Methods for breaking immunological tolerance using multiple guide rnas

Abstract

Methods and compositions are provided for making non-human animals with reduced tolerance of a foreign antigen of interest and making antigen-binding proteins against that foreign antigen of interest using such animals. The methods and compositions employ CRISPR/Cas9 systems using multiple guide RNAs to reduce or eliminate expression of a self-antigen homologous to or sharing an epitope of interest with the foreign antigen of interest or to reduce or eliminate expression of an epitope on the self-antigen that is shared with the foreign antigen of interest.

We claim: 
     
         1 . A method of generating antigen-binding proteins against a foreign antigen of interest, comprising:
 (a) making a genetically modified non-human animal with reduced tolerance of a foreign antigen of interest, comprising:
 (i) introducing into a non-human animal one-cell stage embryo or a non-human animal pluripotent cell that is not a one-cell stage embryo:
 (I) a Cas9 protein; 
 (II) a first guide RNA that hybridizes to a first guide RNA recognition sequence within a target genomic locus, wherein the target genomic locus comprises all or part of a gene encoding a self-antigen homologous to or sharing an epitope of interest with the foreign antigen of interest; and 
 (III) a second guide RNA that hybridizes to a second guide RNA recognition sequence within the target genomic locus; 
 
 wherein the target genomic locus is modified in a pair of corresponding first and second chromosomes to produce a modified non-human animal one-cell stage embryo or a modified non-human animal pluripotent cell with a biallelic modification, wherein expression of the self-antigen is eliminated; and 
 (ii) producing a genetically modified F0 generation non-human animal from the modified non-human animal one-cell stage embryo or the modified non-human animal pluripotent cell, wherein the target genomic locus is modified in the pair of corresponding first and second chromosomes in the genetically modified F0 generation non-human animal such that expression of the self-antigen is eliminated; 
   (b) immunizing the genetically modified F0 generation non-human animal produced in step (a) with the foreign antigen of interest; and   (c) maintaining the genetically modified F0 generation non-human animal under conditions sufficient to initiate an immune response to the foreign antigen of interest, wherein the genetically modified F0 generation non-human animal produces antigen-binding proteins against the foreign antigen of interest.   
     
     
         2 . The method of  claim 1 , wherein the cell in step (a)(i) is the non-human animal pluripotent stem cell, and the producing the genetically modified F0 generation non-human animal in step (a)(ii) comprises:
 (I) introducing the modified non-human animal pluripotent cell into a host embryo; and   (II) implanting the host embryo into a surrogate mother to produce the genetically modified F0 generation non-human animal in which the target genomic locus is modified in the pair of corresponding first and second chromosomes such that expression of the self-antigen is eliminated.   
     
     
         3 . The method of  claim 2 , wherein the pluripotent cell is an embryonic stem (ES) cell. 
     
     
         4 . The method of  claim 1 , wherein the cell in step (a)(i) is the non-human animal one-cell stage embryo, and the producing the genetically modified F0 generation non-human animal in step (a)(ii) comprises implanting the modified non-human animal one-cell stage embryo into a surrogate mother to produce the genetically modified F0 generation non-human animal in which the target genomic locus is modified in the pair of corresponding first and second chromosomes such that expression of the self-antigen is eliminated. 
     
     
         5 . The method of  claim 1 , further comprising making a hybridoma from B cells isolated from the immunized, genetically modified F0 generation non-human animal. 
     
     
         6 . The method of  claim 1 , further comprising obtaining from the immunized, genetically modified F0 generation non-human animal a first nucleic acid sequence encoding an immunoglobulin heavy chain variable domain of one of the antigen-binding proteins against the foreign antigen of interest and/or a second nucleic acid sequence encoding an immunoglobulin light chain variable domain of one of the antigen-binding proteins against the foreign antigen of interest. 
     
     
         7 . The method of  claim 6 , wherein the first nucleic acid sequence and/or the second nucleic acid sequence are obtained from a lymphocyte of the genetically modified F0 generation non-human animal or from a hybridoma produced from the lymphocyte. 
     
