Methods and compositions for targeted genetic modification using paired guide RNAs

Abstract

Provided herein are compositions and methods for forming and promoting double allele targeted modification of the genome in a cell and for producing non-human animals with a modified genome. Also provided herein are compositions and methods for converting the genome modification in a cell that is heterozygous for an allele into a homozygous composition and method for the allele. The method utilizes Cas proteins and two or more guide RNAs targeting different positions in the same genome target locus. Also provided herein are methods for identifying cells with a modified genome.

Description

[0001] Divisional Application Instructions

[0002] This application is a divisional application of an invention patent application with an application date of November 20, 2015, application number 201580063428.3, and invention name “Methods and compositions for targeted genetic modification using paired guide RNAs”.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Patent Application No. 62 / 083,005, filed November 21, 2014, U.S. Patent Application No. 62 / 182,314, filed June 19, 2015, and U.S. Patent Application No. 62 / 211,421, filed August 28, 2015, each of which is incorporated herein by reference in its entirety for all purposes.

[0005] Citation of Submitted Sequence Listing

[0006] As a text file submitted via EFS WEB

[0007] The sequence listing written into file 472225SEQLIST.txt is 32.7 kb, created on November 20, 2015, and is hereby incorporated by reference herein. Background Art

[0008] Although some progress has been made in targeting various genomic loci, there are still many types of genomic loci that cannot be effectively targeted or a variety of genomic modifications that cannot be correctly or effectively achieved using conventional targeting strategies. For example, problems arise when attempting to create large targeted genomic deletions or other large targeted genetic modifications, especially in eukaryotic cells and organisms.

[0009] Specifically, when using conventional targeting strategies, it is difficult to effectively produce cells or animals that are homozygous or compound heterozygous (e.g., hemizygous) for large targeted genomic deletions or other genomic modifications. For example, although F0 generation mice that are heterozygous for large targeted genomic deletions can be obtained by conventional targeting strategies, to produce F2 generation mice that are homozygous for the deletion, it is necessary to further mate these heterozygous mice. These additional mating steps are not only costly, but also time-consuming. Summary of the invention

[0010] The present invention provides methods and compositions for modifying a genome in a cell. In one aspect, the present invention provides a method for modifying a genome in a cell, comprising contacting the genome with: (a) a first Cas protein; (b) a first CRISPR RNA that hybridizes to a first CRISPR RNA recognition sequence in a genomic target locus; (c) a second CRISPR RNA that hybridizes to a second CRISPR RNA recognition sequence in a genomic target locus; (d) tracrRNA; and (e) a targeting vector comprising a nucleic acid insert flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm hybridizes to the 5' target sequence and the 3' homology arm hybridizes to the 3' target sequence, provided that if the cell is a 1-cell embryo, the length of the targeting vector does not exceed 5 kb; wherein the genome comprises a pair of first homologous chromosomes and a second homologous chromosome containing a genomic target locus; and wherein the first Cas protein cleaves at least one of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence to produce at least one double-stranded break in at least one of the first homologous chromosome and the second homologous chromosome. In one aspect, the invention provides a method for performing bi-allelic modification on a genome in a cell, comprising contacting the genome with: (a) a first Cas protein; (b) a first CRISPR RNA that hybridizes to a first CRISPR RNA recognition sequence within a genomic target locus; (c) a second CRISPR RNA that hybridizes to a second CRISPR RNA recognition sequence within the genomic target locus; (d) tracrRNA; and (e) a targeting vector comprising a nucleic acid insert flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm hybridizes to the 5' target sequence and the 3' homology arm hybridizes to the 3' target sequence, provided that if the cell is a 1-cell stage embryo, the length of the targeting vector does not exceed 5 kb; wherein the genome comprises a pair of first homologous chromosomes and a second homologous chromosome comprising the genomic target locus; and wherein the first Cas protein cleaves at least one of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence to generate at least one double-stranded break in at least one of the first homologous chromosome and the second homologous chromosome.

[0011] These methods may also include identifying cells that contain the modified genome. In some methods, the nucleic acid insert comprises a selection box adjacent to a first homology arm that hybridizes to a first target sequence, wherein the first homology arm is a 5' homology arm and the first target sequence is a 5' target sequence, or wherein the first homology arm is a 3' homology arm and the first target sequence is a 3' target sequence, wherein the identification process comprises: (a) obtaining DNA from a cell; (b) exposing the DNA of the cell to a probe that binds within the first target sequence, a probe that binds within the nucleic acid insert, and a probe that binds within a reference gene having a known copy number, wherein each probe generates a detectable signal upon binding; (c) detecting the signal from each probe binding; and (d) d) comparing the signal from the reference gene probe to the signal from the first target sequence probe to determine the copy number of the first target sequence, and comparing the signal from the reference gene probe to the signal from the nucleic acid insert probe to determine the copy number of the nucleic acid insert, wherein the copy number of the nucleic acid insert of one or two and the copy number of the first target sequence of two indicates targeted insertion of the nucleic acid insert at the genomic target locus, and wherein the copy number of the nucleic acid insert of one or more and the copy number of the first target sequence of three or more indicates random insertion of the nucleic acid insert at a genomic locus other than the target genomic locus.

[0012] In some methods, the first Cas protein cleaves at least one of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in each of the first homologous chromosome and the second homologous chromosome to generate at least one double-strand break in each of the first homologous chromosome and the second homologous chromosome. In some methods, the first Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in at least one of the first homologous chromosome and the second homologous chromosome to generate at least two double-strand breaks in at least one of the first homologous chromosome and the second homologous chromosome.

[0013] Some methods further comprise contacting the genome with: a third CRISPR RNA that hybridizes to a third CRISPR RNA recognition sequence within the genomic target locus; and a fourth CRISPR RNA that hybridizes to a fourth CRISPR RNA recognition sequence within the genomic target locus. Optionally, the first CRISPR RNA recognition sequence is separated from the third CRISPR RNA recognition sequence by about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1000 bp, about 1000 bp to about 1100 bp, about 1100 bp to about 1200 bp, about 1200 bp to about 1300 bp, about 1300 bp to about 1400 bp, about 1500 bp to about 1500 bp, about 1600 bp to about 1700 bp, about 1700 bp to about 1800 bp, about 1800 bp to about 1900 bp, about 1900 bp to about 1910 bp, about 1910 bp to about 1920 bp, about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 6 kb, about 6 kb to about 7 kb, about 7 kb to about 8 kb, about 8 kb to about 9 kb, about 9 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, or about 90 kb to about 100 kb. Optionally, the second CRISPR RNA recognition sequence is identical to the fourth CRISPR RNA recognition sequence. The RNA recognition sequences are about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1000 bp. p to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 6 kb, about 6 kb to about 7 kb, about 7 kb to about 8 kb, about 8 kb to about 9 kb, about 9 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, or about 90 kb to about 100 kb.Optionally, the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence are a first pair of CRISPR RNA recognition sequences, and the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence are a second pair of CRISPR The RNA recognition sequences, wherein the first pair is separated from the second pair by about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1 kb, about 1 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, about 2.5 Mb to about From about 3 Mb to about 4 Mb, from about 4 Mb to about 5 Mb, from about 5 Mb to about 10 Mb, from about 10 Mb to about 20 Mb, from about 20 Mb to about 30 Mb, from about 30 Mb to about 40 Mb, from about 40 Mb to about 50 Mb, from about 50 Mb to about 60 Mb, from about 60 Mb to about 70 Mb, from about 70 Mb to about 80 Mb, from about 80 Mb to about 90 Mb, or from about 90 Mb to about 100 Mb.

[0014] In some methods, the first Cas protein cleaves at least two of the first CRISPR RNA recognition sequence, the second CRISPR RNA recognition sequence, the third CRISPR RNA recognition sequence, and the fourth CRISPR RNA recognition sequence to generate at least two double-strand breaks in at least one of the first homologous chromosome and the second homologous chromosome. In some methods, the first Cas protein cleaves at least two of the first CRISPR RNA recognition sequence, the second CRISPR RNA recognition sequence, the third CRISPR RNA recognition sequence, and the fourth CRISPR RNA recognition sequence to generate at least two double-strand breaks in both the first homologous chromosome and the second homologous chromosome.

[0015] In some methods, the nucleic acid insert is inserted between the 5' and 3' target sequences. Optionally, the 5' and 3' target sequences are within the genomic target locus. Optionally, the cell is not a 1-cell stage embryo, and the targeting vector is a large targeting vector (LTVEC) of at least 10 kb.

[0016] In some methods, the genome is simultaneously contacted with the first CRISPR RNA and the second CRISPR RNA, resulting in an increase in the efficiency of double allele modification compared to the genome being contacted with the first CRISPR RNA or the second CRISPR RNA alone. In some methods, the cell is diploid, and the double allele modification results in homozygosity or compound heterozygosity at the genomic target locus. Optionally, the compound heterozygosity is hemizygosity. In some methods, the double allele modification includes a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in the first homologous chromosome. In some methods, the double allele modification includes a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in both the first homologous chromosome and the second homologous chromosome. In some methods, the double allele modification also includes the insertion of a nucleic acid insert between the 5' and 3' target sequences in both the first homologous chromosome and the second homologous chromosome. In some methods, the biallelic modification comprises: (1) a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in both the first homologous chromosome and the second homologous chromosome; and (2) an insertion of a nucleic acid insert between the 5' and 3' target sequences in the first homologous chromosome but not in the second homologous chromosome. In some methods, the biallelic modification comprises: (1) a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in the first homologous chromosome; and (2) a locus disruption between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in the second homologous chromosome. In some methods, the biallelic modification comprises: (1) a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in the first homologous chromosome; (2) an insertion of a nucleic acid insert between the 5' and 3' target sequences in the first homologous chromosome; and (3) a locus disruption between the 5' and 3' target sequences in the second homologous chromosome. In some methods, the biallelic modification comprises: (1) a deletion between a first CRISPR RNA recognition sequence and a second CRISPR RNA recognition sequence in a first homologous chromosome; (2) an insertion of a nucleic acid insert between the 5' and 3' target sequences in the first homologous chromosome, wherein the nucleic acid insert sequence is homologous or orthologous to the deleted sequence.

[0017] In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are separated by about 1 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb. In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are separated by at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb. In some methods, the first CRISP RNA recognition sequence and the second CRISP RNA recognition sequence are separated by about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, or about 900 bp to about 1 kb. In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are less than 25 bp, less than 50 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, less than 500 bp, less than 600 bp, less than 700 bp, less than 800 bp, less than 900 bp, less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, or less than 10 kb apart.

[0018] In some methods, the first and second CRISPR RNA recognition sequences are each located at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, or at least 100 kb from both the 5' and 3' target sequences. In some methods, the first and second CRISPR RNA recognition sequences are each located about 50 bp to about 100 bp, about 200 bp to about 300 bp, about 300 bp to about 400 bp, about 400 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, or about 50 kb to about 100 kb from both the 5' and 3' target sequences. In some methods, the first and second CRISPR RNA recognition sequences are each located greater than 50 bp, greater than 100 bp, greater than 200 bp, greater than 300 bp, greater than 400 bp, greater than 500 bp, greater than 600 bp, greater than 700 bp, greater than 800 bp, greater than 900 bp, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, greater than 10 kb, greater than 20 kb, greater than 30 kb, greater than 40 kb, greater than 50 kb, greater than 60 kb, greater than 70 kb, greater than 80 kb, greater than 90 kb, or greater than 100 kb from both the 5' and 3' target sequences.

