p1 and the non-human MHC region (this region is referred to as "R2a").

[0053] The positional relationship between R1a, R1b, R2a, and R2b is shown in Figure 3(a). The R1a, R1b, R2a, and R2b regions may be any regions from P1 and p1, but are preferably regions near P1 and p1. The targeting vector 3 used in the present invention can be composed of, for example, an R2a sequence, a CAG promoter sequence, a blasticidin resistance gene, a rabbit β-globin-derived poly(A) additional signal sequence, and an R1b sequence.

[0054] (2-2) Second recombination in two-step recombination 1 or first recombination in two-step recombination 2 In the present invention, a vector that causes the second recombination in two-step recombination 1 and the first recombination in two-step recombination 2, i.e., the recombination from (b1) to (c) in Figure 1 or the recombination from (a) to (b2) in Figure 1, is referred to as a targeting vector 4.

[0055] Although the recombination in this section can be performed using a targeting vector utilizing a recombinase as described above, preliminary experiments by the present inventors have shown that recombination occurs more efficiently when a targeting vector utilizing a genome editing tool is used than when a targeting vector utilizing a recombinase is used, and the subsequent production of a non-human mammal is also more effective. Therefore, in the present invention, the second recombination in two-step recombination 1 or the first recombination in two-step recombination 2 is performed using a genome editing tool.

[0056] (iv) Targeting Vector 4 In the case of two-step recombination 1, targeting vector 4 causes translocation recombination between a region from an arbitrary position P2 to the distal end that is proximal to the human MHC region to be recombined and that is targeted for cleavage by the genome editing tool in the chromosome ( FIG. 1(b1)) after translocation recombination due to the action of targeting vector 1 and targeting vector 2, or the action of targeting vector 3, and a region from position p2 corresponding to P2 to the distal end that is proximal to the non-human MHC region in the chromosome of a non-human mammal in which a non-human MHC is present.

[0057] Furthermore, in the case of two-step recombination 2, targeting vector 4 can be used first, followed by targeting vectors 1 to 3. In short, the recombinations shown in Figures 1(a) to 1(c) may be carried out by first performing recombination from P1 and p1 to the distal end, followed by recombination from P2 and p2 to the distal end (in the order of Figures 1(a), (b1), and (c)), or by first performing recombination from P2 and p2 to the distal end, followed by recombination from P1 and p1 to the distal end (in the order of Figures 1(a), (b2), and (c)).

[0058] The targeting vector 4 comprises a homologous sequence to the sequence of a portion of the region R3a proximal to the P2 (between P2 and the centromere), a drug resistance gene 5 (e.g., a blasticidin resistance gene) for drug selection after targeting, and a homologous sequence to the sequence of a portion of the region R4b between the p2 and the non-human MHC region, or comprises a homologous sequence to the sequence of a portion of the region R3b between the P2 and the human MHC region, a drug resistance gene 5 for drug selection after targeting, and a homologous sequence to the sequence of a portion of the region R4a proximal to the p2 (between p2 and the centromere).

[0059] The positional relationship between R3a, R3b, R4a, and R4b is shown in Figure 3(b). The R3a, R3b, R4a, and R4b regions may be any regions relative to P2 and p2, but are preferably regions near P2 and p2. The targeting vector 4 used in the present invention can be composed of, for example, an R3a sequence, a CAG promoter sequence, a blasticidin resistance gene, a rabbit β-globin-derived poly(A) additional signal sequence, and an R4b sequence.

[0060] Figure 1(e) shows an enlarged view of the region from near the MHC region to the distal end. In the present example, targeting vector 4 was designed to induce recombination between the KIFC1 locus and the DAXX locus in human chromosomes, and between the KIFC1 locus and the Daxx locus in non-human chromosomes (Figure 5C). Targeting vector 4 contains a blasticidin (BS) resistance gene.

[0061] In Figure 5C, "PX458.1a-pHLACR2" is a genome editing vector that artificially cuts an arbitrary position P2 of a chromosome to facilitate translocation recombination via the targeting vector 4. PX458.1a-pHLACR2 is a vector that utilizes CRISPR-Cas9 and is composed of, for example, a guide RNA sequence, a Cas9 sequence, and a gene sequence for the fluorescent protein ametrine.

[0062] Like PX458.1a-pHLACR2, "PX458.1a-pH2CR1" is a genome editing vector that artificially cuts an arbitrary position p2 of a chromosome to facilitate translocation recombination via targeting vector 4. PX458.1a-pH2CR1 is a vector that utilizes CRISPR-Cas9 and is composed of, for example, a guide RNA sequence, a Cas9 sequence, and a gene sequence for the fluorescent protein ametrine.

[0063] <Drug resistance genes for drug selection> In the present invention, as described above, drug resistance genes 1 to 5 for drug selection are used in the targeting vector. The combination of each of the drug resistance genes 1 to 5 is arbitrary and is selected appropriately so as to enable selection of the desired drug. Examples of genes used for drug resistance genes 1 to 5 include, but are not limited to, a neomycin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, and a blasticidin resistance gene.

[0064] <Fluorescent Gene for Confirming Recombination or Selection> In the present invention, a predetermined fluorescent gene can be included in the targeting vector to confirm chromosome introduction, confirmation that the introduced chromosome is maintained, or confirmation that drug selection has been performed as intended. The type of fluorescent gene can be selected arbitrarily as long as it allows for confirmation in each process, and it is also possible to use different types of fluorescent gene for each vector. Examples of fluorescent genes include green fluorescent genes (GFP, EGFP, etc.), red fluorescent genes, and yellow fluorescent genes.

[0065] (3) Cells To establish a non-human mammal with a humanized MHC, it is necessary to replace the endogenous MHC gene in the cells of the non-human mammal with a human MHC gene. In the present invention, as described above, conventional homologous recombination or genome editing techniques can be used.

[0066] The cells are not particularly limited as long as they are animal cells, and examples thereof include ES cells, spermatogonial stem cells, fibroblasts, etc., with ES cells being preferred. Examples of cell culture media include Glasgow's Minimal Essential Medium (GMEM), Dulbecco's Modified Eagle's Medium (DMEM), and RPMI 1640 medium. Commonly used animal cell culture additives, such as fetal bovine serum (FBS), non-essential amino acids, antibiotics (e.g., penicillin, streptomycin, etc.), and growth factors / cytokines (e.g., epidermal growth factor, fibroblast growth factor, leukemia inhibitory factor, etc.), can be added to the culture medium as appropriate depending on the cell type.

[0067] After culturing the cells for a predetermined period of time, the cells are recovered by incubating them in a medium containing trypsin. The recovered cells can be subcultured multiple times, if necessary, with or without feeder cells. The cultured cells can be confirmed to be the target cells by using their marker genes as indicators. When the cells are ES cells, for example, Oct3 / 4, alkaline phosphatase, Nanog, etc. can be used as indicators, and they can be detected by any method such as RT-PCR or Western blotting. ES cells can also be determined by colony morphology.

[0068] (4) Production of Non-Human Mammals Non-human mammals can be produced by standard methods such as the production of chimeric animals using ES cells, somatic cell nuclear transfer, and intracytoplasmic sperm injection using spermatogonial stem cells. The non-human mammals to be produced are not particularly limited, and examples include rodents, livestock, and primates. Examples of rodents include mice, rats, guinea pigs, and hamsters. Examples of livestock include cows, horses, pigs, and sheep. Examples of primates include Japanese macaques and common marmosets. Other pet or laboratory animals such as dogs, cats, monkeys, and rabbits can also be used. In the present invention, rodents, especially mice, are preferred.

[0069] In the case of chimeric animals, the established ES cells are first aggregated with an early embryo or injected into a blastocyst. The embryo thus produced is called a chimeric embryo, and this chimeric embryo is then transplanted into the uterus of a pseudopregnant foster mother and allowed to give birth to produce a chimeric animal. Here, the term "embryo" refers to an individual at any stage in ontogeny from fertilization to birth, and includes early embryos such as 2-cell, 4-cell, 8-cell, morula, and blastocyst.