     
         8 . The method of  claim 7 , wherein the genetically modified F0 generation non-human animal comprises a humanized immunoglobulin locus, and wherein the first nucleic acid sequence encodes a human immunoglobulin heavy chain variable domain, and the second nucleic acid sequence encodes a human immunoglobulin light chain variable domain. 
     
     
         9 . The method of  claim 1 , wherein the antigen-binding proteins produced by the genetically modified F0 generation non-human animal against the foreign antigen of interest have a higher titer than antigen-binding proteins produced by a control non-human animal that is wild type at the target genomic locus following immunization of the control non-human animal with the foreign antigen of interest. 
     
     
         10 . The method of  claim 1 , wherein a more diverse repertoire of antigen-binding proteins against the foreign antigen of interest is produced by the genetically modified F0 generation non-human animal following immunization of the genetically modified F0 generation non-human animal with the foreign antigen of interest compared with antigen-binding proteins produced by a control non-human animal that is wild type at the target genomic locus following immunization of the control non-human animal with the foreign antigen of interest. 
     
     
         11 . The method of  claim 1 , wherein the antigen-binding proteins produced by the genetically modified F0 generation non-human animal against the foreign antigen of interest use a greater diversity of heavy chain V gene segments and/or light chain V gene segments compared with antigen-binding proteins produced by a control non-human animal that is wild type at the target genomic locus following immunization of the control non-human animal with the foreign antigen of interest. 
     
     
         12 . The method of  claim 1 , wherein some of the antigen-binding proteins produced by the genetically modified F0 generation non-human animal against the foreign antigen of interest cross-react with the self-antigen. 
     
     
         13 . The method of  claim 1 , wherein the first guide RNA recognition sequence is 5′ of the second guide RNA recognition sequence in the target genomic locus, and
 wherein step (a)(i) further comprises performing a retention assay to determine the copy number is two for a region 5′ and within about 1 kb of the first guide RNA recognition sequence and/or for a region 3′ and within about 1 kb of the second guide RNA recognition sequence. 
 
     
     
         14 . The method of  claim 1 , wherein the foreign antigen of interest is an ortholog of the self-antigen. 
     
     
         15 . The method of  claim 1 , wherein the foreign antigen of interest comprises of all or part of a human protein. 
     
     
         16 . The method of  claim 1 , wherein the target genomic locus is modified to comprise an insertion of one or more nucleotides, a deletion of one or more nucleotides, or a replacement of one or more nucleotides. 
     
     
         17 . The method of  claim 16 , wherein the target genomic locus is modified to comprise a deletion of one or more nucleotides. 
     
     
         18 . The method of  claim 17 , wherein the deletion is a precise deletion without random insertions and deletions (indels). 
     
     
         19 . The method of  claim 1 , wherein the first guide RNA recognition sequence comprises the start codon for the gene encoding the self-antigen or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon, and the second guide RNA recognition sequence comprises the stop codon for the gene encoding the self-antigen or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the stop codon. 
     
     
         20 . The method of  claim 1 , wherein the first and second guide RNA recognition sequences are different, and each of the first and second guide RNA recognition sequences comprises the start codon for the gene encoding the self-antigen or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon. 
     
     
         21 . The method of  claim 1 , wherein the target genomic locus is modified to comprise a biallelic deletion of between about 0.1 kb to about 200 kb. 
     
     
         22 . The method of  claim 1 , wherein the modification comprises a biallelic deletion of all or part of the gene encoding the self-antigen. 
     
     
         23 . The method of  claim 1 , wherein the modification comprises a biallelic disruption of the start codon of the gene encoding the self-antigen. 
     
     
         24 . The method of  claim 1 , wherein the introducing step (a)(i) further comprises introducing into the non-human animal pluripotent cell or the non-human animal one-cell stage embryo:
 (iv) a third guide RNA that hybridizes to a third guide RNA recognition sequence within the target genomic locus; and/or   (v) a fourth guide RNA that hybridizes to a fourth guide RNA recognition sequence within the target genomic locus.   
     