[0019] In some methods, the deleted nucleic acid is about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb. In some methods, the deleted nucleic acid is at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb. Optionally, the deleted nucleic acid is at least 550 kb, at least 600 kb, at least 650 kb, at least 700 kb, at least 750 kb, at least 800 kb, at least 850 kb, at least 900 kb, at least 950 kb, at least 1 Mb, at least 1.5 Mb, or at least 2 Mb.

[0020] In some methods, the targeting vector is a linear form. Optionally, the targeting vector is single-stranded or double-stranded. In some methods, the cell is not a 1-cell stage embryo, and the targeting vector is a large targeting vector (LTVEC) of at least 10kb. In some methods, the cell is not a 1-cell stage embryo, and the targeting vector is a large targeting vector (LTVEC), wherein the sum of the 5' and 3' homology arms of the LTVEC is at least 10kb. Optionally, the LTVEC is about 50 kb to about 300 kb, about 50 kb to about 75 kb, about 75 kb to about 100 kb, about 100 kb to 125 kb, about 125 kb to about 150 kb, about 150 kb to about 175 kb, about 175 kb to about 200 kb, about 200 kb to about 225 kb, about 225 kb to about 250 kb, about 250 kb to about 275 kb, or about 275 kb to about 300 kb. Optionally, the sum of the 5' and 3' homology arms of the LTVEC is about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, about 90 kb to about 100 kb. , about 100 kb to about 110 kb, about 110 kb to about 120 kb, about 120 kb to about 130 kb, about 130 kb to about 140 kb, about 140 kb to about 150 kb, about 150 kb to about 160 kb, about 160 kb to about 170 kb, about 170 kb to about 180 kb, about 180 kb to about 190 kb or about 190 kb to about 200 kb.

[0021] In some methods, cell is non-human cell, non-pluripotent human cell, or the human induced pluripotent stem cell directly derived from the adult cell of differentiation. Optionally, cell is mammalian cell, non-pluripotent human cell, non-human cell, rodent cell, mouse cell and rat cell. Optionally, cell is non-pluripotent cell, non-human pluripotent cell, rodent pluripotent cell, mouse pluripotent cell, rat pluripotent cell, mouse embryonic stem (ES) cell, rat ES cell or the human induced pluripotent stem (iPS) cell directly derived from the adult cell of differentiation. Optionally, cell is non-human mammal 1 cell stage embryo. Optionally, cell is non-human mammal 1 cell stage embryo, and the length of targeting vector is about 50 nucleotides to about 5kb. Optionally, cell is non-human mammal 1 cell stage embryo, and targeting vector is single-stranded DNA, and its length is about 60 to about 200 nucleotides.

[0022] In some methods, the first Cas protein is Cas9. In some methods, the first Cas protein has nuclease activity on both strands of double-stranded DNA.

[0023] In some methods, the first Cas protein is a nickase. Some methods also include contacting the genome with: (f) a second Cas protein that is a nickase; (g) a third CRISPR RNA that hybridizes to a third CRISPR RNA recognition sequence; and (h) a fourth CRISPR RNA that hybridizes to a fourth CRISPR RNA recognition sequence; wherein the first Cas protein cleaves the first strand of the genomic DNA within the first CRISPR RNA recognition sequence and within the second CRISPR RNA recognition sequence, and the second Cas protein cleaves the second strand of the genomic DNA within the third CRISPR RNA recognition sequence and within the fourth CRISPR RNA recognition sequence.

[0024] In some methods, the first CRISPR RNA and tracrRNA are fused together as a first guide RNA (gRNA), and / or the second CRISPR RNA and tracrRNA are fused together as a second gRNA. In some methods, the first CRISPR RNA and tracrRNA are separate RNA molecules, and / or the second CRISPR RNA and tracrRNA are separate RNA molecules.

[0025] In some methods, the contacting process includes introducing a first Cas protein, a first CRISPR RNA, a second CRISPR RNA, and a tracrRNA into a cell. In some methods, (a) the first Cas protein is introduced into the cell in the form of a protein, a messenger RNA (mRNA) encoding the first Cas protein, or a DNA encoding the first Cas protein; (b) the first CRISPR RNA is introduced into the cell in the form of RNA or in the form of a DNA encoding the first CRISPR RNA; (c) the second CRISPR RNA is introduced into the cell in the form of RNA or in the form of a DNA encoding the second CRISPR RNA; and / or (d) tracrRNA is introduced into the cell in the form of RNA or in the form of a DNA encoding tracrRNA. In some methods, the first Cas protein, the first CRISPR RNA, and the tracrRNA are introduced into the cell as a first protein-RNA complex, and / or the first Cas protein, the second CRISPR RNA, and the tracrRNA are introduced into the cell as a second protein-RNA complex. In some methods, (a) DNA encoding the first Cas protein is effectively connected to the first promoter in the first expression construct; (b) DNA encoding the first CRISPR RNA is effectively connected to the second promoter in the second expression construct; (c) DNA encoding the second CRISPR RNA is effectively connected to the third promoter in the third expression construct; and / or (d) DNA encoding tracrRNA is effectively connected to the fourth promoter in the fourth expression construct; wherein the first promoter, the second promoter, the third promoter and the fourth promoter are active in the cell. Optionally, the first expression construct, the second expression construct, the third expression construct and / or the fourth expression construct are components of a single nucleic acid molecule. In some methods, (a) DNA encoding a first Cas protein is effectively linked to a first promoter in a first expression construct; (b) DNA encoding a first CRISPR RNA and tracrRNA is fused together in DNA encoding a first guide RNA (gRNA) and effectively linked to a second promoter in a second expression construct; and / or (c) DNA encoding a second CRISPR RNA and tracrRNA is fused together in DNA encoding a second gRNA and effectively linked to a third promoter in a third expression construct; wherein the first promoter, the second promoter, and the third promoter are active in the cell. Optionally, the first expression construct, the second expression construct, and / or the third expression construct are components of a single nucleic acid molecule.

[0026] In some methods, the cell has been modified to reduce non-homologous end joining (NHEJ) and / or increase gene conversion or homology-directed repair (HDR). Optionally, the cell has been modified to reduce the expression or activity of DNA-PK and / or reduce the expression or activity of PARP1. Optionally, the cell has been modified to reduce the expression or activity of ligase IV. Optionally, the reduction in expression or activity is inducible, reversible, time-specific and / or spatially specific.

[0027] In some methods, (1) the cell is not a 1-cell stage embryo, and the targeting vector is a large targeting vector, wherein the sum of the 5' and 3' homology arms is at least 10 kb; (2) the first CRISPR RNA recognition sequence and the second CRISPR The RNA recognition sequences are each located greater than 200 bp, greater than 300 bp, greater than 400 bp, greater than 500 bp, greater than 600 bp, greater than 700 bp, greater than 800 bp, greater than 900 bp, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, greater than 10 kb, greater than 20 kb, greater than 30 kb, greater than 40 kb, greater than 50 kb, greater than 60 kb, greater than 70 kb, greater than 80 kb, greater than 90 kb, or greater than 100 kb from both the 5' and 3' target sequences; (3) the first Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in at least one of the first homologous chromosome and the second homologous chromosome to generate at least two double-stranded breaks in at least one of the first homologous chromosome and the second homologous chromosome; and (4) the biallelic modification comprises the first CRISPR RNA recognition sequence in the first homologous chromosome. A deletion between a CRISPR RNA recognition sequence and a second CRISPR RNA recognition sequence and insertion of a nucleic acid insert between the 5' and 3' target sequences in a first homologous chromosome, wherein the nucleic acid insert sequence is homologous or orthologous to the deleted sequence.

[0028] The present invention also provides a method for producing an F0 generation non-human animal, comprising: (a) introducing non-human ES cells into a non-human host embryo, wherein the non-human ES cells are produced by any of the above methods; and (b) gestating the non-human host embryo in a surrogate mother; wherein the surrogate mother produces an F0 generation non-human animal comprising a double allele modification. Some methods include: (a) contacting a genome in a non-human ES cell with: (i) a first Cas protein; (ii) a first CRISPR RNA that hybridizes to a first CRISPR RNA recognition sequence within a genomic target locus; (iii) a second CRISPR RNA that hybridizes to a second CRISPR RNA recognition sequence within the genomic target locus; (iv) a tracrRNA; and (v) a targeting vector comprising a nucleic acid insert flanked by 5' homology arms and 3' homology arms, wherein the genome comprises a pair of first and second homologous chromosomes comprising the genomic target locus; and wherein the first Cas protein cleaves at least one of the first and second CRISPR RNA recognition sequences to generate at least one double-stranded break in at least one of the first and second homologous chromosomes; (b) identifying a non-human ES cell comprising a biallelic modification; (c) introducing the non-human ES cell comprising the biallelic modification into a non-human host embryo; and (d) gestating the non-human host embryo in a surrogate mother; wherein the surrogate mother produces an F0 generation non-human animal comprising the biallelic modification.

[0029] In some methods, the first Cas protein cleaves at least one of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in each of the first homologous chromosome and the second homologous chromosome to generate at least one double-strand break in each of the first homologous chromosome and the second homologous chromosome. In some methods, the first Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in at least one of the first homologous chromosome and the second homologous chromosome to generate at least two double-strand breaks in at least one of the first homologous chromosome and the second homologous chromosome.

[0030] In some methods, the non-human animal is a mouse, the non-human ES cell is a mouse ES cell, and the non-human host embryo is a mouse host embryo. In some methods, the non-human animal is a rat, the non-human ES cell is a rat ES cell, and the non-human host embryo is a rat host embryo.

[0031] In some methods, biallelic modification results in homozygosity or compound heterozygosity at the genomic target locus. Optionally, the compound heterozygosity is hemizygosity.

[0032] In some methods, the first CRISPR RNA and tracrRNA are fused together as a first guide RNA (gRNA), and / or the second CRISPR RNA and tracrRNA are fused together as a second gRNA. In some methods, the first CRISPR RNA and tracrRNA are separate RNA molecules, and / or the second CRISPR RNA and tracrRNA are separate RNA molecules.

[0033] In some methods, the contacting process includes introducing a first Cas protein, a first CRISPR RNA, a second CRISPR RNA, and a tracrRNA into a cell. In some methods, (a) the first Cas protein is introduced into the cell in the form of a protein, a messenger RNA (mRNA) encoding the first Cas protein, or a DNA encoding the first Cas protein; (b) the first CRISPR RNA is introduced into the cell in the form of RNA or in the form of a DNA encoding the first CRISPR RNA; (c) the second CRISPR RNA is introduced into the cell in the form of RNA or in the form of a DNA encoding the second CRISPR RNA; and / or (d) tracrRNA is introduced into the cell in the form of RNA or in the form of a DNA encoding tracrRNA. In some methods, the first Cas protein, the first CRISPR RNA, and the tracrRNA are introduced into the cell as a first protein-RNA complex, and / or the first Cas protein, the second CRISPR RNA, and the tracrRNA are introduced into the cell as a second protein-RNA complex. In some methods, (a) DNA encoding the first Cas protein is effectively connected to the first promoter in the first expression construct; (b) DNA encoding the first CRISPR RNA is effectively connected to the second promoter in the second expression construct; (c) DNA encoding the second CRISPR RNA is effectively connected to the third promoter in the third expression construct; and / or (d) DNA encoding tracrRNA is effectively connected to the fourth promoter in the fourth expression construct; wherein the first promoter, the second promoter, the third promoter and the fourth promoter are active in the cell. Optionally, the first expression construct, the second expression construct, the third expression construct and / or the fourth expression construct are components of a single nucleic acid molecule. In some methods, (a) DNA encoding a first Cas protein is effectively linked to a first promoter in a first expression construct; (b) DNA encoding a first CRISPR RNA and tracrRNA is fused together in DNA encoding a first guide RNA (gRNA) and effectively linked to a second promoter in a second expression construct; and / or (c) DNA encoding a second CRISPR RNA and tracrRNA is fused together in DNA encoding a second gRNA and effectively linked to a third promoter in a third expression construct; wherein the first promoter, the second promoter, and the third promoter are active in the cell. Optionally, the first expression construct, the second expression construct, and / or the third expression construct are components of a single nucleic acid molecule.