[0070] For ease of explanation, the following description will use ES cells as an example of cells. Methods for producing aggregates using ES cells and embryos include known techniques such as microinjection and aggregation. When using the microinjection method, ES cells are injected into recovered embryos to produce cell aggregates. When using the aggregation method, ES cells are aggregated with normal embryos from which the zona pellucida has been removed. The ES cells used here are not particularly limited, and examples include C57BL / 6-derived RENKA, XO, a Y-chromosome-deficient strain of TT2 cells derived from an F1 hybrid of C57BL / 6 and CBA, and ES cells established from an F1 hybrid of C57BL / 6 and DBA / sCrSlc.

[0071] On the other hand, pseudopregnant female animals to be used as foster parents can be obtained by mating a female animal with a normal estrous cycle with a male animal that has been castrated by vasoligation or the like. A chimeric embryo produced by the above-mentioned method can be implanted into the uterus of the pseudopregnant animal thus produced, and then the animal is allowed to give birth, thereby producing a chimeric animal.

[0072] From these chimeric animals, animals derived from ES cell-transplanted embryos are selected. The selected chimeric animals are mated with animals of an inbred strain. The resulting offspring will then exhibit the coat color of the animal derived from the ES cells, confirming that the ES cells have been introduced into the germline of the chimeric animal. Whether the resulting offspring possess a humanized MHC gene can be determined by digesting the DNA with a restriction enzyme and detecting DNA fragments of the desired size, or by analyzing the DNA using PCR.

[0073] 3. Production of non-human mammalian chromosomes with deleted or disrupted MHC genes, and cells containing the chromosomes in a homozygous or heterozygous arrangement. In MHC-humanized (heterozygous) ES cells, one allele of the MHC region is humanized. Using a mouse as an example of a non-human mammal 1, all deletions of genomic sequences or gene disruption by CRISPR / Cas9 targeting sequences within the mouse MHC region will occur on the allele containing the mouse MHC region (Figure 17). Therefore, disruption and deletion of mouse MHC genes in MHC-humanized (heterozygous) ES cells can produce mouse MHC-gene-deficient alleles. Since MHC-humanized (heterozygous) mice can be produced from MHC-humanized (heterozygous) ES cells, mouse MHC-gene-deficient mice can be produced by producing mouse MHC-gene-deficient MHC-humanized (heterozygous) ES cells.

[0074] In the examples, mouse MHC gene-deficient alleles were generated using MHC-humanized (heterozygous) ES cells in which the MHC region from Daxx to Mog of CBAxC57BL / 6 F1 mouse-derived XO ES cells was humanized. First, examining the distribution of MHC genes on the genome, it was found that 34 of the 39 genes are present in five clusters without any non-MHC genes (Figure 18). These clusters are designated d1, d2, d3, d4, and d5, in descending order of size.

[0075] It can be seen that the five MHC genes, H2-K1, H2-Oa, H2-M5, H2-M3, and H2-M2, do not form a cluster and exist in isolation. These isolated MHC genes are classified as d6. In the present invention, to delete or disrupt MHC genes, the sequences of the d1 to d5 MHC genes are deleted using two CRISPR / Cas9s designed to sandwich the entire or part of the locus of all MHC genes in the cluster, and the isolated MHC genes classified as d6 can be destroyed using CRISPR / Cas9s designed to disrupt each MHC gene. For example, to delete the d1 MHC gene cluster, CRISPR / Cas9 is designed to target both sides of the d1 cluster, i.e., the region between the H2-M10.2 locus located most centromeric in the d1 cluster and d3 in FIG. 18, and the region between the H2-M10.6 locus located most telomeric in the d1 cluster and d6 (H2-M5 gene), to induce deletion. CRISPR / Cas9 is designed in the same way for other clusters. Since the genomic base sequences upstream and downstream of each MHC cluster often differ between non-human mammals 1 (e.g., mice) and mammals 2 (e.g., humans), these different base sequences are targeted in the design of CRISPR / Cas9. This allows only the mouse MHC gene cluster (MHC gene cluster 1) to be disrupted or deleted from heterologous cells in which MHC has been humanized.

[0076] For the clusters d1 to d5, CRISPR / Cas9 is designed to sandwich each cluster as described above. Furthermore, when deleting or disrupting each gene group from d1 to d6, the gene group to be deleted or disrupted at one time is not limited to one, and two or more can be combined. For example, a combination of d1 and d3, a combination of d2 and d3 (a combination of two clusters), a combination of d1, d3, and d2 (a combination of three clusters), MHC genes classified into d5 and d6 (a combination of one cluster and an MHC gene present in isolation), etc. can be appropriately combined. When deleting or disrupting each gene group from d1 to d6, the order in which the genes are disrupted or deleted is arbitrary. For example, d1 is deleted followed by d2, and d2 is deleted followed by d3, and the deletion or disruption can be performed in order from d1 to d6, or in any order. The clusters to be deleted or destroyed may be part or all of them.

[0077] Alternatively, one cluster can be divided into subgroups and subjected to multiple deletion or deletion steps. For example, in cluster d1 shown in Figure 18, H2-M10.2 to H2-M11 can be divided into subgroups d1a and H2-M9 to H2-M10.6 can be divided into subgroups d1b, and clusters in each subgroup can be deleted or deleted.

[0078] On the other hand, since the gene group classified as d6 does not form a cluster, CRISPR / Cas9 is designed to disrupt the gene for each gene. The d6MHC gene group can be divided into subgroups and each can be disrupted separately. Here, some of the MHC genes of non-human mammal 1 (e.g., mouse) may remain in the chromosome having the MHC region of mammal 2 (e.g., human). In this case, all of the MHC gene group is disrupted or deleted from the wild-type allele, and the MHC genes of non-human mammal 1 remaining in the chromosome having the MHC region of mammal 2 are disrupted. In Figure 18, in addition to the human MHC region, mouse-derived H2-M3 and H2-M2 genes remain, so these remaining genes are also disrupted or deleted.

[0079] In this way, by deleting or disrupting the d1 to d6 MHC gene cluster, it is possible to delete all mouse MHC genes. In the present invention, clusters d1 to d6 can be arbitrarily selected and a portion of these clusters can be deleted or disrupted, or all of d1 to d6 can be deleted or disrupted.

[0080] In one embodiment of the present invention, d1 to d6 clones are isolated by sequentially deleting the MHC genes contained in each cluster from d1 to d6, as shown in Figure 19. The d1 clone is a clone obtained by deleting the MHC genes in the d1 cluster of an MHC humanized (heterozygous) ES cell, the d2 clone is a clone obtained by deleting the MHC genes in the d2 cluster from the d1 clone, the d3 clone is a clone obtained by deleting the MHC genes in the d3 cluster from the d2 clone, the d4 clone is a clone obtained by deleting the MHC genes in the d4 cluster from the d3 clone, the d5 clone is a clone obtained by deleting the MHC genes in the d5 cluster from the d4 clone and simultaneously disrupting the H2-M5 gene, and the d6 clone is a clone obtained by disrupting the H2-K1, H2-Oa, H2-M3, and H2-M2 genes of the d5 clone. In producing the d6 clone, simultaneous disruption of four genes results in low gene disruption efficiency. Therefore, in some cases, a d6a clone was isolated by disrupting only H2-K1 and H2-M3 of the d5 clone, and then H2-Oa and H2-M2 were disrupted to isolate the d6 clone.

[0081] 4. Non-human mammals having chromosomes in which MHC genes are deleted or disrupted As described above, non-human mammals can be produced using ES cells having chromosomes in which all MHC genes are deleted or disrupted. For example, non-human mammals can be produced by transplanting ES cells having chromosomes in which all MHC genes are deleted or disrupted into early embryos, or by somatic cell nuclear transfer of ES cells having chromosomes in which all MHC genes are deleted or disrupted. Furthermore, chromosomes in which MHC genes are deleted or disrupted can be introduced from the above-mentioned cells into other cells by microcell fusion or other methods.

[0082] EXAMPLES The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.