     
         25 . The method of  claim 1 , wherein the cell in step (a)(i) is the non-human animal pluripotent stem cell, and the Cas9 protein, the first guide RNA, and the second guide RNA are each introduced into the non-human animal pluripotent stem cell in the form of DNA. 
     
     
         26 . The method of  claim 1 , wherein the cell in step (a)(i) is the non-human animal pluripotent stem cell, and the Cas9 protein, the first guide RNA, and the second guide RNA are each introduced into the non-human animal pluripotent stem cell by electroporation or nucleofection. 
     
     
         27 . The method of  claim 1 , wherein the cell in step (a)(i) is the non-human animal one-cell stage embryo, and the Cas9 protein, the first guide RNA, and the second guide RNA are each introduced into the non-human animal one-cell stage embryo in the form of RNA. 
     
     
         28 . The method of  claim 1 , wherein the cell in step (a)(i) is the non-human animal one-cell stage embryo, and the Cas9 protein, the first guide RNA, and the second guide RNA are introduced into the non-human animal one-cell stage embryo by pronuclear injection or cytoplasmic injection. 
     
     
         29 . The method of  claim 1 , wherein an exogenous repair template is not introduced in step (a)(i). 
     
     
         30 . The method of  claim 1 , wherein the introducing step (a)(i) further comprises introducing into the non-human animal pluripotent cell or the non-human animal one-cell stage embryo an exogenous repair template comprising a 5′ homology arm that hybridizes to a 5′ target sequence at the target genomic locus and a 3′ homology arm that hybridizes to a 3′ target sequence at the target genomic locus, provided that if the cell in step (a)(i) is the non-human animal one-cell stage embryo, the exogenous repair template is no more than about 5 kb in length. 
     
     
         31 . The method of  claim 30 , wherein the exogenous repair template further comprises a nucleic acid insert flanked by the 5′ homology arm and the 3′ homology arm. 
     
     
         32 . The method of  claim 31 , wherein the nucleic acid insert is homologous or orthologous to the target genomic locus. 
     
     
         33 . The method of  claim 30 , wherein the exogenous repair template is between about 50 nucleotides to about 1 kb in length. 
     
     
         34 . The method of  claim 33 , wherein the exogenous repair template is between about 80 nucleotides to about 200 nucleotides in length. 
     
     
         35 . The method of  claim 30 , wherein the exogenous repair template is a single-stranded oligodeoxynucleotide. 
     
     
         36 . The method of  claim 30 , wherein the cell in step (a)(i) is the non-human animal pluripotent cell, and wherein:
 (a) the exogenous repair template is a large targeting vector (LTVEC) that is at least 10 kb in length; or   (b) the exogenous repair template is an LTVEC, wherein the sum total of the 5′ and 3′ homology arms of the LTVEC is at least 10 kb in length.   
     
     
         37 . The method of  claim 30 , wherein the target genomic locus is modified to comprise a deletion of one or more nucleotides, and
 wherein the deleted nucleic acid sequence consists of the nucleic acid sequence between the 5′ and 3′ target sequences.   
     
     
         38 . The method of  claim 30 , wherein the exogenous repair template comprises a nucleic acid insert flanked by the 5′ homology arm and the 3′ homology arm,
 wherein the nucleic acid insert is homologous or orthologous to the deleted nucleic acid sequence, 
 wherein the target genomic locus is modified to comprise a deletion of one or more nucleotides, and 
 wherein the nucleic acid insert replaces the deleted nucleic acid sequence. 
 
     
     
         39 . The method of  claim 1 , wherein the non-human animal comprises a humanized immunoglobulin locus. 
     
     
         40 . The method of  claim 1 , wherein the non-human animal is a rodent. 
     
     
         41 . The method of  claim 40 , wherein the rodent is a mouse. 
     
     
         42 . The method of  claim 41 , wherein the mouse strain comprises a BALB/c strain. 
     