[0034] In some methods, the first Cas protein is Cas9. In some methods, the first Cas protein has nuclease activity on both strands of double-stranded DNA.

[0035] In some methods, the first Cas protein is a nickase. Some methods also include contacting the genome with: (f) a second Cas protein that is a nickase; (g) a third CRISPR RNA that hybridizes to a third CRISPR RNA recognition sequence; and (h) a fourth CRISPR RNA that hybridizes to a fourth CRISPR RNA recognition sequence; wherein the first Cas protein cleaves the first strand of the genomic DNA within the first CRISPR RNA recognition sequence and within the second CRISPR RNA recognition sequence, and the second Cas protein cleaves the second strand of the genomic DNA within the third CRISPR RNA recognition sequence and within the fourth CRISPR RNA recognition sequence.

[0036] In some methods, the cell has been modified to reduce non-homologous end joining (NHEJ) and / or increase gene conversion or homology-directed repair (HDR). Optionally, the cell has been modified to reduce the expression or activity of DNA-PK and / or reduce the expression or activity of PARP1. Optionally, the cell has been modified to reduce the expression or activity of ligase IV. Optionally, the reduction in expression or activity is inducible, reversible, time-specific and / or spatially specific.

[0037] The present invention also provides a method for producing an F0 generation non-human animal, comprising implanting a genetically modified 1-cell stage embryo produced by any of the above methods into a surrogate mother; wherein the surrogate mother produces an F0 generation non-human animal comprising biallelic gene modifications.

[0038] The present invention also provides a method for modifying a genome in a cell that is heterozygous for a first allele, comprising contacting the genome with: (a) a first Cas protein; (b) tracrRNA; (c) a first CRISPR RNA hybridized to a first CRISPR RNA recognition sequence in a second allele, wherein the first allele is on a first homologous chromosome and the second allele is at a corresponding locus on a second homologous chromosome; and (d) a second CRISPR RNA hybridized to a second CRISPR RNA recognition sequence in the second allele; wherein the first Cas protein cleaves at least one of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence to produce at least one double-strand break and an end sequence that undergoes recombination, wherein the recombination occurs between the first allele and the second allele to form a modified genome that is homozygous for the first allele. Some methods also include identifying a cell that is homozygous for the first allele.

[0039] In some methods, the first Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence. In some methods, the first Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence to produce at least two double-strand breaks and terminal sequences where recombination occurs. In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are located in the second allele but not the first allele. In some methods, the Cas protein and the first CRISPR RNA do not exist at the same time in nature.

[0040] In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are separated by about 1 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb. In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are separated by at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb. In some methods, the first CRISP RNA recognition sequence and the second CRISP RNA recognition sequence are separated by about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, or about 900 bp to about 1 kb. In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are less than 25 bp, less than 50 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, less than 500 bp, less than 600 bp, less than 700 bp, less than 800 bp, less than 900 bp, less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, or less than 10 kb apart.

[0041] In some methods, the sequence difference between the first allele and the second allele spans about 100 bp to about 200 bp, about 200 bp to about 400 bp, about 400 bp to about 600 bp, about 600 bp to about 800 bp, about 800 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, about 4 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 4 kb, about 0 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb. In some methods, the sequence difference between the first allele and the second allele spans at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 800 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, 20 kb, at least 30 kb, at least kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb or at least 500 kb.

[0042] In some methods, the first allele comprises a targeted modification and the second allele is a wild-type allele. In some methods, the first allele is a wild-type allele and the second allele comprises a pathogenic mutation.

[0043] In some methods, the recombination comprises gene conversion. In some methods, the recombination comprises loss of heterozygosity (LOH).

[0044] In some methods, the cell is a non-human cell, a non-pluripotent human cell, or a human induced pluripotent stem cell cell directly derived from a differentiated adult cell. Optionally, the cell is a mammalian cell, a non-pluripotent human cell, a non-human cell, a rodent cell, a mouse cell, or a rat cell. Optionally, the cell is a non-pluripotent cell, a non-human pluripotent cell, a rodent pluripotent cell, a mouse pluripotent cell, a rat pluripotent cell, a mouse embryonic stem (ES) cell, a rat ES cell, or a human induced pluripotent stem (iPS) cell directly derived from a differentiated adult cell.

[0045] In some methods, the first Cas protein is Cas9. In some methods, the first Cas protein has nuclease activity on both strands of double-stranded DNA.

[0046] In some methods, the first Cas protein is a nickase. Some methods also include contacting the genome with: (f) a second Cas protein that is a nickase; (g) a third CRISPR RNA that hybridizes to a third CRISPR RNA recognition sequence; and (h) a fourth CRISPR RNA that hybridizes to a fourth CRISPR RNA recognition sequence; wherein the first Cas protein cleaves the first strand of the genomic DNA within the first CRISPR RNA recognition sequence and within the second CRISPR RNA recognition sequence, and the second Cas protein cleaves the second strand of the genomic DNA within the third CRISPR RNA recognition sequence and within the fourth CRISPR RNA recognition sequence.

[0047] In some methods, the first CRISPR RNA and tracrRNA are fused together as a first guide RNA (gRNA), and / or the second CRISPR RNA and tracrRNA are fused together as a second gRNA. In some methods, the first CRISPR RNA and tracrRNA are separate RNA molecules, and / or the second CRISPR RNA and tracrRNA are separate RNA molecules.

[0048] In some methods, the contacting process includes introducing a first Cas protein, a first CRISPR RNA, a second CRISPR RNA, and a tracrRNA into a cell. In some methods, (a) the first Cas protein is introduced into the cell in the form of a protein, a messenger RNA (mRNA) encoding the first Cas protein, or a DNA encoding the first Cas protein; (b) the first CRISPR RNA is introduced into the cell in the form of RNA or in the form of a DNA encoding the first CRISPR RNA; (c) the second CRISPR RNA is introduced into the cell in the form of RNA or in the form of a DNA encoding the second CRISPR RNA; and / or (d) tracrRNA is introduced into the cell in the form of RNA or in the form of a DNA encoding tracrRNA. In some methods, the first Cas protein, the first CRISPR RNA, and the tracrRNA are introduced into the cell as a first protein-RNA complex, and / or the first Cas protein, the second CRISPR RNA, and the tracrRNA are introduced into the cell as a second protein-RNA complex. In some methods, (a) DNA encoding the first Cas protein is effectively connected to the first promoter in the first expression construct; (b) DNA encoding the first CRISPR RNA is effectively connected to the second promoter in the second expression construct; (c) DNA encoding the second CRISPR RNA is effectively connected to the third promoter in the third expression construct; and / or (d) DNA encoding tracrRNA is effectively connected to the fourth promoter in the fourth expression construct; wherein the first promoter, the second promoter, the third promoter and the fourth promoter are active in the cell. Optionally, the first expression construct, the second expression construct, the third expression construct and / or the fourth expression construct are components of a single nucleic acid molecule. In some methods, (a) DNA encoding a first Cas protein is effectively linked to a first promoter in a first expression construct; (b) DNA encoding a first CRISPR RNA and tracrRNA is fused together in DNA encoding a first guide RNA (gRNA) and effectively linked to a second promoter in a second expression construct; and / or (c) DNA encoding a second CRISPR RNA and tracrRNA is fused together in DNA encoding a second gRNA and effectively linked to a third promoter in a third expression construct; wherein the first promoter, the second promoter, and the third promoter are active in the cell. Optionally, the first expression construct, the second expression construct, and / or the third expression construct are components of a single nucleic acid molecule.

[0049] In some methods, the cell has been modified to reduce non-homologous end joining (NHEJ) and / or increase gene conversion or homology-directed repair (HDR). Optionally, the cell has been modified to reduce the expression or activity of DNA-PK and / or reduce the expression or activity of PARP1. Optionally, the cell has been modified to reduce the expression or activity of ligase IV. Optionally, the reduction in expression or activity is inducible, reversible, time-specific and / or spatially specific.

[0050] The present invention also provides a method for modifying a genome in a cell that is heterozygous for a first allele, comprising contacting the genome with: (a) a first Cas protein; (b) tracrRNA; and (c) a first CRISPR RNA hybridized with a first non-allele-specific CRISPR RNA recognition sequence, wherein the first allele is on a first homologous chromosome, and the CRISPR RNA recognition sequence is located near the centromere of the locus corresponding to the first allele on a second homologous chromosome; and wherein the first Cas protein cuts the first CRISPR RNA recognition sequence to produce a double-strand break, and the cell is modified to become homozygous for the first allele. Some methods also include identifying cells that are homozygous for the first allele. In some methods, the Cas protein and the first CRISPR RNA do not exist simultaneously under natural conditions.

[0051] Such methods may also include contacting the genome with a second CRISPR RNA that hybridizes to a second non-allele-specific CRISPR RNA recognition sequence, the CRISPR RNA recognition sequence being located near the centromere of a locus corresponding to the first allele on a second homologous chromosome, wherein the first Cas protein cleaves at least one of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence to produce at least one double-stranded break. In some methods, the first Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence.

[0052] In some methods, loss of heterozygosity occurs on the telomeric side of the double-strand break.

[0053] In some methods, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are located on the second homologous chromosome instead of the first homologous chromosome. In some methods, the first CRISPR RNA recognition site is about 100 bp to about 1 kb, about 1 kb to about 10 kb, about 10 kb to about 100 kb, about 100 kb to about 1 Mb, about 1 Mb to about 10 Mb, about 10 Mb to about 20 Mb, about 20 Mb to about 30 Mb, about 30 Mb to about 40 Mb, about 40 Mb to about 50 Mb, about 50 Mb to about 60 Mb, about 60 Mb to about 70 Mb, about 70 Mb to about 80 Mb, about 80 Mb to about 90 Mb, or about 90 Mb to about 100 Mb from the centromere. In some methods, the first allele is about 100 bp to about 1 kb, about 1 kb to about 10 kb, about 10 kb to about 100 kb, about 100 kb to about 1 Mb, about 1 Mb to about 10 Mb, about 10 Mb to about 20 Mb, about 20 Mb to about 30 Mb, about 30 Mb to about 40 Mb, about 40 Mb to about 50 Mb, about 50 Mb to about 60 Mb, about 60 Mb to about 70 Mb, about 70 Mb to about 80 Mb, about 80 Mb to about 90 Mb, or about 90 Mb to about 100 Mb from the first CRISPR RNA recognition site. In some methods, the second homologous chromosome region replaced by loss of heterozygosity is about 100 bp to about 1 kb, about 1 kb to about 10 kb, about 10 kb to about 100 kb, about 100 kb to about 1 Mb, about 1 Mb to about 10 Mb, about 10 Mb to about 20 Mb, about 20 Mb to about 30 Mb, about 30 Mb to about 40 Mb, about 40 Mb to about 50 Mb, about 50 Mb to about 60 Mb, about 60 Mb to about 70 Mb, about 70 Mb to about 80 Mb, about 80 Mb to about 90 Mb, or about 90 Mb to about 100 Mb.