[0083] 1. Preparation of targeting vector <Method>

[0084] Preparation of TV3R-dHLA pEF-GFP (Addgene plasmid #11154; http: / / n2t.net / addgene:11154; RRID:Addgene_11154) was digested with restriction enzymes EcoRI-HF (New England BioLabs) and NotI-HF (New England BioLabs), and a hygromycin resistance gene was cloned downstream of the human elongation factor 1α-derived promoter sequence using an In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech) in accordance with the attached protocol to prepare pEF-Hyg (SEQ ID NO: 1). Next, the upstream side of the human elongation factor 1α-derived promoter sequence encoded by pEF-Hyg was digested with the restriction enzyme SalI, and a synthetic DNA fragment FRT-BGHpA-3′Neo-SA-loxP (Thermo Fisher Scientific) (SEQ ID NO: 2) was cloned using the In-Fusion® HD Cloning Kit to produce TV3.

[0085] Next, a sequence containing the 5' and 3' homologous regions of the targeting vector TV3R-dHLA was amplified using the genome of MRC5 cells as a template and the following primer set with PrimeSTAR (registered trademark) GXL DNA Polymerase (Takara) according to the attached protocol.

[0086] Furthermore, the entire homologous region was amplified by nested-PCR using the following primer set, and cloned into pGEM (registered trademark)-T Easy Vector (Promega) to prepare pGEMTe-dHLA.

[0087] Next, pGEMTe-dHLA was amplified using PrimeSTAR® GXL DNA Polymerase with the following primer set: TV3 was digested with SalI-HF (New England BioLabs) and HindIII-HF (New England BioLabs) to prepare a fragment containing FRT-BGHpA-3′Neo-SA-loxP. This fragment was then cloned using the In-Fusion® HD Cloning Kit to produce TV3-dHLA.

[0088] Furthermore, a synthetically prepared DNA fragment P2A-mRuby2 (SEQ ID NO: 9) (Thermo Fisher Scientific) was amplified using the following primer set with PrimeSTAR (registered trademark) GXL DNA Polymerase. The amplified product and TV3-dHLA were digested with the restriction enzyme BssHII (New England BioLabs) and then ligated using a Ligation-Convenience Kit (Nippon Gene) to prepare TV3R-dHLA.

[0089] Preparation of TV4-dH2 pEF-GFP (Addgene plasmid #11154; http: / / n2t.net / addgene:11154; RRID:Addgene_11154) was digested with restriction enzymes EcoRI-HF and NotI-HF, and the EGFP gene was cloned downstream of the human elongation factor 1α-derived promoter sequence using the In-Fusion (registered trademark) HD ​​Cloning Kit in accordance with the attached protocol to prepare pEF-EGFP2 (SEQ ID NO: 12). Next, pEF-EGFP2 was digested with the restriction enzyme BsrGI (New England BioLabs), and the sequence P2A-5′Neo-SD-loxP-SA-T2A-Puro (Thermo Fisher Scientific) (SEQ ID NO: 13), which had been synthesized as two DNA fragments, was cloned into the digest using the In-Fusion® HD Cloning Kit to produce TV4dF.

[0090] TV4dF was digested with the restriction enzyme SalI-HF, and the following annealed oligo DNA was ligated to this using a Ligation-Convenience Kit to prepare TV4.

[0091] Next, a sequence containing the 5' homologous region and 3' homologous region of the targeting vector TV4-dH2 was amplified using the C57BL / 6 mouse genome as a template and the following primer set with PrimeSTAR (registered trademark) GXL DNA Polymerase according to the attached protocol.

[0092] Furthermore, the entire homologous region was amplified by nested-PCR using the following primer set, and cloned into pGEM (registered trademark)-T Easy Vector (Promega) to prepare pGEMTe-dH2.

[0093] Next, pGEMTe-dH2 was amplified using the following primer set with PrimeSTAR® GXL DNA Polymerase, and a fragment containing P2A-5′Neo-SD-loxP-SA-T2A-Puro, prepared by digesting TV4 with restriction enzymes SalI-HF and HindIII-HF, was cloned using the In-Fusion® HD Cloning Kit to produce TV4-dH2.

[0094] Preparation of TV-TI1 / 2-BS The Hprt expression vector TV-TI1 / 2-Hprt (SEQ ID NO: 22), which uses pGEM (registered trademark)-T Easy Vector as the vector backbone and has the 5' and 3' homologous regions used in TV-TI1 / 2-BS, was digested with restriction enzymes XhoI (New England BioLabs) and XbaI (New England BioLabs), and a synthetically prepared DNA fragment (IDT) (SEQ ID NO: 23) containing a blasticidin resistance gene was cloned into this using the In-Fusion (registered trademark) HD ​​Cloning Kit to prepare TV-TI1 / 2-BS.

[0095] Preparation of genome editing vectors (1) PX458a-dHLACR1 and PX458a-dH2CR1 gRNAs were designed for the following sequences, and pSpCas9(BB)-2A-GFP(PX458) (Addgene plasmid #48138; http: / / n2t.net / addgene:48138; RRID: Addgene 48138) was modified to express gRNA and Cas9. PX458a was prepared by replacing the EGFP gene with the fluorescent gene ametrine (SEQ ID NO: 24).

[0096] Preparation of genome editing vector (2) The gRNAs of PX458.1a-pHLACR2 and PX458.1a-pH2CR1 were designed for the following sequences, and PX458.1a with a modified gRNA scaffold sequence of PX458a was used for expression of gRNA and Cas9 (SEQ ID NO: 27).

[0097] Preparation of drug-resistant mouse embryonic fibroblasts (MEFs) Drug-resistant MEFs were generated to allow ES cells to be cultured on feeder cells during drug selection. First, MEFs were prepared from embryonic day 14.5 embryos and infected with a lentivirus expressing a gene (HBPN) consisting of hygromycin resistance, blasticidin resistance, puromycin resistance, and neomycin resistance genes linked by a 2A sequence. To improve the proliferation capacity of MEFs, HBPN-expressing MEFs were cultured under hypoxic conditions of 5% oxygen / 5% CO2, and then treated with mitomycin C to be used as drug-resistant MEFs.

[0098] Cloning of MRC5-A9 fusion cells hChA9 MRC5 cells transfected with a Neo resistance gene using lentivirus and A9 cells were fused using Genom ONE-CF (Ishihara Sangyo Kaisha), and Ouabain-resistant / G418-resistant cells were isolated as fusion cells. Human cells are Ouabain-sensitive, while mouse cells are Ouabain-insensitive. Fusion cells can be selected by adding G418 and Ouabain to the culture medium. Fusion cells carrying human chromosome 6 were identified by genomic PCR using the following primer set, which was designed for the intergenic region between KIFC1 and DAXX:

[0099] For detection of human chromosome 6 by FISH, a DNA fragment artificially synthesized from sequences specific to the centromere of human chromosome 6 was used to prepare a probe, with reference to the report by Jabs et al. (Am. J. Hum. Genet. 41: 374-390, 1987).

[0100] The synthesized sequence is as follows:

[0101] Cloning of TV3RhChA9. A clone (TV3RhChA9) targeting TV3R-dHLA in MRC5-A9 fusion cells was generated by transfecting hChA9 with PX458a-dHLACR1 and the targeting vector TV3R-dHLA using PEI max (Polysciences). PX458a-dHLACR1 was co-transfected to increase the efficiency of homologous recombination of the targeting vector. The day after transfection, vector-transfected cells were sorted using the expression of the fluorescent gene mRuby2 on TV3R-dHLA as an indicator, and mRuby2-positive, hygromycin-resistant clones were isolated. Genomic DNA was prepared from the isolated clones, and recombinant clones were identified by PCR. The primers used for PCR are as follows. Similar analysis was also performed when recloning of isolated clones was performed.

[0102]

[0103] Cloning of TV4RENKA TV4RENKA is an ES cell clone created by transfecting PX458a-dH2CR1 and the targeting vector TV4-dH2 into RENKA using Lipofectamine 3000 (Thermo Fisher Scientific). PX458a-dH2CR1 was co-introduced to increase the efficiency of homologous recombination of the targeting vector. EGFP-positive clones were isolated, and recombined clones were identified by genomic PCR. Clones can also be isolated in the same manner for XO and BDF1.