     
         43 . The method of  claim 42 , wherein the mouse strain comprises BALB/c, C57BL/6, and 129 strains. 
     
     
         44 . The method of  claim 43 , wherein the mouse strain is 50% BALB/c, 25% C57BL/6, and 25% 129. 
     
     
         45 . The method of  claim 41 , wherein the MHC haplotype of the mouse is MHC b/d . 
     
     
         46 . The method of  claim 41 , wherein the mouse comprises in its germline human unrearranged variable region gene segments inserted at an endogenous mouse immunoglobulin locus. 
     
     
         47 . The method of  claim 46 , wherein the human unrearranged variable region gene segments are heavy chain gene segments, and the mouse immunoglobulin locus is a heavy chain locus, and/or wherein the human unrearranged variable region gene segments are kappa or lambda light chain segments, and the mouse immunoglobulin locus is a light chain locus. 
     
     
         48 . The method of  claim 46 , wherein the mouse comprises in its germline human unrearranged variable region gene segments operably linked to a mouse constant region gene, wherein the mouse lacks a human constant region gene, and wherein the mouse constant region gene is at an endogenous mouse immunoglobulin locus. 
     
     
         49 . The method of  claim 46 , wherein the mouse comprises:
 (a) a hybrid heavy chain locus comprising an insertion of human immunoglobulin heavy chain V, D, and J gene segments, wherein the human heavy chain immunoglobulin V, D, and J gene segments are operably linked to a mouse immunoglobulin heavy chain gene, wherein the mouse immunoglobulin heavy chain gene is at an endogenous mouse immunoglobulin locus; and   (b) a hybrid light chain locus comprising an insertion of human immunoglobulin light chain V and J gene segments, wherein the human V and J gene segments are operably linked to a mouse immunoglobulin light chain constant region gene sequence;   wherein (a) rearranges to form a hybrid heavy chain sequence comprising a human variable region operably linked to a mouse constant region, and (b) rearranges to form a hybrid light chain sequence comprising a human variable region operably linked to a mouse constant region, and wherein the mouse is incapable of forming an antibody that comprises a human variable region and a human constant region.   
     
     
         50 . The method of  claim 41 , wherein the mouse comprises in its germline a humanized immunoglobulin light chain variable locus comprising no more than one or no more than two rearranged human light chain V/J sequences operably linked to a mouse light chain constant region, and wherein the mouse further comprises a humanized immunoglobulin heavy chain variable locus comprising at least one unrearranged human V, at least one unrearranged human D, and at least one unrearranged human J segment operably linked to a mouse heavy chain constant region gene. 
     
     
         51 . The method of  claim 50 , wherein the mouse comprises a humanized heavy chain immunoglobulin variable locus and a humanized light chain immunoglobulin variable locus, wherein the mouse expresses a single light chain. 
     
     
         52 . The method of  claim 51 , wherein the mouse comprises:
 (a) a single rearranged human immunoglobulin light chain variable region (V L /J L ) that encodes a human V L  domain of an immunoglobulin light chain, wherein the single rearranged human V L /J L  region is selected from a human Vκ1-39/Jκ5 gene segment or a human Vκ3-20/Jκ1 gene segment; and   (b) a replacement of endogenous heavy chain variable (V H ) gene segments with one or more human VH gene segments, wherein the human V H  gene segments are operably linked to an endogenous heavy chain constant (C H ) region gene, and the human V H  gene segments are capable of rearranging and forming a human/mouse chimeric heavy chain gene.   
     