[0054] In some methods, the cell is a non-human cell, a non-pluripotent human cell, or a human induced pluripotent stem cell cell directly derived from a differentiated adult cell. Optionally, the cell is a mammalian cell, a non-pluripotent human cell, a non-human cell, a rodent cell, a mouse cell, a rat cell, a non-pluripotent cell, a non-human pluripotent cell, a rodent pluripotent cell, a mouse pluripotent cell, a rat pluripotent cell, a mouse embryonic stem (ES) cell, a rat ES cell, or a human induced pluripotent stem (iPS) cell directly derived from a differentiated adult cell or a non-human mammal 1-cell stage embryo.

[0055] In some methods, the first Cas protein is Cas9. In some methods, the first Cas protein has nuclease activity on both strands of double-stranded DNA. In some methods, the first Cas protein is a nickase. Optionally, the first Cas protein is a nickase, and wherein the method further comprises contacting the genome with a second Cas protein as a nickase, a third CRISPR RNA hybridized with a third CRISPR RNA recognition sequence, and a fourth CRISPR RNA hybridized with a fourth CRISPR RNA recognition sequence, wherein the first Cas protein cuts the first strand of genomic DNA within the first CRISPR RNA recognition sequence and within the second CRISPR RNA recognition sequence, and the second Cas protein cuts the second strand of genomic DNA within the third CRISPR RNA recognition sequence and within the fourth CRISPR RNA recognition sequence.

[0056] In some methods, the first CRISPR RNA and tracrRNA are fused together as a first guide RNA (gRNA), and / or the second CRISPR RNA and tracrRNA are fused together as a second gRNA. In some methods, the first CRISPR RNA and tracrRNA are separate RNA molecules, and / or the second CRISPR RNA and tracrRNA are separate RNA molecules.

[0057] In some methods, the contacting process includes introducing a first Cas protein, a first CRISPR RNA, a second CRISPR RNA, and a tracrRNA into a cell. In some methods, the first Cas protein is introduced into the cell in the form of a protein, a messenger RNA (mRNA) encoding the first Cas protein, or a DNA encoding the first Cas protein. Optionally, the DNA encoding the first Cas protein is effectively connected to the first promoter in the first expression construct, wherein the first promoter is active in the cell. In some methods, the first CRISPR RNA is introduced into the cell in the form of RNA or in the form of DNA encoding the first CRISPR RNA. Optionally, the DNA encoding the first CRISPR RNA is effectively connected to the second promoter in the second expression construct, wherein the second promoter is active in the cell. In some methods, the second CRISPR RNA is introduced into the cell in the form of RNA or in the form of DNA encoding the second CRISPR RNA. Optionally, the DNA encoding the second CRISPR RNA is effectively connected to the third promoter in the third expression construct, wherein the third promoter is active in the cell. In some methods, tracrRNA is introduced into the cell in the form of RNA or in the form of DNA encoding tracrRNA. Optionally, the DNA encoding tracrRNA is effectively connected to a fourth promoter in a fourth expression construct, wherein the fourth promoter is active in the cell. Optionally, the first expression construct, the second expression construct, the third expression construct and / or the fourth expression construct are components of a single nucleic acid molecule.

[0058] Optionally, the DNA encoding the first Cas protein is effectively connected to the first promoter in the first expression construct; the DNA encoding the first CRISPR RNA and tracrRNA is fused together in the DNA encoding the first guide RNA (gRNA) and effectively connected to the second promoter in the second expression construct; and / or the DNA encoding the second CRISPR RNA and tracrRNA is fused together in the DNA encoding the second gRNA and effectively connected to the third promoter in the third expression construct; wherein the first promoter, the second promoter and the third promoter are active in the cell. Optionally, the first expression construct, the second expression construct and / or the third expression construct are components of a single nucleic acid molecule.

[0059] Optionally, the first Cas protein, the first CRISPR RNA and the tracrRNA are introduced into the cell as a first protein-RNA complex, and / or the first Cas protein, the second CRISPR RNA and the tracrRNA are introduced into the cell as a second protein-RNA complex.

[0060] In some methods, the cell has been modified to reduce non-homologous end joining (NHEJ) and / or increase gene conversion or homology-directed repair (HDR). Optionally, the cell has been modified to reduce the expression or activity of one or more of the following: DNA-PK, PARP1, and ligase IV. Optionally, the reduction in expression or activity is inducible, reversible, time-specific, and / or spatially specific.

[0061] In some methods, the first allele comprises a mutation. Optionally, the mutation is a targeted modification. In some methods, the first allele is a wild-type allele, and the corresponding locus on the second homologous chromosome comprises a mutation.

[0062] The present invention also provides a method for identifying targeted insertion of a nucleic acid insert at a target genomic locus in a diploid cell that is not a 1-cell stage embryo, comprising: (a) obtaining DNA from a cell, wherein the cell has been contacted with a large targeting vector (LTVEC), the LTVEC comprising a nucleic acid insert flanked by a first homology arm that hybridizes to a first target sequence and a second homology arm that hybridizes to a second target sequence, wherein the nucleic acid insert comprises a selection box adjacent to the first homology arm; (b) exposing the DNA of the cell to a probe that binds within the first target sequence, a probe that binds within the nucleic acid insert, and a probe that binds within a reference gene having a known copy number, wherein each probe produces a detectable (c) detecting the signal from each probe binding; and (d) comparing the signal from the reference gene probe to the signal from the first target sequence probe to determine the copy number of the first target sequence, and comparing the signal from the reference gene probe to the signal from the nucleic acid insert probe to determine the copy number of the nucleic acid insert, wherein the copy number of the nucleic acid insert is one or two and the copy number of the first target sequence is two indicating targeted insertion of the nucleic acid insert at the target genomic locus, and wherein the copy number of the nucleic acid insert is one or more and the copy number of the first target sequence is three or more indicating random insertion of the nucleic acid insert at a genomic locus other than the target genomic locus.

[0063] In some methods, the signal from the first target sequence probe binding is used to determine the threshold cycle (Ct) value of the first target sequence, the signal from the reference gene probe binding is used to determine the threshold cycle (Ct) value of the reference gene, and the copy number of the first target sequence is determined by comparing the first target sequence Ct value to the reference gene Ct value. In some methods, the signal from the nucleic acid insert probe binding is used to determine the threshold cycle (Ct) value of the nucleic acid insert, and the copy number of the nucleic acid insert is determined by comparing the first target sequence Ct value to the reference gene Ct value.

[0064] In some methods, the selection cassette comprises a drug resistance gene.

[0065] In some methods, the nucleic acid insert is at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb. In some methods, the distance between the sequence to which the probe binds in the first target sequence and the selection box is no more than 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb or 5 kb.

[0066] Some methods further include determining the copy number of the second target sequence. Optionally, step (b) further includes exposing the DNA of the cell to a probe that binds to the second target sequence, step (c) further includes detecting a signal from the binding of the second target sequence probe, and step (d) further includes comparing the signal from the reference gene probe with the signal from the second target sequence probe to determine the copy number of the second target sequence.

[0067] Some methods also include determining the copy number of one or more additional sequences within the nucleic acid insert. Optionally, step (b) also includes exposing the DNA of the cell to one or more additional probes that bind to the nucleic acid insert, step (c) also includes detecting signals from the binding of the one or more additional probes, and step (d) also includes comparing the signal from the reference gene probe with the signal from the one or more additional nucleic acid insert probes to determine the copy number of the one or more additional sequences within the nucleic acid insert. Optionally, the one or more additional sequences within the nucleic acid insert include sequences adjacent to the second target sequence.

[0068] In some methods, the LTVEC is designed to delete endogenous sequences from the target genomic locus, or the cell has been contacted with a Cas protein, a first CRISPR RNA that hybridizes to a first CRISPR RNA recognition sequence within the target genomic locus, a second CRISPR RNA that hybridizes to a second CRISPR RNA recognition sequence within the target genomic locus, and tracrRNA. Optionally, such methods also include determining the copy number of the endogenous sequence at the target genomic locus. Optionally, step (b) also includes exposing the DNA of the cell to a probe that binds to an endogenous sequence at the target genomic locus, step (c) also includes detecting a signal from the binding of the endogenous sequence probe, and step (d) also includes comparing the signal from the reference gene probe with the signal from the endogenous sequence probe to determine the copy number of the endogenous sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 A schematic diagram of the deletion of the mouse Lrp5 ectodomain using LTVEC and one or two 5' region (A, B, B2), middle region (C, D) and 3' region (E2, E, F) gRNAs, and simultaneous replacement of the ectodomain with the corresponding human LRP5 form is shown. The LTVEC is shown at the top of the figure and the mouse Lrp5 locus is shown at the bottom of the figure. The positions of the Cas9 cleavage sites directed by the eight guide RNAs are shown by the vertical arrows below the mouse gene sequence. The horizontal arrows represent PCR primers for the mouse and human sequences.

[0070] Figure 2A A general schematic diagram of using LTVEC and two guide RNAs (guide RNA A and B) to delete a mouse gene and simultaneously replace the gene with the corresponding human form is shown. Figure 2A At the top, the mouse loci are shown Figure 2A The positions of the Cas9 cutting sites directed by the two guide RNAs are shown by arrows below the mouse gene sequence.

[0071] FIG. 2B to FIG. 2E Unique biallelic modifications (allele types) that occur at higher frequencies when two guide RNAs are used are shown. Thick lines with diagonal hatching represent mouse genes, dashed lines represent deletions in mouse genes, and thick black lines represent insertions in human genes. Figure 2B The homozygous collapsed allele (CRISPR-induced large deletion) is shown. Figure 2C Homozygous targeted alleles are shown. Figure 2D Hemizygous targeted alleles are shown. Figure 2E Compound heterozygous alleles are shown.

[0072] Figure 3A and 3B The results of PCR assays confirming the genotypes of selected clones are shown. Figure 3A Shown are the results of a long-range PCR assay of selected ES cell clones using primers m-lr-f and m-5'-r, which form a junction between the human insert and sequences outside of the sequence homologous to the 5' homology arm, thereby providing correct targeting. Figure 3B PCR assay results for 5'DelJ, 5'Ins J, Del A+F, and Del A+E2 are shown. 5'Del J shows the PCR product obtained using m-5'-f and m-5-r primers, which amplify the wild-type sequence around the gRNA A cleavage site to determine the retention or loss of this sequence. 5'Ins J shows the PCR product obtained using m-5'-f and h-5'-r primers, which form a connection between the human insert and the mouse genome. This assay will give positive results in both targeted and random integration clones. Del A+F shows the expected amplicon size (359bp) and actual bands for large deletions mediated by dual gRNA A and F cleavage in clones BO-F10 and AW-A8. Del A+E2 shows the same concept for clone BA-A7. NT indicates no template, + / + indicates parental VGF1 hybrid ES cell wild-type control, H / + indicates heterozygous humanized genotype, H / Δ indicates hemizygous humanized genotype, H / H indicates homozygous humanized genotype, and Δ / Δ indicates homozygous deletion genotype.