[0104] The primers used for PCR are as follows:

[0105] Cloning of TV3-4RENKA TV3-4RENKA is an ES cell clone created by introducing a modified human chromosome 6 from TV3RhChA9 into TV4RENKA using the retro-MMCT method (Suzuki, T. et al. (2016) PloS One, 11(6): e0157187.). Cells that were mRuby2-positive and hygromycin-resistant were cloned, and a clone with 41 chromosomes was isolated. FISH analysis was performed using Cy3-labeled mouse Cot-1 and Cy5-labeled RP11-54H13.

[0106] Cloning of TV3-4RENKATl3 / 4 A Cre expression vector was introduced into TV3-4RENKA using Lipofectamine 3000 to induce chromosomal translocation between loxP sites. Clones that underwent chromosomal translocation were isolated using G418 resistance as an indicator and designated "TV3-4RENKATl3 / 4." After confirming recombination by genomic PCR, FISH analysis confirmed that a translocation had occurred between mouse chromosome 17 and human chromosome 6.

[0107] The primers used for PCR are as follows:

[0108] In FISH analysis, detection was performed using Cy3-labeled RP11-54H13, DY490-labeled RP23-147G23 and Cy5-labeled RP23-119G24, or Cy3-labeled human Cot-1 and DY490-labeled mouse Cot-1.

[0109] Cloning of TV3-4RENKAT1b For cloning of TV3-4RENKAT1b, TV-T11 / 2-BS, PX458.1a-pHLACR2, and PX458.1a-pH2CR1 were co-transfected into TV3-4RENKAT13 / 4 using Lipofectamine 3000. Ametrine-positive cells were sorted the day after transfection, and blasticidin-resistant clones were isolated. Recombination was confirmed by genomic PCR, and then replacement of the MHC region was confirmed by FISH analysis.

[0110] The primers used for PCR are as follows:

[0111] In the FISH analysis, detection was carried out using Cy3-labeled RP11-147G23, DY490-labeled human Cot-1, and Cy5-labeled RP23-306H20.

[0112] Cloning of TV3-4RENKATlbdh6 To isolate TV3-4RENKATlbdh6, which lacks the remaining human chromosome 6 from TV3-4RENKATlb, mRuby2-negative cells were sorted and seeded on feeder cells to isolate clones. Furthermore, FISH analysis of clones with a karyotype analysis of 40 chromosomes (39 in the case of XO) was performed to confirm the loss of the remaining human chromosome 6. FISH analysis was performed using Cy3-labeled RP11-147G23, DY490-labeled human Cot-1, and Cy5-labeled RP23-361C19. TV3-4XOTlbdh6 and TV3-4BDF1Tlbdh6 were also isolated using the same procedure as TV3-4RENKATlbdh6.

[0113] 2. Generation of MHC-humanized mice MHC-humanized chimeric mice were generated by the aggregation chimera method using MHC-humanized ES cells and ICR-derived embryos. In the case of chimeric mice generated using RENKA-derived MHC-humanized ES cells, sperm cells were isolated from the testes and then subjected to microinsemination into eggs from B6 / DBA F1 mice or ICR mice. Furthermore, in the case of female chimeric mice generated using XO ES cell-derived MHC-humanized ES cells, they were mated with C57BL / 6 male mice to establish MHC-humanized chimeric mice (MHC-humanized heterozygous mice). The establishment of MHC-humanized mice was confirmed by EGFP fluorescence of the offspring and genomic PCR.

[0114] The primer set used for genomic PCR is as follows: DAXX: MICA: MOG: S76: DAXX (confirmed with 3 primers):

[0115] 3. Results and Discussion Construction of Vectors for MHC Region Replacement First, we constructed the vectors necessary for the series of recombination procedures. Considering the impact of translocation replacement on the expression of surrounding genes, we designed targeting vectors TV3R-dHLA and TV4-dH2 to recombine the TV3R or TV4 sequence between the MOG and GABBR1 loci, which have relatively large intergenic sequences distal to the MHC region in humans or mice (Figures 5A and 5B). Furthermore, to increase the efficiency of homologous recombination, we constructed CRISPR vectors PX458a-dHLACR1 and PX458a-dH2CR1, which induce DNA double-strand breaks at their respective target sites.

[0116] TV3R encodes a loxP sequence necessary for translocation induction, the 3'-side sequence of the Neo resistance gene that confers G418 resistance during translocation recombination, an FRT sequence that removes sequences within the targeting vector after translocation induction, a hygromycin resistance gene that is used for drug selection during targeting, and the red fluorescent gene mRuby2 that detects the presence of the targeted chromosome.

[0117] On the other hand, TV4 encodes the loxP sequence required for translocation induction, the 5' sequence of the Neo resistance gene for G418 resistance during translocation recombination, the FRT sequence for removing a sequence within the targeting vector after translocation induction, the puromycin resistance gene for drug selection during targeting, and the green fluorescent gene EGFP for detecting the presence of the targeted chromosome. The TV3R and TV4 sequences are designed so that the EGFP expression unit and the rearranged Neo resistance gene remain on mouse chromosome 17 after translocation induction, making it possible to select MHC-humanized mouse chromosome 17-bearing cells using drugs and fluorescence.

[0118] Next, we constructed the targeting vector TV-Tl 1 / 2-BS to induce translocation recombination between the DAXX and KIFC1 loci, which have relatively large intergenic sequences proximal to the MHC region in humans and mice, and the CRISPR vectors PX458.1a-pHLACR2 and PX458.1a-pH2CR1 to cleave the respective target sites and increase recombination efficiency (Figure 5C). TV-Tl 1 / 2-BS contains a blasticidin resistance gene expression cassette, so that recombined cells can be selected for blasticidin resistance. Furthermore, the blasticidin resistance gene was designed to be recombined onto the remaining human chromosome 6, which is ultimately removed from the ES cells. This prevents the introduction of an exogenous gene expression cassette into the proximal MHC region of chromosome 17 in MHC-humanized mice, minimizing the impact on gene expression around the humanized MHC region.

[0119] To introduce human chromosome 6 from normal human fibroblast MRC5 cells into mouse embryonic stem cells (ES cells), it was necessary to create fusion cells between MRC5 cells and A9 mouse fibroblasts with high chromosome donation capacity. Therefore, to select the fusion cells, a neomycin resistance gene was first introduced into MRC5 cells using a lentivirus (Figure 4, Step 1 (circled number 1). From Step 1 onwards, each step is numbered with a circle, as in Step 1). Next, the neomycin-resistant MRC5 cells were fused with A9 cells, and the fusion cell line hChA9, which was resistant to both ouabain and G418, drugs that selectively kill human cells, was isolated (Figure 4, Step 2).

[0120] The presence of sequences within the human HLA region in the isolated clones was confirmed by genomic PCR, and signals were detected in clones #1, 5, and 13 (Fig. 6A). Furthermore, to confirm the presence of human chromosome 6 in hChA9#5, FISH analysis was performed using a probe specific for the human chromosome 6 centromere sequence, confirming the presence of two copies of human chromosome 6 in this fusion cell clone (Fig. 6B).

[0121] TV3R targeting: The targeting vector TV3R-dHLA was recombined into the distal end of the human chromosome 6 MHC region of hChA9#5, introducing the sequence required for MHC region replacement (Figure 4, Step 3). Hygromycin-resistant clones were isolated and analyzed by genomic PCR, resulting in the isolation of multiple targeted cells, TV3RhChA9 (Figure 7A). To eliminate the possibility of clone contamination, TV3RhChA9#5-45(128) was recloned to isolate TV3RhChA9#5-45a(128) (Figure 7B). FISH analysis of TV3RhChA9#5-45a(128) confirmed that the majority of cells retained a single copy of human chromosome 6 (Figure 7C). Therefore, TV3RhChA9#5-45a(128) was used as a modified human chromosome 6 donor cell.

[0122] TV4 Targeting TV4-dH2 was targeted to the distal side of the MHC region on chromosome 17 of the B6-derived mouse ES cell RENKA, to introduce a sequence required for swapping the MHC region ( FIG. 4 , Step 4).