     
         53 . The method of  claim 51 , wherein the mouse expresses a population of antibodies, and the mouse's germline includes only a single immunoglobulin kappa light chain variable region gene that is a rearranged human germline kappa light chain variable region gene,
 wherein the mouse is either heterozygous for the single immunoglobulin kappa light chain variable region gene in that it contains only one copy, or is homozygous for the single immunoglobulin kappa light chain variable region gene in that it contains two copies, the mouse being characterized by active affinity maturation so that:
 (i) each immunoglobulin kappa light chain of the population comprises a light chain variable domain that is encoded by the rearranged human germline kappa light chain variable region gene, or by a somatically mutated variant thereof; 
 (ii) the population includes antibodies comprising the immunoglobulin kappa light chains whose light chain variable domain is encoded by the rearranged human germline kappa light chain variable region gene and antibodies comprising the immunoglobulin kappa light chains whose light chain variable domain is encoded by the somatically mutated variants thereof; and 
 (iii) the mouse generates a diverse collection of somatically mutated high affinity heavy chains that successfully pair with the immunoglobulin kappa light chains to form the antibodies of the population. 
   
     
     
         54 . The method of  claim 51 , wherein the mouse is heterozygous or homozygous in its germline for:
 (a) an insertion at an endogenous mouse κ immunoglobulin light chain variable region locus of a rearranged Vκ/Jκ sequence comprising:
 (i) a single human germline Vκ sequence, which single human germline Vκ sequence is present in SEQ ID NO: 148 or SEQ ID NO: 149; and 
 (ii) a single human germline Jκ sequence, wherein the rearranged Vκ/Jκ sequence is operably linked to the endogenous mouse κ constant region; and 
   (b) an insertion at an endogenous mouse immunoglobulin heavy chain variable region locus of a plurality of human immunoglobulin heavy chain variable region gene segments, wherein the human immunoglobulin heavy chain variable region gene segments are operably linked to an endogenous mouse immunoglobulin heavy chain constant region, and the human immunoglobulin heavy chain variable region gene segments are capable of rearranging and forming a rearranged human/mouse chimeric immunoglobulin heavy chain gene.   
     
     
         55 . The method of  claim 46 , wherein the mouse comprises a modification of an immunoglobulin heavy chain locus, wherein the modification reduces or eliminates endogenous ADAM6 function,
 wherein the mouse comprises an ectopic nucleic acid sequence encoding a mouse ADAM6 protein, an ortholog thereof, a homolog thereof, or a fragment thereof, wherein the ADAM6 protein, ortholog thereof, homolog thereof, or fragment thereof is functional in a male mouse, and   wherein the ectopic nucleic acid sequence encoding the mouse ADAM6 protein, ortholog thereof, homolog thereof, or fragment thereof is present at the human heavy chain variable region locus.   
     
     
         56 . The method of  claim 1 , wherein the non-human animal is a mouse that is at least partially derived from a BALB/c strain, and the mouse comprises a humanized immunoglobulin locus,
 wherein the foreign antigen of interest is all or part of a human protein that is orthologous to the self-antigen,   wherein the first guide RNA recognition sequence comprises the start codon for the gene encoding the self-antigen or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon and the second guide RNA recognition sequence comprises the stop codon for the gene encoding the self-antigen or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the stop codon, and   wherein the modification comprises a biallelic deletion of all or part of the gene encoding the self-antigen, whereby expression of the self-antigen is eliminated.   
     
     
         57 . The method of  claim 1 , wherein the non-human animal is a mouse that is at least partially derived from a BALB/c strain, and the mouse comprises a humanized immunoglobulin locus,
 wherein the foreign antigen of interest is all or part of a human protein that is orthologous to the self-antigen,   wherein the first guide RNA recognition sequence comprises the start codon for the gene encoding the self-antigen and the second guide RNA recognition sequence comprises the stop codon for the gene encoding the self-antigen or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon, and   wherein the modification comprises biallelic disruption of the start codon for the gene encoding the self-antigen, whereby expression of the self-antigen is eliminated.   
     