[0073] FIG. 4A to FIG. 4C Mouse ES cell clone AW-D9 (targeted with Lrp5 humanized LTVEC combined with Cas9 and two gRNAs) is shown. Figure 4A ) and BA-D5( Figure 4C ) and clone BS-C4 targeted with LTVEC alone ( Figure 4B ) were analyzed by fluorescence in situ hybridization (FISH). The arrow indicates the location of the hybridization signal on band B of chromosome 19. The red signal indicates hybridization only with the mouse probe (dashed arrow, Figure 4B ). The mixed yellow signal indicates hybridization with both the red mouse probe and the green human probe. One chromosome 19 band B has a red signal (dashed arrow) and the other chromosome 19 band B has a yellow signal (solid arrow), confirming the correct locus and heterozygous genotype of the targeted BS-C4 clone ( Figure 4B ). Both chromosome 19 bands B have yellow signals (solid arrows, Figure 4A and Figure 4C ), confirming the correct loci and homozygous genotypes of targeted AW-D9 and BS-C4 clones.

[0074] Figure 5 Shown is a schematic diagram of the results of an assay for chromosome 19 designed to examine gene conversion or mitotic recombination events mediated by two guide RNAs by analyzing loss of heterozygosity (LOH) in VGF1 hybrid ES cells. The approximate location of the qPCR chromosome copy number (CCN) assay is indicated by the arrow. The approximate location of the structural variant (SV) polymorphism PCR assay is indicated by the mountain shape, on which its distance from the Lrp5 locus is given (in Mb). Single nucleotide variant (SNV) The approximate location of the allele typing assay is indicated by an arrow, with its distance from the Lrp5 locus given in Mb. The locations of the gRNA recognition sites for F, E2, D, B2, and A are indicated by the oblique arrows above the Lrp5 gene illustration.

[0075] Figure 6 A schematic diagram showing the use of LTVEC and one or two 5' region (A, B), middle region (C, D) and 3' region (E, E2) gRNAs to delete the region from exon 2 to the stop codon in the mouse C5 (Hc) gene and replace the region with the corresponding human C5 form. LTVEC is shown at the top of the figure and the mouse C5 (Hc) locus is shown at the bottom of the figure. The positions of the Cas9 cleavage sites directed by the six guide RNAs are shown by arrows below the mouse gene sequence.

[0076] Fig. 7A and Figure 7B The mouse ES cell clone Q-E9 ( Fig. 7A ) and O-E3( Figure 7B ) were analyzed by fluorescence in situ hybridization (FISH). The arrow indicates the location of the hybridization signal on band B of chromosome 2. The red signal indicates hybridization only with the mouse probe (dashed arrow, Fig. 7A ). The mixed yellow signal indicates hybridization with both the red mouse probe and the green human probe (solid arrow). One chromosome 2 band B has a red signal (dashed arrow) and the other chromosome 2 band B has a yellow signal (solid arrow), confirming the correct locus and heterozygous genotype for the targeted Q-E9 clone ( Fig. 7A ). Both chromosome 2 bands B have yellow signals (solid arrows, Figure 7B ), confirming the correct locus and homozygous genotype of the targeted O-E3 clone.

[0077] Figure 8Schematic diagram of using LTVEC and one or two 5' region (A, B), middle region (D, C) and 3' region (E, F) gRNAs to delete the mouse Ror1 gene and simultaneously replace the gene with the corresponding human ROR1 form. LTVEC is shown at the top of the figure and the mouse Ror1 locus is shown at the bottom of the figure. The positions of the Cas9 cleavage sites directed by the six guide RNAs are shown by arrows below the mouse gene sequence.

[0078] Fig. 9 Schematic diagram of using LTVEC and one or two 5' region (A, A2, B), middle region (C, D) and 3' region (E2, E, F) gRNAs to delete the mouse Trpa1 gene and simultaneously replace the gene with the corresponding human TRPA1 form. LTVEC is shown at the top of the figure and the mouse Trpa1 locus is shown at the bottom of the figure. The positions of the Cas9 cleavage sites directed by the eight guide RNAs are shown by arrows below the mouse gene sequence.

[0079] FIG. 10A to FIG. 10E Results of structural variation (SV) assays of clones BR-B4, BP-G7, BO-G11, BO-F10, B0-A8, and BC-H9 are shown, with VGF1 (F1H4), 129, and B6 DNA used as controls. These assays were performed at the following distances from the telomere to the Lrp5 locus: 13.7 Mb ( Fig. 10A )、20.0Mb( Fig. 10B )、36.9Mb( Fig. 10C )、48.3Mb( Fig. 10D ) and 56.7Mb( Fig.10E ). The positions of the PCR products of the B6 and 129 alleles are indicated by arrows.

[0080] FIG. 11A to FIG. 11C The centromere side of Lrp5 is shown at 0.32 Mb ( Fig.11A ), 1.2Mb on the telomeric side of Lrp5 ( Fig. 11B ) and 57.2Mb on the telomeric side of Lrp5 ( Fig. 11C ). The values ​​on each axis represent relative fluorescence intensity. These figures represent four repetitions of each sample, and the results are shown with solid dots (B6 allele), hollow dots (129 allele) and oblique lines (B6 / 129 alleles).

[0081] FIG. 12A to FIG. 12C Schematic diagram showing a possible mechanism for mitotic recombination during the G2 phase of the cell cycle that can produce homozygous events and widespread gene conversion detected by loss of heterozygosity. Fig. 12ADuplicated homologous chromosomes are shown showing two chromatids in a hybrid 129 / B6 ES cell that is heterozygous for targeted humanization on the 129 homolog. Double arrows indicate possible double-strand breaks generated by dual gRNA-guided Cas9 cleavage, which promotes reciprocal exchanges through homologous recombination between chromatids on homologous chromosomes, which are shown as crossovers on the centromeric side of the targeted allele, ultimately resulting in Fig. 12B The hybridized chromatids shown in . Fig. 12C It is shown that after mitosis and cell division, four types of chromosomes may be separated into daughter cells. The chromosomes that retain heterozygosity, i.e., parental heterozygotes (Hum / +, upper left) and heterozygotes (Hum / +, upper right) through equal exchange, cannot be distinguished by LOH determination. The other two types show loss of heterozygosity, wherein humanized homozygotes (Hum / Hum, such as clone BO-A8, lower left) show telomere side B6 allele loss, and wild-type homozygotes (+ / +, lower right) show telomere side 129 allele loss. The latter, i.e., wild-type homozygotes, will be lost because they do not retain the drug resistance box of the humanized allele.

[0082] Fig.13 Shown is a targeting vector (LTVEC) with a homology arm size of 35kb and 31kb or a targeting vector (sTVEC) with a homology arm size of 5kb per homology arm and one or two 5' regions (A, B), middle regions (C, D) and 3' regions (E, E2) gRNAs to delete the region from exon 2 to the stop codon in the mouse C5 (Hc) gene, while replacing the schematic diagram of the region with the corresponding human C5 form. The two targeting vectors are shown at the top of the figure, and the mouse C5 (Hc) locus is shown at the bottom of the figure. The position of the Cas9 cleavage site guided by six guide RNAs is shown by the vertical arrows below the mouse gene sequence, and the primers for screening are shown by horizontal arrows. The position of the allele gain (GOA) assay to determine the number of insert copies and the allele loss (LOA) assay to determine the mouse sequence of the targeted deletion is shown by a triangle.

[0083] Fig.14 A schematic diagram of using LTVEC and two 5' region (A, B) gRNAs to delete the first five exons of the mouse Cmah gene while replacing this portion with a lacZ reporter gene and a hygromycin resistance selection cassette is shown. LTVEC is shown at the top of the figure, and the mouse Cmah locus is shown at the bottom of the figure. The positions of the Cas9 cleavage sites guided by the two guide RNAs are shown by the vertical arrows below the mouse gene sequence, and the positions of the GOA assays for determining the number of insert copies and the LOA assays for determining the mouse sequence of the targeted deletion are shown by triangles.

[0084] Fig.15 A schematic diagram of the cleavage event and resulting excision product (SEQ ID NO: 112) when the mouse Cmah locus (SEQ ID NO: 109) is targeted with two 5' region gRNAs (A and B; SEQ ID NOS: 107 and SEQ ID NOS: 108, respectively). The gRNA sequence hybridizing to the Cmah locus is shown in bold, the Cas9 protein is represented by a dotted oval, the Cas9 cleavage site is shown by a vertical arrow, and the protospacer adjacent motif (PAM) is boxed. The approximate positions of the LOA assay forward primer, probe, and reverse primer are shown by the horizontal bars and arrows above the figure. The 5' and 3' fragments generated after cleavage and excision are SEQ ID NOS: 110 and SEQ ID NOS: 111, respectively.

[0085] FIG. 16A to FIG. 16E Shown are possible mechanisms explaining the results observed in CRISPR / Cas9-assisted humanization experiments performed in F1 hybrid mouse ES cells, which have one haploid chromosome set derived from the 129S6 / SvEvTac mouse cell line and one haploid chromosome set derived from the C57BL / 6NTac (B6) mouse cell line. Fig.16A Reciprocal chromatid exchange by mitotic crossing over is shown, in which heterozygous modification occurs on chromosome 129 either before genome duplication or after genome duplication, followed by gene conversion between sister chromatids. Fig. 16B Reciprocal chromatid exchange by mitotic crossing over is shown, in which a single chromatid 129 is modified following genome duplication. Fig. 16C Reciprocal chromatid exchange by mitotic crossover is shown, where LTVEC targeting has not yet occurred, but Cas9 cleavage has occurred on chromosome 129 or B6 (B6 cleavage is shown). Fig.16D Chromatid duplication by breakage-induced replication is shown, with heterozygous modification occurring on chromosome 129 either before or after genome duplication, followed by gene conversion between sister chromatids. Fig.16E Chromatid copying by break-induced replication is shown, where a single chromatid 129 is modified following genome duplication. Fig.16F Chromatid copying by break-induced replication is shown, where LTVEC targeting has not yet occurred, but Cas9 cleavage has occurred on chromosome 129 or B6 (B6 cleavage is shown).

[0086] 17A to 17CScreening strategies for targeted modifications are shown. Fig.17A A standard modification of allele (MOA) screening strategy is shown to detect heterozygous targeting by a large targeting vector (LTVEC) in which the endogenous sequence in the mouse chromosome is deleted and replaced by the Neo-SDC insert. Probes mTU and mTD are directed against the upstream and downstream regions of the endogenous sequence targeted for deletion. Fig. 17B Shows the use of Retention assays (retU and retD probes) were combined with modification of allele (MOA) assays (mTGU, mTM, and mTGD probes for loss of allele (LOA) assays, and hTU and hTD probes for gain of allele (GOA) assays) to screen for CRISPR / Cas9-assisted humanization. Fig. 17C Shows the use of The retention assay (retU and retD probes) was combined with the loss-of-allele (LOA) assay (mTGU, mTM, and mTGD probes) to screen for CRISPR / Cas9-assisted deletions by paired guide RNAs (gU and gD).