[0123] Analysis of EGFP-positive clones by genomic PCR confirmed the presence of many recombinant clones (Figure 8A). Because EGFP within the TV4 sequence can be used to confirm germline transmission if it is present only in the MHC-humanized allele, clone TV4RENKA#17, which has 40 chromosomes and in which only one allele is targeted, was used in subsequent experiments (Figure 8).

[0124] Introduction of Modified Human Chromosome 6 The modified human chromosome 6 of TV3RhChA9#5-45a(128) was introduced into TV4RENKA#17 (Figure 4, Step 5). Because ES cells typically have low chromosome introduction efficiency and fused cells tend to have lower chromosome donation capacity than A9 cells, it is possible that chromosome introduction may not be possible using conventional chromosome introduction methods using polyethylene glycol (PEG-MMCT method). Therefore, the modified human chromosome 6 was introduced using a highly efficient chromosome introduction method (retro-MMCT method) that utilizes ecotropic envelope proteins derived from murine leukemia virus.

[0125] Hygromycin-resistant, mRuby2-positive ES cells were cloned, and chromosome samples were prepared for karyotype analysis. Normal mouse cells have 40 chromosomes (39 for XO), but TV3-4RENKA#17 / 5-45a-3 was confirmed to have 41 chromosomes (40 for XO). Furthermore, since it was confirmed that the chromosome retained the submetacentric chromosome characteristic of human chromosome 6, further FISH analysis revealed that this chromosome contained the human MHC region. Therefore, it was considered that the introduction of modified human chromosome 6 was successful (Figure 9).

[0126] Induction of chromosomal translocation using Cre / loxP The recombinase Cre can induce translocation between loxP sequences on independent chromosomes. Therefore, to induce a translocation between the modified human chromosome 6 and the distal MHC region of the modified mouse chromosome 17 carried by TV3-4RENKA#17 / 5-45a-3, the recombinase Cre was transiently expressed in TV3-4RENKA#17 / 5-45a-3 (Figure 4, step 6). When translocation recombination is induced between the loxP sequences present in the TV3 and TV4 sequences, the Neo resistance gene is reconstructed (Figure 10A), and clones that became G418 resistant were isolated. Among these clones, translocation recombination occurred in 41 chromosomes (40 in XO) and was confirmed by genomic PCR and FISH. This clone was designated "TV3-4RENKA T13 / 4#17-1" and used in subsequent experiments (Fig. 10B-D).

[0127] Induction of chromosomal translocation using CRISPR / Cas9 Translocation on the proximal side of the MHC region was planned to be induced using the recombinase Dre, as was the case on the distal side, so initially ES cells were prepared in which the Dre recognition sequence rox had been recombined into the proximal side. However, translocation using Dre was inefficient, and the expression of the HPRT gene, which was planned to be used as a selection marker, was highly required in ES cells, so it was found that the expression of the HPRT gene reconstructed by recombination translocation could not withstand selective culture using HAT. For this reason, the modification of mouse chromosome 17 was changed to only the introduction of TV4, and disruption of the Hprt gene in ES cells was discontinued, resulting in the creation of TV3-4RENKA T13 / 4#17-1.

[0128] Therefore, we decided to induce translocation on the proximal side of the MHC region using CRISPR / Cas9. Specifically, a CRISPR vector that cleaves the KIFC1-DAXX intergenic region on mouse chromosome 17 and human chromosome 6, respectively, and the translocation induction targeting vector TV-Tl1 / 2-BS were introduced into TV3-4RENKA Tl3 / 4#17-1, and a clone that became blasticidin resistant was isolated (Figure 4, Step 7). Of these, clone TV3-4RENKA Tlb#38 had 41 chromosomes (40 in XO), and translocation recombination was confirmed by genomic PCR. Furthermore, analysis by the FISH method revealed that the modified human chromosome 6 was translocated with the modified chromosome 17, not with the wild-type chromosome 17, so TV3-4RENKA Tlb#38 was used in subsequent experiments (Figure 11).

[0129] Isolation of MHC-humanized ES cells To ensure the undifferentiated nature of MHC-humanized ES cells, we initially planned to introduce the MHC-humanized mouse chromosome 17 generated up to this point into ES cells with a low passage number, thereby deleting the endogenous mouse chromosome 17 and generating MHC-humanized ES cells (Figure 4, Step 8'). However, this procedure required the generation of ES-A9 fusion cells and the introduction of the MHC-humanized chromosome into the ES cells, and we found that chromosomal abnormalities occurred frequently during this process, which limited the procedure. Therefore, we attempted to generate chimeric mice by isolating a large number of MHC-humanized ES clones from TV3-4RENKA Tlb#38 by spontaneously shedding human chromosomes (Figure 4, Step 8).

[0130] It has been reported that human chromosomes are prone to shedding in mouse cells. Therefore, TV3-4RENKA Tlb#38 was cultured for several days in the absence of a selective agent to induce shedding of the remaining human chromosome 6. To isolate clones lacking the remaining human chromosome 6, we initially planned to use 6-thioguanine, which can select for HPRT gene-deficient cells, as a selective agent. However, as mentioned above, it was found that the Hprt gene is difficult to use as a selection marker in ES cells.

[0131] Therefore, we decided to isolate clones by sorting mRuby2-negative cells using the expression of the fluorescent gene mRuby2, which was designed to remain on the remaining human chromosome 6. As a result, we successfully isolated the TV3-4RENKAT1b#38dh6 clone, which had lost the remaining human chromosome 6 (Figure 12). Of these, TV3-4RENKAT1b#3, which had 40 chromosomes (39 in XO) and in which MHC-humanized mouse chromosome 17 was confirmed by FISH, was used for subsequent experiments. Furthermore, we performed whole-genome sequencing of TV3-4RENKAT1bdh6#3 using the mouse genome sequence and sequences within the human MHC region as references, and identified the haplotype of the introduced human MHC gene cluster (Figure 12C).

[0132] Establishment of MHC-humanized mice Chimeric mice were generated from TV3-4RENKAT1b#38dh6#3, and three mice with a coat color chimerism rate of 50% to 60% were obtained (FIG. 13A) (FIG. 4, step 9).

[0133] ES-derived cells carrying MHC-humanized chromosomes are engineered to express EGFP (Figure 13B). Therefore, we analyzed the contribution of MHC-humanized ES cells to the testes using EGFP expression as an indicator, and found that nearly 100% were EGFP-positive (Figure 13C). This suggested that there was a high probability of obtaining offspring if testicular cells were subjected to ICSI / ROSI. Therefore, we performed ICSI using testicular cells from chimeric mice (Figure 4, Step 10).

[0134] As a result, we succeeded in generating MHC-humanized mice that glow green throughout their bodies through germline transmission of MHC-humanized ES cells (Figures 14A and 14B). Furthermore, genomic PCR confirmed that EGFP-positive individuals possessed the human MHC region sequence (Figure 14C). These results demonstrate that we have successfully established MHC-humanized (heterozygous) mice.

[0135] Generation of MHC-humanized mice by natural mating of chimeric mice. Based on the results obtained so far, we found that MHC-humanized chimeric mice generated from B6-derived ES cells have low fertility. Therefore, we also performed MHC humanization on XO ES cells (B6 / CBA F1) that develop into females to generate chimeric mice (Figure 15A) (Figure 4, Step 9), which were then mated with wild-type mice. As a result, we successfully obtained MHC-humanized heterozygous mice by natural mating (Figure 15B-D) (Figure 4, Step 10).

[0136] 4. Analysis of Human MHC Gene Expression in MHC-Humanized (Heterozygous) Mice <Method> Analysis of Human MHC (also known as HLA) Protein Expression. To prepare splenocytes, the excised spleen was first minced with a knife, and then the tissue fragments were ground using the frosted section of a glass slide. The cells were then hemolyzed with 0.83% ammonium chloride and passed through a nylon mesh to prepare splenocytes. For flow cytometry analysis, cells were stained with Pacific Blue-labeled anti-mouse CD19 antibody (Biolegend), APC-labeled anti-HLA class I antibody (MBL) or APC-labeled anti-HLA-DR, DP, and DQ antibodies (Biolegend) using the APC Conjugation Kit-Lightning-Link (Abcam), and analyzed using a FACS Aria III (BD). In the Western blot analysis, detection was performed using anti-HLA-A, B, C antibody (MBL), anti-HLA-A antibody (Abcam), anti-HLA-DRA antibody (Abcam), or HRP-labeled anti-ACTB antibody (Genscript).