     
         58 . The method of  claim 56 , wherein the mouse comprises:
 (a) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein, an ortholog thereof, a homolog thereof, or a fragment thereof, wherein the ADAM6 protein, ortholog thereof, homolog thereof, or fragment thereof is functional in a male mouse;   (b) a hybrid heavy chain locus comprising an insertion of human immunoglobulin heavy chain V, D, and J gene segments, wherein the human heavy chain immunoglobulin V, D, and J gene segments are operably linked to a mouse immunoglobulin heavy chain gene, wherein the mouse immunoglobulin heavy chain gene is at an endogenous mouse immunoglobulin locus; and   (c) a hybrid light chain locus comprising an insertion of human immunoglobulin light chain V and J gene segments, wherein the human V and J gene segments are operably linked to a mouse immunoglobulin light chain constant region gene sequence;   wherein (b) rearranges to form a hybrid heavy chain sequence comprising a human variable region operably linked to a mouse constant region, and (c) rearranges to form a hybrid light chain sequence comprising a human variable region operably linked to a mouse constant region, and wherein the mouse is incapable of forming an antibody that comprises a human variable region and a human constant region.   
     
     
         59 . The method of  claim 57 , wherein the mouse comprises:
 (a) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein, an ortholog thereof, a homolog thereof, or a fragment thereof, wherein the ADAM6 protein, ortholog thereof, homolog thereof, or fragment thereof is functional in a male mouse;   (b) a hybrid heavy chain locus comprising an insertion of human immunoglobulin heavy chain V, D, and J gene segments, wherein the human heavy chain immunoglobulin V, D, and J gene segments are operably linked to a mouse immunoglobulin heavy chain gene, wherein the mouse immunoglobulin heavy chain gene is at an endogenous mouse immunoglobulin locus; and   (c) a hybrid light chain locus comprising an insertion of human immunoglobulin light chain V and J gene segments, wherein the human V and J gene segments are operably linked to a mouse immunoglobulin light chain constant region gene sequence;   wherein (b) rearranges to form a hybrid heavy chain sequence comprising a human variable region operably linked to a mouse constant region, and (c) rearranges to form a hybrid light chain sequence comprising a human variable region operably linked to a mouse constant region, and wherein the mouse is incapable of forming an antibody that comprises a human variable region and a human constant region.   
     
     
         60 . The method of  claim 56 , wherein the mouse is heterozygous or homozygous in its germline for:
 (a) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein, an ortholog thereof, a homolog thereof, or a fragment thereof, wherein the ADAM6 protein, ortholog thereof, homolog thereof, or fragment thereof is functional in a male mouse;   (b) an insertion at an endogenous mouse κ immunoglobulin light chain variable region locus of a rearranged Vκ/Jκ sequence comprising:
 (i) a single human germline Vκ sequence, which single human germline Vκ sequence is present in SEQ ID NO: 148 or SEQ ID NO: 149; and 
 (ii) a single human germline Jκ sequence, wherein the rearranged Vκ/Jκ sequence is operably linked to the endogenous mouse κ constant region; and 
   (c) an insertion at an endogenous mouse immunoglobulin heavy chain variable region locus of a plurality of human immunoglobulin heavy chain variable region gene segments, wherein the human immunoglobulin heavy chain variable region gene segments are operably linked to an endogenous mouse immunoglobulin heavy chain constant region, and the human immunoglobulin heavy chain variable region gene segments are capable of rearranging and forming a rearranged human/mouse chimeric immunoglobulin heavy chain gene.   
     
     
         61 . The method of  claim 57 , wherein the mouse is heterozygous or homozygous in its germline for:
 (a) an ectopic nucleic acid sequence encoding a mouse ADAM6 protein, an ortholog thereof, a homolog thereof, or a fragment thereof, wherein the ADAM6 protein, ortholog thereof, homolog thereof, or fragment thereof is functional in a male mouse;   (b) an insertion at an endogenous mouse κ immunoglobulin light chain variable region locus of a rearranged Vκ/Jκ sequence comprising:
 (i) a single human germline Vκ sequence, which single human germline Vκ sequence is present in SEQ ID NO: 148 or SEQ ID NO: 149; and 
 (ii) a single human germline Jκ sequence, wherein the rearranged Vκ/Jκ sequence is operably linked to the endogenous mouse κ constant region; and 
   (c) an insertion at an endogenous mouse immunoglobulin heavy chain variable region locus of a plurality of human immunoglobulin heavy chain variable region gene segments, wherein the human immunoglobulin heavy chain variable region gene segments are operably linked to an endogenous mouse immunoglobulin heavy chain constant region, and the human immunoglobulin heavy chain variable region gene segments are capable of rearranging and forming a rearranged human/mouse chimeric immunoglobulin heavy chain gene.   
     