[0087] Fig.18 A schematic diagram (not drawn to scale) of a targeting vector of approximately 900 kb of a mouse immunoglobulin heavy chain locus in which the variable region gene segments are replaced by human equivalents (triangles) and a Pgk-Neo insert flanked by loxP sites (effectively linked to the phosphoglycerate kinase I promoter of the neomycin phosphotransferase gene). Two gRNAs are used to cut the mouse immunoglobulin heavy chain locus at the 5' end, and two gRNAs are used to cut the locus at the 3' end, and the targeting vector deletes the mouse immunoglobulin heavy chain locus and replaces the locus with the Pgk-Neo insert. The location of the Cas9 cleavage site guided by the four guide RNAs is shown by the vertical arrow below the target locus. The circled horizontal lines indicate the sequences used for modification of allele (MOA) assay (hIgH31, hIgH1, mIgHA1, mIgHA7, and hIgH9) and retention assay (5'IgH arm 1, 5'IgH arm 2, mIgM-398, and mIgM-1045). Probe.

[0088] definition

[0089] The terms "protein", "polypeptide" and "peptide" used interchangeably herein include polymeric forms of amino acids of any length, including coded amino acids and non-coded amino acids, as well as chemically or biochemically modified or derivatized amino acids. These terms also include modified polymers, such as polypeptides with modified peptide backbones.

[0090] The terms "nucleic acid" and "polynucleotide" used interchangeably herein include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or their analogs or modified forms. These terms include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural or derived nucleotide bases.

[0091] "Codon optimization" generally includes the process of modifying a nucleic acid sequence to enhance expression in a specific host cell by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of a host cell while maintaining the native amino acid sequence. For example, the nucleic acid encoding the Cas protein may be modified to replace a codon with a higher frequency of use in a given prokaryotic cell or eukaryotic cell (including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells or any other host cell) compared to a naturally occurring nucleic acid sequence. Codon usage tables are readily available, such as provided at the "Codon Usage Database". These tables can be adapted in a variety of ways. See Nakamura et al. (2000) Nucleic Acids Research 28: 292 (Nakamura et al., 2000, "Nucleic Acids Research", Vol. 28, p. 292), which is incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available for codon optimization of specific sequences for expression in a specific host (see, e.g., Gene Forge).

[0092] "Operably linked" or "operably linked" includes the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that the two components function normally and at least one of the components has the potential to mediate a function imposed on at least one of the other components. For example, a promoter may be operably linked to a coding sequence if the promoter controls the transcription level of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors.

[0093] "Complementarity" of nucleic acids means that the nucleotide sequence in one strand of nucleic acid forms hydrogen bonds with another sequence on the opposite nucleic acid strand due to the orientation of its nucleobase groups. Complementary bases in DNA are usually A and T and C and G. In RNA, they are usually C and G and U and A. Complementarity can be complete or substantial / sufficient. Complete complementarity between two nucleic acids means that the two nucleic acids can form a duplex, where each base in the duplex binds to a complementary base according to the Watson-Crick pairing principle. "Substantial" or "sufficient" complementarity means that the sequence in one strand is not completely and / or completely complementary to the sequence in the opposite strand, but sufficient bonding occurs between the bases on the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequence and standard mathematical calculations to predict the Tm (melting temperature) of the hybridized strand, or by empirically determining the Tm using conventional methods. The Tm includes the temperature at which 50% denaturation occurs in a population of hybrid complexes formed between two nucleic acid strands. At temperatures below Tm, the formation of hybrid complexes is favored, while at temperatures above Tm, the melting or separation of the two chains in the hybrid complex is favored. Tm can be estimated for nucleic acids with known G+C content in 1M NaCl aqueous solution, for example using Tm=81.5+0.41(%G+C), while other known Tm calculation methods take into account nucleic acid structural features.

[0094] "Hybridization conditions" include the cumulative environment in which one nucleic acid strand bonds to a second nucleic acid strand through complementary strand interactions and hydrogen bonding to produce a hybrid complex. Such conditions include the chemical composition of the aqueous or organic solution containing the nucleic acid and its concentration (e.g., salts, chelating agents, formamide) and the temperature of the mixture. Other factors (e.g., the length of incubation time or the size of the reaction chamber) may have an impact on the environment. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2.sup.nd ed., pp. 1.90-1.91, 9.47-9.51, 1 1.47-11.57 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), which is incorporated herein by reference in its entirety for all purposes.

[0095] Hybridization requires that the two nucleic acids contain complementary sequences, but mismatches are allowed between bases. The conditions suitable for hybridization between two nucleic acids depend on the length and complementarity of the nucleic acids, and these variables are well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of the hybrid of the nucleic acids with these sequences. For hybridization between nucleic acids with short sequence segment complementarity (e.g., complementarity within 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides), the position of mismatch becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of hybridizable nucleic acids is at least about 10 nucleotides. The exemplary minimum length of hybridizable nucleic acids includes at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides, and at least about 30 nucleotides. In addition, the temperature and washing solution salt concentration can be adjusted as needed according to factors such as the length and complementarity of the complementary region.

[0096] The sequence of the polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to achieve specific hybridization. In addition, the polynucleotide can hybridize in one or more segments so that the intervening or adjacent segments do not participate in hybridization events (e.g., loop structures or hairpin structures). The polynucleotide (e.g., gRNA) can have at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence complementarity in the target region within the target nucleic acid sequence it targets. For example, a gRNA in which 18 of the 20 nucleotides are complementary to the target region and thus specifically hybridizes will have 90% complementarity. In this example, the remaining non-complementary nucleotides can be clustered or interspersed in complementary nucleotides and do not need to be adjacent to each other or adjacent to complementary nucleotides.

[0097] The percent complementarity between specific sequence segments of nucleic acid sequences within a nucleic acid can generally be determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program known in the art (Altschul et al. (1990) J. Mol. Biol. 215:403-410; Zhang and Madden (1997) Genome Res. 7:649-656) or using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison, Wisconsin, USA). Wis.)), these programs use the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0098] The methods and compositions provided herein employ a variety of different components. It has been confirmed throughout this specification that some components may have active variants and fragments. Such components include, for example, Cas proteins, CRISPR RNA, tracrRNA, and guide RNA. The biological activity of each of these components is described elsewhere herein.

[0099] In the context of two polynucleotides or polypeptide sequences, "sequence identity" or "identity" refers to the residues that are identical in the two sequences when aligned within a specified comparison window to achieve maximum correspondence. When using percentage sequence identity to refer to proteins, it should be recognized that non-identical residue positions usually differ in conservative amino acid substitutions, in which amino acid residues are replaced with other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and thus do not change the functional properties of the molecule. When the sequence difference is conservative substitution, the percentage sequence identity can be adjusted upward to correct the conservative nature of the substitution. The difference is that sequences with such conservative substitutions are referred to as having "sequence similarity" or "similarity". The manner in which this adjustment is performed is well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, if an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of 0, a conservative substitution is given a score between 0 and 1. The score for conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0100] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences within a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) to ensure optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0101] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 using the following parameters: % identity and % similarity for nucleotide sequences using gap weight (GAP Weight) 50 and length weight 3 and nwsgapdna.cmp scoring matrix; % identity or % similarity for amino acid sequences using gap weight 8 and length weight 2 and BLOSUM62 scoring matrix; or any equivalent programs thereof. "Equivalent programs" include any sequence comparison programs that produce such alignments for any two sequences under consideration that have identical nucleotide or amino acid residue matches and identical sequence identity percentages when compared to the corresponding alignments produced by GAP version 10.

[0102] The term "in vitro" includes artificial environments (e.g., test tubes) and processes or reactions that occur within artificial environments. The term "in vivo" includes natural environments (e.g., cells or organisms or bodies) and processes or reactions that occur within natural environments. The term "ex vivo" includes cells that have been removed from an individual's body and processes or reactions that occur within such cells.

[0103] A composition or method that "comprises" or "includes" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" a certain protein may include the protein alone or in combination with other ingredients.

[0104] The specification of a range of values ​​includes all integers within or defining the range, and all sub-ranges defined by the integers within the range.

[0105] Unless otherwise apparent from the context, the term "about" encompasses those values ​​that fall within the standard tolerance of measurement error (eg, SEM) for the stated value.

[0106] Unless the context clearly indicates otherwise, the singular forms of the quantifiers "a", "an", and "the" include plural referents. For example, the term "Cas protein" or "at least one Cas protein" may include multiple Cas proteins, including mixtures thereof. DETAILED DESCRIPTION

[0107] I. Overview

[0108] The present invention provides methods and compositions for modifying the genome in a cell.These methods and compositions use CRISPR / Cas systems, using two guide RNAs (gRNAs) to target different sites in a single genome target locus.For example, these methods and compositions can use CRISPR / Cas systems, using two guide RNAs (gRNAs) to produce paired double-strand breaks at different sites in a single genome target locus.Or, these methods and compositions can use CRISPR / Cas systems, using two guide RNAs (gRNAs) to produce paired single-strand breaks at different sites in a single genome target locus.In some methods, two or more (e.g., three or four) guide RNAs can be used, for example, to produce two or more single-strand breaks or double-strand breaks at different sites in a single genome target locus.

[0109] Some methods promote biallelic genetic modification and include genome shearing, thereby making large nucleic acid sequences missing from the chromosome between two cleavage sites. Other methods promote biallelic genetic modification and include the disappearance of intracellular nucleic acid sequences and are replaced by exogenous nucleic acid sequences simultaneously. As further summarized in detail below, these methods using two gRNAs improve the efficiency of producing such cells or animals with biallelic targeted genetic modification by promoting the production of cells and animals in a single targeting step. Therefore, the number of animals and matings necessary for producing animals with biallelic targeted genetic modification is reduced.

[0110] Other methods include gene conversion or loss of heterozygosity, whereby a genome that is heterozygous for an allele is modified to become homozygous for the allele by cutting at sites determined by two gRNAs in the corresponding alleles on the corresponding homologous chromosomes. As further detailed below, the use of two gRNAs in these methods increases the frequency of gene conversion and enables gene conversion on large segments of chromosomal DNA.

[0111] II. CRISPR / Cas System

[0112] The methods and compositions disclosed herein can utilize clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems or components of such systems to modify the genome within a cell. The CRISPR / Cas system includes transcripts and other elements that participate in the expression of Cas genes or guide the activity of Cas genes. The CRISPR / Cas system may be a type I, type II, or type III system. The methods and compositions disclosed herein employ a CRISPR / Cas system to perform site-directed cleavage of nucleic acids by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein).

[0113] Some CRISPR / Cas systems used in the methods disclosed herein are non-naturally occurring. A "non-naturally occurring" system includes any system that indicates human intervention, such as one or more components of the system being altered or mutated from its naturally occurring state, at least substantially free of at least one other component with which it is naturally associated in nature, or associated with at least one other component that is not naturally associated with it. For example, some CRISPR / Cas systems employ non-naturally occurring CRISPR complexes that include gRNA and Cas proteins that do not occur together in nature.

[0114] A. Cas RNA-guided endonucleases

[0115] Cas proteins generally include at least one RNA recognition or binding domain. Such domains can interact with guide RNA (gRNA, described in more detail below). Cas proteins may also include nuclease domains (e.g., DNA enzyme or RNA enzyme domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. The nuclease domain has catalytic activity for nucleic acid cutting. Cutting includes the breaking of covalent bonds of nucleic acid molecules. Cutting can produce blunt ends or staggered ends, and it can be single-stranded or double-stranded.