[0137] To prepare lung fibroblasts, the excised lung tissue was first minced with a knife and treated with 0.1% collagenase A (Roche) for 1 hour. The cells were then released by pipetting and passed through a nylon mesh to prepare lung fibroblasts. The lung fibroblasts were cultured in DMEM supplemented with 10% FCS.

[0138] Results: Human MHC gene expression in splenocytes was analyzed at the protein level. First, splenocytes were prepared from wild-type (Wt) and MHC-humanized (heterozygous) mice (Het) established from XO ES cells, and human MHC protein expression was analyzed by Western blotting using anti-HLA-A, B, C, anti-HLA-A, and anti-HLA-DRA antibodies. As a result, human MHC expression was specifically detected in Het-derived splenocytes (Figure 16A).

[0139] Next, the expression of HLA proteins on the cell surface was analyzed. Spleen cells are mainly composed of B cells and T cells, and MHC class I should be expressed in all cells, while MHC class II should be expressed in B cells. Therefore, analysis was performed by flow cytometry using an antibody against CD19, a B cell marker, an anti-HLA class I antibody that recognizes human MHC class I, and an anti-HLA-DP, DQ, DR antibody that recognizes human MHC class II. HLA class I was detected in all Het-derived spleen cells, and HLA-DP, DQ, DR were specifically expressed in Het-derived B cells (Figure 16B). [Example 2] Generation of cells and animals with chromosomes deleted or deleted in MHC regions

[0140] In MHC humanized (heterozygous) ES cells, the MHC region of one allele is humanized, so any deletion of genomic sequences or gene disruption using CRISPR / Cas9 targeting sequences within the mouse MHC region will occur entirely on the allele containing the mouse MHC region (Figure 17). Therefore, disruption or deletion of mouse MHC genes in MHC humanized (heterozygous) ES cells can produce mouse MHC gene-deficient alleles. Since MHC humanized (heterozygous) mice can be produced from MHC humanized (heterozygous) ES cells, mouse MHC gene-deficient mice can be produced by producing mouse MHC gene-deficient MHC humanized (heterozygous) ES cells.

[0141] <Outline of this Example> In this example, mouse MHC gene-deficient alleles were generated using MHC-humanized (heterozygous) ES cells in which the MHC region from Daxx to Mog of CBAxC57BL / 6 F1 mouse-derived XO ES cells was humanized. First, examining the distribution of MHC genes on the genome, it was found that 35 of the 39 genes exist in five clusters without any non-MHC genes (Figure 18). These clusters are designated d1, d2, d3, d4, and d5 in descending order of size. It can be seen that the five clusters, H2-K1, H2-Oa, H2-M5, H2-M3, and H2-M2, do not form clusters and exist independently. These isolated MHC genes were classified as d6, and the MHC genes present within the cluster were deleted using two CRISPR / Cas9 sequences designed to sandwich the cluster, while the isolated MHC genes were disrupted using CRISPR / Cas9 sequences designed to disrupt the protein-coding sequence of each MHC gene.

[0142] Specifically, d1 to d6 clones were isolated by sequentially deleting the MHC genes as shown in Figure 19. The d1 clone was a clone in which the MHC genes in the d1 cluster of MHC humanized (heterozygous) ES cells had been deleted, the d2 clone was a clone in which the MHC genes in the d2 cluster had been deleted from the d1 clone, the d3 clone was a clone in which the MHC genes in the d3 cluster had been deleted from the d2 clone, the d4 clone was a clone in which the MHC genes in the d4 cluster had been deleted from the d3 clone, the d5 clone was a clone in which the MHC genes in the d5 cluster had been deleted from the d4 clone and the H2-M5 gene had been simultaneously disrupted, and the d6 clone was a clone in which the H2-K1, H2-Oa, H2-M3, and H2-M2 genes of the d5 clone had been disrupted. In producing the d6 clone, simultaneous disruption of four genes results in low gene disruption efficiency. Therefore, in some cases, a d6a clone was isolated by disrupting only H2-K1 and H2-M3 of the d5 clone, and then H2-Oa and H2-M2 were disrupted to isolate the d6 clone.

[0143] <Method> Preparation of genome editing vector For expression of gRNA and Cas9, pSpCas9(BB)-2A-GFP(PX458) (Addgene plasmid #48138; http: / / n2t.net / addgene:48138; RRID: Addgene 48138) was modified and the marker fluorescent gene was replaced with ametrine, PX458.1a, was used.

[0144] The following vector was used to delete the d1 cluster: The sequence of the gRNA is as follows:

[0145] The following vector was used to delete the d2 cluster: The sequence of the gRNA is as follows:

[0146] The following vector was used to delete the d3 cluster: The sequence of the gRNA is as follows:

[0147] The following vector was used to delete the d4 cluster: The sequence of the gRNA is as follows:

[0148] The following vectors were used to delete the d5 cluster and disrupt H2-M5: The sequences of the gRNAs are as follows:

[0149] The following vectors were used to disrupt H2-K1, H2-Oa, H2-M3, and H2-M2 contained in d6. The sequences of the gRNAs are as follows:

[0150] Isolation of d1 clones To create d1 clones in which the mouse MHC genes contained in d1 had been deleted, MHC-humanized (heterozygous) ES cells were transfected with PX458.1a-M10.2upCR1 and PX458.1a-M10.6downCR1 using Lipofectamine 3000 (Thermo Fisher Scientific), and the following day, vector-introduced cells were sorted using the expression of the fluorescent gene ametrine as an indicator. Two or three days after sorting, the cells were replated for clonal isolation. Genomic DNA was prepared from the isolated clones, and clones in which the d1 cluster had been deleted were identified by PCR. The primers used for PCR are as follows:

[0151] Among the PCR-positive clones, clones with normal chromosome numbers were subjected to FISH analysis, which confirmed the deletion of the d1 cluster. For FISH analysis, Cy3-labeled RP23-147G23 was used to detect mouse chromosome 17, DY490-labeled RP23-355M14 to detect the d1 cluster, and Cy5-labeled RP11-54H13 to detect the human MHC region.

[0152] Isolation of d2 Clones To generate d2 clones in which the mouse MHC genes contained in d1 and d2 had been deleted, PX458.1a-H2D1upCR1 and PX458.1a-Q10downCR1 were transfected into d1 clones using Lipofectamine 3000. The following day, the vector-transfected cells were sorted using the expression of the fluorescent gene ametrine as an indicator. Two or three days after sorting, the cells were replated for clonal isolation. Genomic DNA was prepared from the isolated clones, and clones in which the d2 cluster had been deleted were identified by PCR. The primers used for PCR are as follows:

[0153] Among the PCR-positive clones, clones with normal chromosome numbers were subjected to FISH analysis, which confirmed the deletion of the d2 cluster. For FISH analysis, Cy3-labeled RP23-147G23 was used to detect mouse chromosome 17, DY490-labeled RP23-101J16 to detect the d2 cluster, and Cy5-labeled RP11-54H13 to detect the human MHC region.

[0154] Isolation of d3 clones: To generate d3 clones with deleted mouse MHC genes contained in d1, d2, and d3, PX458.1a-T24upCR1 and PX458.1a-T3downCR1 were transfected into d2 clones using Lipofectamine 3000. The following day, vector-transfected cells were sorted using the expression of the fluorescent gene ametrine as an indicator. Two or three days after sorting, the cells were replated for clonal isolation. Genomic DNA was prepared from the isolated clones, and clones with deleted d3 clusters were identified by PCR. The primers used for PCR are as follows:

[0155] Among the PCR-positive clones, clones with normal chromosome numbers were subjected to FISH analysis, which confirmed the deletion of the d3 cluster. For FISH analysis, Cy3-labeled RP23-147G23 was used to detect mouse chromosome 17, DY490-labeled RP23-268P19 to detect the d3 cluster, and Cy5-labeled RP11-54H13 to detect the human MHC region.