     
         62 . The method of  claim 1 , wherein the non-human animal pluripotent cell is a hybrid cell or the non-human mammalian one-cell stage embryo is a hybrid one-cell stage embryo, and wherein the method further comprises:
 (a′) comparing the sequence of the pair of corresponding first and second chromosomes within the target genomic locus, and selecting a target region within the target genomic locus prior to the contacting step (a) based on the target region having a higher percentage of sequence identity between the pair of corresponding first and second chromosomes relative to all or part of the remainder of the target genomic locus, wherein the target region comprises:
 the first guide RNA recognition sequence and at least 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6, kb, 7 kb, 8 kb, 9 kb, or 10 kb of flanking sequence on the 5′ side, the 3′ side, or each side of the first guide RNA recognition sequence, and/or 
 the second guide RNA recognition sequence and at least 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6, kb, 7 kb, 8 kb, 9 kb, or 10 kb of flanking sequence on the 5′ side, the 3′ side, or each side of the second guide RNA recognition sequence. 
   
     
     
         63 . The method of  claim 62 , wherein the target region has a higher percentage of sequence identity between the pair of corresponding first and second relative to the remainder of the target genomic locus. 
     
     
         64 . The method of  claim 63 , wherein the target region has at least 99.9% sequence identity between the pair of corresponding first and second chromosomes, and the remainder of the target genomic locus has no more than 99.8% sequence identity between the pair of corresponding first and second chromosomes. 
     
     
         65 . A method of making a genetically modified non-human animal with reduced tolerance of a foreign antigen of interest, comprising:
 (a) introducing into a non-human animal one-cell stage embryo or a non-human animal pluripotent cell that is not a one-cell stage embryo:
 (i) a Cas9 protein; 
 (ii) a first guide RNA that hybridizes to a first guide RNA recognition sequence within a target genomic locus, wherein the target genomic locus comprises all or part of a gene encoding a self-antigen homologous to or sharing an epitope of interest with the foreign antigen of interest; and 
 (iii) a second guide RNA that hybridizes to a second guide RNA recognition sequence within the target genomic locus; 
   wherein the target genomic locus is modified in a pair of corresponding first and second chromosomes to produce a modified non-human animal one-cell stage embryo or a modified non-human animal pluripotent cell with a biallelic modification, wherein expression of the self-antigen is eliminated; and   (b) producing a genetically modified F0 generation non-human animal from the modified non-human animal one-cell stage embryo or the modified non-human animal pluripotent cell, wherein the target genomic locus is modified in the pair of corresponding first and second chromosomes in the genetically modified F0 generation non-human animal such that expression of the self-antigen is eliminated.   
     
     
         66 . The method of  claim 65 , wherein the cell in step (a) is the non-human animal pluripotent stem cell, and the producing the genetically modified F0 generation non-human animal in step (b) comprises:
 (I) introducing the modified non-human animal pluripotent cell into a host embryo; and   (II) implanting the host embryo into a surrogate mother to produce the genetically modified F0 generation non-human animal in which the target genomic locus is modified in the pair of corresponding first and second chromosomes such that expression of the self-antigen is eliminated.   
     
     
         67 . The method of  claim 66 , wherein the pluripotent cell is an embryonic stem (ES) cell. 
     
     
         68 . The method of  claim 65 , wherein the cell in step (a) is the non-human animal one-cell stage embryo, and the producing the genetically modified F0 generation non-human animal in step (b) comprises implanting the modified non-human animal one-cell stage embryo into a surrogate mother to produce the genetically modified F0 generation non-human animal in which the target genomic locus is modified in the pair of corresponding first and second chromosomes such that expression of the self-antigen is eliminated.