[0116] Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 ( CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966, and homologs or modified forms thereof.

[0117] In some cases, the Cas protein is derived from a type II CRISPR / Cas system. For example, the Cas protein can be a Cas9 protein or be derived from a Cas9 protein. These Cas9 proteins generally share four key motifs with a conserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, and motif 3 is a HNH motif. The Cas9 protein can be from, for example, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus lusacidocaldarius, Bacillus pseudomycoides, Bacillus selenate-reducing. selenitireducens), Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptorbecscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum), Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas shaloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleuschthonoplastes, Oscillatoria sp., Petrotoga sp. mobilis), Thermosipho africanus, or Acaryochloris marina. Additional examples of Cas9 family members include those described in WO 2014 / 131833, which is incorporated herein by reference in its entirety. In a specific example, the Cas9 protein is from Streptococcus pyogenes (S.pyogenes) or a Cas9 protein derived therefrom. The amino acid sequence of the Cas9 protein from Streptococcus pyogenes can be found, for example, in the SwissProt database under accession number Q99ZW2. .

[0118] The Cas protein may be a wild-type protein (i.e., a protein existing in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. The Cas protein may also be an active variant or fragment of a wild-type or modified Cas protein. The active variant or fragment may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a wild-type or modified Cas protein or a portion thereof, wherein the active variant retains the ability to cut at the desired cleavage site, thereby retaining the nick-inducing activity or the double-strand break-inducing activity. Assays for nick-inducing activity or double-strand break-inducing activity are known, and generally measure the overall activity and specificity of the Cas protein to a DNA substrate comprising a cleavage site.

[0119] The Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity and / or enzyme activity. The Cas protein can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted or inactivated, or the Cas protein can be truncated to remove domains that are not essential for the function of the protein, or to optimize (e.g., enhance or reduce) the activity of the Cas protein.

[0120] Some Cas proteins include at least two nuclease domains, such as a DNA enzyme domain. For example, the Cas9 protein may include a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC domain and the HNH domain can each cut different strands of double-stranded DNA, thereby producing double-strand breaks in the DNA. See, for example, Jinek et al. (2012) Science 337: 816-821 (Jinek et al., 2012, Science, Vol. 337, pp. 816-821), which is incorporated herein by reference in its entirety for all purposes.

[0121] One or both of these nuclease domains can be deleted or mutated so that they are no longer functional or have reduced nuclease activity. If one of the nuclease domains is deleted or mutated, the resulting Cas protein (e.g., Cas9) can be referred to as a nickase, and a single-strand break can be generated at the CRISPR RNA recognition sequence in double-stranded DNA, but a double-strand break (i.e., it can cut complementary strands or non-complementary strands, but cannot cut both at the same time) will not be generated. If both nuclease domains are deleted or mutated, the resulting Cas protein (e.g., Cas9) will have the ability to cut both strands of double-stranded DNA with a reduction. The example of a mutation that Cas9 is converted into a nickase is the D10A (aspartic acid to alanine at the 10th position of Cas9) mutation in the RuvC domain of Cas9 from Streptococcus pyogenes. Similarly, H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from Streptococcus pyogenes can convert Cas9 into a nickase. Other examples of mutations that convert Cas9 into nickases include corresponding mutations of Cas9 from Streptococcus thermophilus (S.thermophilus). See, for example, Sapranauskas et al. (2011) Nucleic Acids Research 39: 9275-9282 (Sapranauskas et al., 2011, "Nucleic Acids Research", Vol. 39, pp. 9275-9282) and WO 2013 / 141680, each of which is incorporated herein by reference in its entirety for all purposes. Such mutations can be generated using well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis or full gene synthesis. Other examples of mutations that form nickases can be found in, for example, WO / 2013 / 176772A1 and WO / 2013 / 142578A1, each of which is incorporated herein by reference in its entirety for all purposes.

[0122] Cas protein can also be a fusion protein. For example, Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. See WO 2014 / 089290, which is incorporated herein by reference in its entirety for all purposes. Cas protein can also be fused to a heterologous polypeptide to provide enhanced or reduced stability. The fused domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or inside the Cas protein.

[0123] An example of a Cas fusion protein is a Cas protein fused to a heterologous polypeptide providing subcellular localization. Such sequences may include, for example, a nuclear localization signal (NLS) such as SV40 NLS for targeting the nucleus, a mitochondrial localization signal for targeting mitochondria, an ER retention signal, etc. See, for example, Lange et al. (2007) J. Biol. Chem. 282: 5101-5105 (Lange et al., 2007, Journal of Biological Chemistry, Vol. 282, pp. 5101-5105), which is incorporated herein by reference in its entirety for all purposes. For example, the Cas protein may be fused to one or more nuclear localization signals (e.g., two or three nuclear localization signals). Such subcellular localization signals may be located at any position within the N-terminus, C-terminus, or Cas protein. NLS may include a stretch of basic amino acids and may be a monotype (monopartite) sequence or a bitype (bipartite) sequence.

[0124] Cas protein may also include a cell penetration domain. For example, the cell penetration domain may be derived from HIV-1TAT protein, TLM cell penetration motif from human hepatitis B virus, MPG, Pep-1, VP22, cell penetration peptides from herpes simplex virus, or polyarginine peptide sequences. See, for example, WO2014 / 089290, which is incorporated herein by reference in its entirety for all purposes. The cell penetration domain may be located at any position within the N-terminus, C-terminus, or Cas protein.

[0125] The Cas protein may also contain a heterologous polypeptide that facilitates tracing or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples of fluorescent proteins include green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRedmonomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly (NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP) and calmodulin.

[0126] Cas protein can be provided in any form. For example, Cas protein can be provided in the form of protein, such as Cas protein complexed with gRNA. As another option, Cas protein can be provided in the form of nucleic acid encoding Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA. Optionally, the nucleic acid encoding Cas protein can be codon optimized to be effectively translated into protein in a specific cell or organism. For example, the nucleic acid encoding Cas protein can be modified to replace with a codon with a higher frequency of use in bacterial cells, yeast cells, non-pluripotent human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells or any other target host cell compared to the naturally occurring polynucleotide sequence. When the nucleic acid encoding Cas protein is introduced into the cell, the Cas protein can be expressed transiently, conditionally or constitutively in the cell.

[0127] The nucleic acid encoding the Cas protein can be stably integrated into the genome of the cell and effectively connected to a promoter active in the cell. Alternatively, the nucleic acid encoding the Cas protein can be effectively connected to a promoter in an expression construct. The expression construct includes any nucleic acid construct that can guide the expression of a target gene or other nucleic acid sequence (e.g., a Cas gene) and can transfer this target nucleic acid sequence to a target cell. For example, the nucleic acid encoding the Cas protein can be in a targeting vector comprising a nucleic acid insert and / or a vector comprising a DNA encoding a gRNA. Alternatively, it can be in a vector or plasmid separated from a targeting vector comprising a nucleic acid insert, and / or in a vector or plasmid separated from a vector comprising a DNA encoding a gRNA. Promoters that can be used for expression constructs include, for example, promoters active in rats, eukaryotes, mammals, non-human mammals, humans, rodents, mice, or hamster pluripotent cells. Promoters active in 1-cell embryos can also be used. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Examples of other promoters are described elsewhere herein.

[0128] B. Guide RNA (gRNA)

[0129] "Guide RNA" or "gRNA" includes an RNA molecule that binds to the Cas protein and targets the Cas protein to a specific location within the target DNA. The guide RNA may contain two segments: a "DNA targeting segment" and a "protein binding segment". A "segment" includes a segment, part or region of a molecule, such as a contiguous nucleotide segment in an RNA. Some gRNAs contain two separate RNA molecules: an "activator-RNA" and a "targeting factor-RNA". Other gRNAs are single RNA molecules (single RNA polynucleotides), which may also be referred to as "single-molecule gRNA", "single guide RNA" or "sgRNA". See, for example, WO / 2013 / 176772A1, WO / 2014 / 065596A1, WO / 2014 / 089290A1, WO / 2014 / 093622A2, WO / 2014 / 099750A2, WO / 2013142578A1, and WO2014 / 131833A1, each of which is incorporated herein by reference in its entirety for all purposes. The terms "guide RNA" and "gRNA" are inclusive and include bimolecular gRNA and unimolecular gRNA.

[0130] An exemplary bimolecular gRNA comprises a crRNA-like ("CRISPR RNA" or "targeting factor-RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA" or "activator-RNA" or "tracrRNA" or "scaffold") molecule. The crRNA comprises the DNA targeting segment (single strand) of the gRNA and a stretch of nucleotides that forms half of the dsRNA duplex of the protein binding segment of the gRNA.

[0131] The corresponding tracrRNA (activator-RNA) comprises a section of nucleotides that form the other half of the dsRNA duplex of the protein binding segment of the gRNA. A section of nucleotides of crRNA is complementary to and hybridized with a section of nucleotides of tracrRNA to form a dsRNA duplex of the protein binding domain of the gRNA. Therefore, each crRNA can be said to have a corresponding tracrRNA. TracrRNA can be in any form (e.g., full-length tracrRNA or active partial tracrRNA) and has different lengths. The form of tracrRNA may include a primary transcript or a processed form. For example, in Streptococcus pyogenes (S.pyogenes), different forms of tracrRNA include 171-nucleotides, 89-nucleotides, 75-nucleotides, and 65-nucleotide forms. See, e.g., Deltcheva et al. (2011) Nature 471:602-607 and WO 2014 / 093661, each of which is incorporated herein by reference in its entirety for all purposes.

[0132] The crRNA and the corresponding tracrRNA hybridize to form gRNA. The crRNA additionally provides a single-stranded DNA targeting segment that hybridizes with the CRISPR RNA recognition sequence. If used for intracellular modification, the exact sequence of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which these RNA molecules will be used. See, e.g., Mali et al. (2013) Science 339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al. (2013) Nat. Biotechnol. 31:227-229; Jiang et al. (2013) Nat. Biotechnol. 31:233-239; and Cong et al. (2013) Science 339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al. (2013) Nat. Biotechnol. 31:227-229; Jiang et al. (2013) Nat. Biotechnol. 31:233-239; and Cong et al. (2013) Science 339:823-826; al. (2013) Science 339:819-823, each of which is incorporated herein by reference in its entirety for all purposes.

[0133] The DNA targeting segment (crRNA) of a given gRNA comprises a nucleotide sequence complementary to the sequence in the target DNA. The DNA targeting segment of gRNA interacts with the target DNA in a sequence-specific manner by hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the DNA targeting segment can vary, and determines the position in the target DNA that will interact with the gRNA and the target DNA. The DNA targeting segment of an individual gRNA can be modified to hybridize with any desired sequence in the target DNA. Naturally occurring crRNAs vary with the Cas9 system and organism, but generally comprise a targeting segment of 21 to 72 nucleotides long, which is flanked by two forward repeats (DR) of 21 to 46 nucleotides long (see, for example, WO2014 / 131833, which is incorporated herein by reference in its entirety for all purposes). For Streptococcus pyogenes, DR is 36 nucleotides long, and the targeting segment is 30 nucleotides long. The DR located at 3' is complementary and hybridized with the corresponding tracrRNA, and then bound to the Cas9 protein.