[0156] Isolation of d4 clones: To generate d4 clones with deleted mouse MHC genes contained in d1, d2, d3, and d4, PX458.1a-H20bupCR1 and PX458.1a-H2Eb2CR1 were transfected into d3 clones using Lipofectamine 3000. The following day, vector-transfected cells were sorted using the expression of the fluorescent gene ametrine as an indicator. Two or three days after sorting, cells were replated for clonal isolation. Genomic DNA was prepared from the isolated clones, and clones with deleted d4 clusters were identified by PCR. The primers used for PCR are as follows: PCR-positive clones were confirmed to have normal chromosome numbers and then used to generate d5 clones.

[0157] Isolation of d5 clones: To generate d5 clones by deleting the mouse MHC genes contained in d1, d2, d3, d4, and d5 and disrupting the H2-M5, d4 clones were transfected with PX458.1a-H2DMaupCR1, PX458.1a-DMb1downCR1, and PX458.1a-H2-M5CR1 using Lipofectamine 3000. The following day, vector-transfected cells were sorted using the expression of the fluorescent gene ametrine as an indicator. Two or three days after sorting, cells were replated for clonal isolation. Genomic DNA was prepared from the isolated clones, and clones with deleted d5 clusters were identified by PCR. The primers used for PCR are as follows:

[0158] PCR-positive clones were further analyzed for gene disruption of H2-M5. First, using the genomic DNA of each clone as a template, a sequence containing the target site of PX458.1a-H2-M5CR1 was amplified with the following primers:

[0159] The PCR product was purified using Sephadex G-50 (Cytiva) or FavorPrep GEL / PCR Purification Mini Kit (Favorgen), and the disruption of the target gene was confirmed by sequence analysis using the following primers. Clones in which deletion of the d5 cluster and disruption of H2-M5 were confirmed were confirmed to have a normal chromosome number, and then used to prepare d6 clones.

[0160] Isolation of d6 clones To create d6 clones in which all mouse MHC genes contained in d1 to d6 were deleted or disrupted, d5 clones were transfected with PX458.1a-H2M2CR1, PX458.1a-H2M3CR1, PX458.1a-H20aCR1, and PX458.1a-H2K1CR1 using Lipofectamine 3000, and the next day, the vector-introduced cells were sorted using the expression of the fluorescent gene ametrine as an indicator. The cells were reseeded on the second or third day after sorting to perform clone isolation. Since simultaneous gene disruption of the above four genes reduces the efficiency of d6 clone isolation, in some cases, d6a clones were isolated by gene disruption of H2-K1 and H2-M3 from the d5 clone, and then H2-Oa and H2-M2 were gene-disrupted to create d6 clones.

[0161] Genomic DNA was prepared from the isolated clones, and sequences containing the target sites of the gRNAs designed for each MHC gene were amplified using the following primers: H2K1

[0162] H2-M3

[0163] H2-M2

[0164] H2-Oa

[0165] Each PCR product was purified using Sephadex G-50 or FavorPrep GEL / PCR Purification Mini Kit, and the disruption of the target gene was confirmed by sequence analysis using the following primers.

[0166] H2K1

[0167] H2-M3

[0168] H2-M2

[0169] H2-Oa

[0170] For SNP analysis upstream and downstream of the H2-M2 gRNA target site, the genomic sequence containing the SNP site was amplified and confirmed by sequence analysis. Each PCR product was purified using Sephadex G-50 or FavorPrep GEL / PCR Purification Mini Kit. The primer sequences used for amplification and sequence analysis of the genomic sequence are as follows:

[0171] Amplification of genomic sequences containing a SNP site (GRCm38 / mm10, chr17:37466727) upstream of the H2-M2 gRNA target site Sequence analysis The SNP sequence is A in CBA and C in C57BL / 6.

[0172] Amplification of genomic sequences containing SNP sites (GRCm38 / mm10, chr17: 37491000, 37491007) downstream of the H2-M2 gRNA target site Sequence analysis The SNP sequences are 37491000, which is C in CBA and T in C57BL / 6, and 37491007, which is C in CBA and A in C57BL / 6.

[0173] Clones that demonstrated gene disruption and SNP maintenance were confirmed to have normal chromosome numbers. Analysis of d5 homozygous mice. For analysis of CD4-positive T cells, spleen cells or thymocytes from wild-type or d5 homozygous mice were prepared and stained with anti-CD3, CD4, and CD8 antibodies (Biolegend). The proportions of CD4- and CD8-positive cells among CD3-positive cells were analyzed using a FACSAria III (BD Biosciences).

[0174] For class switching analysis, 11- to 15-week-old wild-type (n = 3) or d5 homozygous mice (n = 1) were subcutaneously immunized with 10 μg recombinant SARS-CoV-2 S Protein S1+S2 (Biolegend) and 50 μg poly(I:C) (Invivogen). Three weeks later, they were boosted with 10 μg recombinant SARS-CoV-2 S Protein S1+S2 and 50 μg poly(I:C). Serum was collected one week after the booster immunization and analyzed for anti-S1 IgG1 and IgG2 by ELISA.

[0175] Analysis of d6 chimeric mice MHC gene cluster deletion chromosomes were identified by the deletion of the d5 cluster using the following primers: The size of the PCR product was 447 bp for the wild type, and 633 bp for the d5 deletion in the ES clone used to generate the d6 mice.

[0176] <Results> Isolation of d1 clones gRNAs were designed within the upstream portion of the H2-M10.2 locus and the protein coding sequence of the H2-M10.6 locus, and these two gRNAs were used to induce deletions in the d1 cluster of MHC humanized (heterozygous) ES cells using CRISPR / Cas9, resulting in the isolation of d1 clones (Figure 20). Genomic PCR was performed using a primer set designed to generate PCR amplification products when the genomic sequence sandwiched between the two CRISPR / Cas9 target sequences was deleted, and multiple PCR-positive clones were obtained. Among the PCR-positive clones, d1 clone #26 was confirmed to have a normal chromosome number (39 in the case of XO ES cells), and FISH analysis using a probe that recognizes the d1 cluster confirmed the deletion of the d1 cluster.

[0177] Isolation of d2 clones gRNA was designed within the protein coding sequence of the upstream portion of the H2-D1 locus and the H2-Q10 locus, and these two gRNAs were used to induce deletion in the d2 cluster of d1 clone #26 using CRISPR / Cas9 to isolate d2 clones (Figure 21). Genomic PCR was performed using a primer set designed to generate PCR amplification products when the genomic sequence sandwiched between the two CRISPR / Cas9 target sequences was deleted, and multiple PCR-positive clones were obtained. Among the PCR-positive clones, d2 clone #85 was confirmed to have a normal chromosome number, and FISH analysis using a probe that recognizes the d2 cluster confirmed the deletion of the d2 cluster.

[0178] Isolation of d3 clones gRNA was designed in the upstream portion of the H2-T24 locus and the downstream portion of the H2-T3 locus, and these two gRNAs were used to induce deletion in the d3 cluster of d2 clone #85 by CRISPR / Cas9 to isolate d3 clones (Figure 22). When genomic PCR was performed using a primer set designed to generate PCR amplification products when the genomic sequence sandwiched between the two CRISPR / Cas9 target sequences was deleted, multiple PCR-positive clones were obtained. Among the PCR-positive clones, d3 clone #26, which was confirmed to have a normal chromosome number, was analyzed by FISH using a probe that recognizes the d3 cluster, and deletion of the d3 cluster was confirmed.

[0179] Isolation of d4 clones gRNAs were designed within the upstream portion of the H2-Ob locus and the protein coding sequence of the H2-Eb2 locus, and deletions were induced in the d4 cluster of d3 clone #26 using CRISPR / Cas9 with these two gRNAs to isolate d4 clones (Figure 23). Genomic PCR was performed using a primer set designed to generate PCR amplification products when the genomic sequence sandwiched between the two CRISPR / Cas9 target sequences was deleted, and multiple PCR-positive clones were obtained. Furthermore, it was confirmed that the PCR-positive d4 clone #72 had a normal chromosome number.

[0180] Isolation of d5 clones gRNA was designed within the protein coding sequence of the H2-DMa locus and downstream of the H2-DMb1 locus, and deletions were induced in the d5 cluster of d4 clone #72 using CRISPR / Cas9 with these two gRNAs. At the same time, gene disruption of H2-M5 was performed using gRNA designed within the protein coding region of H2-M5, and d5 clones were isolated (Figure 24). Genomic PCR was performed using a primer set designed to generate PCR amplification products when the genomic sequence sandwiched between the two CRISPR / Cas9 target sequences was deleted, and multiple PCR-positive clones were obtained. Next, sequence analysis was performed on the PCR-positive clones to determine whether the H2-M5 gene had been disrupted. As a result, a deletion of 13 bases was confirmed in d5 clone #22. Further chromosome number analysis was performed on clones in which gene disruption of H2-M5 was confirmed, and it was confirmed that d5 clone #22 had a normal chromosome number.

[0181] Isolation of d6 clones Using gRNAs designed within the protein-coding sequences of H2-K1, H2-Oa, H2-M2, and H2-M3, gene disruption of d5 clone #22 was performed by CRISPR / Cas9, and d6 clones were isolated (Figure 25).

[0182] Sequence analysis was performed to confirm whether each gene had been disrupted by CRISPR / Cas9, and clones in which all four genes had been disrupted were identified. Because H2-M2 and H2-M3 are located outside the MHC humanized region, two copies of the gene exist in the genome. Because mutations occur separately in each allele, sequence analysis may detect two different sequences. The ES cells used in this example were derived from an F1 cross between CBA and C57BL / 6, and the human MHC region is present in the CBA-derived chromosome. Therefore, gene disruption was confirmed for H2-M3 using sequence primers capable of specifically detecting the C57BL / 6 allele using SNPs. For H2-M2, no SNPs were present near the CRISPR / Cas9 target site, making allele-specific sequence analysis difficult. Therefore, sequence analysis was performed using primers that detect both alleles, and only one sequence was detected in d6 clone #198 shown in Figure 25. This may be because the same mutation is present in both alleles, or because the introduction of multiple CRISPR / Cas9s may have induced large deletions between adjacent CRISPR / Cas9 target sequences, leaving only one allele that can be amplified by PCR.

[0183] Therefore, we analyzed whether SNPs near H2-M2 could be detected to confirm whether large deletions had occurred (Figure 26). As a result, in clone #198, SNPs upstream and downstream of H2-M2 were detected, confirming that no large deletions had been induced. Since #198 also had a normal chromosome number, it is believed that we have successfully produced ES cells with alleles that disrupt all 39 mouse MHC genes.

[0184] Generation of d5 clone-derived mice. The d5 ES clone isolated during the process of deleting mouse MHC had 35 of the 39 mouse MHC genes disrupted or deleted, and did not possess mouse MHC class II, which functions in antigen presentation. Therefore, d5 homozygous mice were generated using the d5 ES clone, and analysis was performed to determine whether mouse MHC class II function was actually lost. First, T cell differentiation in wild-type and d5 homozygous mice was analyzed. Because d5 homozygous mice are unable to present antigens via MHC class II, it is thought that CD4+ T cells do not differentiate. Therefore, analysis of the proportion of CD4+ T cells in the spleen and thymus of d5 homozygous mice revealed a significant decrease (Figure 27). Furthermore, to analyze antibody class switching in d5 homozygous mice, d5 homozygous mice were immunized with the spike protein of SARS-CoV-2 and anti-S1 IgG1 and IgG2 in the serum were analyzed. It was found that IgG1 and IgG2 that recognize the spike protein were extremely low or absent in d5 homozygous mice. These results suggest that functional CD4+ T cells are significantly reduced or absent in d5 homozygous mice because they do not express mouse MHC class II, which functions in antigen presentation.

[0185] To establish mice in which all mouse MHC genes were disrupted or deleted, chimeric mice were generated from d6 ES clones and germline-transmitted to generate d6 heterozygous mice (Fig. 28A, B). These d6 heterozygous mice were then mated with each other, and the resulting offspring were analyzed by genomic PCR. These offspring were confirmed to be MHC gene-deficient mice with a homozygous MHC-deficient chromosome (Fig. 28C, D).

Claims

1. A chromosome of a non-human mammal in which all or part of the major histocompatibility complex (MHC) genes are deleted or destroyed.

2. The chromosome of claim 1, wherein the non-human mammal is a rodent.

3. The chromosome according to claim 2, wherein the rodent is a mouse.

4. A cell containing the chromosome described in claim 1 in a homozygous or heterozygous form.

5. The cell of claim 4 which is an embryonic stem cell.

6. A non-human mammal having the chromosome described in claim 1.

7. A method for producing a cell having a chromosome having the major histocompatibility complex (MHC) gene group of a non-human mammal (non-human mammal 1) and a chromosome in which all or part of the MHC gene group 1 has been destroyed or deleted, the method comprising the step of disrupting or deleting all or part of the MHC gene group (MHC gene group 1) derived from a non-human mammal 1, from a cell having a heterozygous chromosome in which all or part of the major histocompatibility complex (MHC) gene group of a mammal 2 out of a pair of chromosomes of the non-human mammal 1 has been replaced with an MHC gene group of a mammal 2 other than the non-human mammal 1.

8. The method of claim 7, wherein the cell is an embryonic stem cell.

9. The method according to claim 7, wherein the step of disrupting or deleting the MHC gene group 1 is carried out by genome editing technology using CRISPR / Cas9.

10. The method of claim 7, wherein the non-human mammal 1 is a rodent.

11. The method of claim 10, wherein the rodent is a mouse.

12. The step of disrupting or deleting all or a part of the MHC gene group 1 comprises: a d1 gene group consisting of H2-M10.2, H2-M10.1, H2-M10.3, H2-M10.4, H2-M11, H2-M9, H2-M1, H2-M10.5 and H2-M10.6 genes; a d2 gene group consisting of H2-D1, H2-Q1, H2-Q2, H2-Q4, H2-Q5, H2-Q6, H2-Q7 and H2-Q10 genes; a d3 gene group consisting of H2-T24, H2-T23, H2-T22, GM11127, H2-BI, H2-T10, GM7030, GM8909 and H2-T3 genes; 12. The method according to claim 11, comprising a step of disrupting or deleting all or a part of the d1 gene group, d2 gene group, d3 gene group, d4 gene group, d5 gene group and d6 gene group from a cell having mouse MHC gene groups consisting of a d4 gene group consisting of Ob, H2-Ab1, H2-Aa, H2-Eb1 and H2-Eb2 genes, a d5 gene group consisting of H2-DMa, H2-DMb2 and H2-DMb1 genes, and a d6 gene group consisting of H2-K1, H2-Oa, H2-M5, H2-M3 and H2-M2 genes, by genome editing technology using CRISPR / Cas9.

13. The method described in claim 12, wherein the genome editing vector used in the genome editing technology using CRISPR / Cas9 is a combination of vectors consisting of gRNAs having the base sequences shown in SEQ ID NOs: 53 to 67.

14. A method for producing a non-human mammal 1 having a chromosome in which all or part of the MHC gene group 1 has been destroyed or deleted, comprising the steps of: using cells obtained by the method according to claim 7 to produce a non-human mammal 1 having, in a pair of chromosomes of the non-human mammal 1, a chromosome having the MHC gene group 2 of the mammal 2 and a chromosome in which all or part of the MHC gene group 1 has been destroyed or deleted; and mating the non-human mammal 1.

15. A method for producing said cells, comprising the step of recovering said cells having a chromosome in which all or part of MHC gene group 1 has been disrupted or deleted from a non-human mammal 1 produced by the method according to claim 14.

16. A method for producing a chromosome in which MHC gene group 1 is disrupted or deleted, comprising the step of recovering said chromosome from a non-human mammal 1 produced by the method according to claim 14, or from a cell produced by the method according to claim 15.

17. A genome editing vector for use in the genome editing technique using CRISPR / Cas9 described in claim 13, comprising a combination of vectors consisting of gRNAs having the base sequences shown in SEQ ID NOs: 53 to 67.

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