[0134] The length of the DNA targeting segment can be from about 12 nucleotides to about 100 nucleotides. For example, the length of the DNA targeting segment can be from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt. Alternatively, the length of the DNA targeting segment can be about 19 nt to about 20 nt, about 19 nt to about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 19 nt to about 70 nt, about 19 nt to about 80 nt, about 19 nt to about 90 nt, about 19 nt to about 100 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, about 20 nt to about 60 nt, about 20 nt to about 70 nt, about 20 nt to about 80 nt, about 20 nt to about 90 nt, or about 20 nt to about 100 nt.

[0135] The length of the nucleotide sequence of the DNA targeting segment that is complementary to the nucleotide sequence of the target DNA (CRISPR RNA recognition sequence) can be at least about 12 nt. For example, the DNA targeting sequence (i.e., the sequence within the DNA targeting segment that is complementary to the CRISPR RNA recognition sequence within the target DNA) can have a length of at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt. Alternatively, the length of the DNA targeting sequence can be about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 45 nt, about 12 nt to about 40 nt, about 12 nt to about 35 nt, about 12 nt to about 30 nt, about 12 nt to about 25 nt, about 12 nt to about 20 nt, about 12 nt to about 19 nt, about 19 nt to about 20 nt, about 19 nt to about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, or about 20 nt to about 60 nt. In some cases, the DNA targeting sequence can have a length of at least about 20 nt.

[0136] TracrRNA can be in any form (e.g., full-length tracrRNA or active partial tracrRNA) and have different lengths. They may include primary transcripts or processed forms. For example, tracrRNA (as part of a single guide RNA or as a separate molecule belonging to a part of a bimolecular gRNA) may include or consist of the following parts: all or part of a wild-type tracrRNA sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracrRNA sequence). Examples of wild-type tracrRNA sequences from Streptococcus pyogenes include forms of 171 nucleotides, 89 nucleotides, 75 nucleotides, and 65 nucleotides. See, for example, Deltcheva et al. (2011) Nature 471: 602-607 (Deltcheva et al., 2011, Nature, Vol. 471, pp. 602-607) and WO 2014 / 093661, each of which is incorporated herein by reference in its entirety for all purposes. Examples of tracrRNAs within single guide RNAs (sgRNAs) include tracrRNA segments present in sgRNAs in the form of +48, ​​+54, +67, and +85, wherein "+n" indicates that up to +n nucleotides of wild-type tracrRNA are included in the sgRNA. See US 8,697,359, which is incorporated herein by reference in its entirety for all purposes.

[0137] The percent complementarity between the DNA targeting sequence and the CRISPR RNA recognition sequence within the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%). In some cases, the percent complementarity between the DNA targeting sequence and the CRISPR RNA recognition sequence within the target DNA is at least 60% within about 20 contiguous nucleotides. For example, within 14 contiguous nucleotides at the 5' end of the CRISPR RNA recognition sequence within the complementary strand of the target DNA, the percent complementarity between the DNA targeting sequence and the CRISPR RNA recognition sequence within the target DNA is 100%, and as low as 0% within the remaining contiguous nucleotides. In this case, the DNA targeting sequence can be considered to be 14 nucleotides long. As another example, within the seven adjacent nucleotides of the 5' end of the CRISPR RNA recognition sequence in the complementary strand of the target DNA, the percentage of complementarity between the DNA targeting sequence and the CRISPR RNA recognition sequence in the target DNA is 100%, and is as low as 0% within the remaining adjacent nucleotides. In this case, the DNA targeting sequence can be considered to be 7 nucleotides long.

[0138] The protein binding segment of the gRNA may include two segments of nucleotides that are complementary to each other. The complementary nucleotides of the protein binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein binding segment of the target gRNA interacts with the Cas protein, and the gRNA guides the bound Cas protein to a specific nucleotide sequence within the target DNA via the DNA targeting segment.

[0139] The guide RNA may include modifications or sequences that provide additional desired characteristics (e.g., modified or regulated stability; subcellular targeting; tracking with fluorescent markers; binding sites for proteins or protein complexes; etc.). Examples of such modifications include, for example, a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (i.e., a 3' poly (A) tail); a riboswitch sequence (e.g., to achieve regulated stability and / or regulated accessibility of a protein and / or protein complex); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin); a modification or sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides a tracer (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.); and combinations thereof.

[0140] The gRNA may include a nucleic acid sequence encoding crRNA and tracrRNA. For example, the gRNA may include: (a) a chimeric RNA having a nucleic acid sequence of 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 1); or (b) a chimeric RNA having a nucleic acid sequence of 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG-3' (SEQ ID NO: 2).

[0141] In some cases, the crRNA comprises 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3' (SEQ ID NO:3); 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG (SEQ ID NO:4); or 5'-GAGUCCGAGCAGAAGAAGAAGUUUUA-3' (SEQ ID NO:5).

[0142] In some cases, the tracrRNA comprises 5'-AAGGCUAGUCCG-3' (SEQ ID NO:6) or 5'-AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG CUUUU-3' (SEQ ID NO:7).

[0143] The guide RNA can be provided in any form. For example, the gRNA can be provided in the form of RNA (as two molecules (separate crRNA and tracrRNA) or as one molecule (sgRNA)), and is optionally provided in the form of a complex with the Cas protein. The gRNA can also be provided in the form of a DNA encoding RNA. The DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or a separate RNA molecule (e.g., a separate crRNA and tracrRNA). In the latter case, the DNA encoding the gRNA can be provided as a separate DNA molecule encoding crRNA and tracrRNA, respectively.

[0144] When the DNA encoding the gRNA is introduced into the cell, the gRNA can be expressed transiently, conditionally or constitutively in the cell. The DNA encoding the gRNA can be stably integrated in the genome of the cell and effectively connected to a promoter active in the cell. As another option, the DNA encoding the gRNA can be effectively connected to a promoter in an expression construct. For example, the DNA encoding the gRNA can be in a targeting vector comprising a nucleic acid insert and / or a vector comprising a nucleic acid encoding a Cas protein. Alternatively, it can be in a vector or plasmid separated from a targeting vector comprising a nucleic acid insert, and / or in a vector or plasmid separated from a vector comprising a nucleic acid encoding a Cas protein. Promoters that can be used in such expression constructs include, for example, promoters active in rats, eukaryotes, mammals, non-human mammals, humans, rodents, mice, or hamster pluripotent cells. Promoters active in 1-cell embryos can also be used. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. In some cases, the promoter is an RNA polymerase III promoter, such as a human U6 promoter, a rat U6 polymerase III promoter, or a mouse U6 polymerase III promoter. Examples of other promoters are described elsewhere herein.

[0145] Alternatively, gRNA can be prepared by various other methods. For example, gRNA can be prepared by in vitro transcription using, for example, T7 RNA polymerase (see, for example, WO 2014 / 089290 and WO 2014 / 065596, each of which is incorporated herein by reference in its entirety for all purposes). Guide RNA can also be a synthetically produced molecule prepared by chemical synthesis.

[0146] C. CRISPR RNA recognition sequence

[0147] The term "CRISPR RNA recognition sequence" includes such a nucleic acid sequence present in the target DNA, to which the DNA targeting segment of the gRNA will bind as long as sufficient binding conditions exist. For example, the CRISPR RNA recognition sequence includes a sequence to which the guide RNA is designed to be complementary, wherein hybridization between the CRISPR RNA recognition sequence and the DNA targeting sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required, as long as there is complementarity sufficient to cause hybridization and promote the formation of the CRISPR complex. The CRISPR RNA recognition sequence also includes the cleavage site of the Cas protein described in more detail below. The CRISPR RNA recognition sequence may comprise any polynucleotide, which may be located, for example, in the nucleus or cytoplasm of a cell, or in an organelle of the cell such as a mitochondria or chloroplast.

[0148] The CRISPR RNA recognition sequence in the target DNA can be targeted by the Cas protein or gRNA (i.e., bound to, hybridized with, or complementary to it). Suitable DNA / RNA binding conditions include physiological conditions that are usually present in cells. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art (see, for example, Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001) (Sambrook et al., "Molecular Cloning Laboratory Guide", 3rd Edition, Cold Spring Harbor Laboratory Press, 2001), which is incorporated herein by reference in its entirety for all purposes. The target DNA strand that is complementary and hybridized with the Cas protein or gRNA may be referred to as a "complementary strand", and the target DNA strand that is complementary to the "complementary strand" (and therefore not complementary to the Cas protein or gRNA) may be referred to as a "non-complementary strand" or "template strand".

[0149] Cas protein can cut nucleic acid at a site within or outside the nucleic acid sequence present in the target DNA to be bound to the DNA targeting segment of gRNA. "Cut site" includes the nucleic acid position where the Cas protein produces a single-strand break or a double-strand break. For example, the formation of a CRISPR complex (comprising a gRNA that hybridizes with a CRISPR RNA recognition sequence and is compounded with a Cas protein) can result in one or two strands of nucleic acid sequence present in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) being bound to the target DNA to be bound to the DNA targeting segment of gRNA. If the cut site is located outside the nucleic acid sequence to be bound to the DNA targeting segment of gRNA, the cut site is still considered to be within the "CRISPR RNA recognition sequence". The cut site may be located on only one strand of nucleic acid or on both strands. The cut site may be located at the same position on both strands of nucleic acid (producing a blunt end), or may be located at different sites on each strand (producing staggered ends). Staggered ends can be generated, for example, by using two Cas proteins, each Cas protein generating single-strand breaks at different cleavage sites on each strand, thereby generating double-strand breaks. For example, a first nickase can form a single-strand break on the first strand of a double-stranded DNA (dsDNA), and a second nickase can form a single-strand break on the second strand of the dsDNA, so that an overhang sequence is formed. In some cases, the CRISPR RNA recognition sequence of the nickase on the first strand is separated from the CRISPR RNA recognition sequence of the nickase on the second strand by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, or 1,000 base pairs.

[0150] Site-specific cleavage of the target DNA by Cas9 can occur at a position determined by (i) base pairing complementarity between the gRNA and the target DNA, and (ii) a short motif in the target DNA, called a protospacer adjacent motif (PAM). The PAM can be flanked by a CRISPR RNA recognition sequence. Optionally, the CRISPR RNA recognition sequence can be flanked by a PAM on the 3' end. For example, the cleavage site of Cas9 can be about 1 to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence. In some cases (e.g., when using Cas9 from Streptococcus pyogenes or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-N1GG-3', wherein N1 is any DNA nucleotide and is immediately adjacent to the 3' of the CRISPR RNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5'-CCN2-3', wherein N2 is any DNA nucleotide and is immediately adjacent to the 5' of the CRISPR RNA recognition sequence of the complementary strand of the target DNA. In some such cases, N1 and N2 may be complementary, and the N1-N2 base pair may be any base pair (eg, N1=C and N2=G; N1=G and N2=C; N1=A and N2=T; or N1=T and N2=A).

[0151] Examples of CRISPR RNA recognition sequences include DNA sequences complementary to the DNA targeting segment of the gRNA, or such DNA sequences other than the PAM sequence. For example, the target motif may be a 20-nucleotide DNA sequence immediately preceding the NGG motif recognized by the Cas protein, such as GN 19 NGG (SEQ ID NO: 8) or N 20 NGG (SEQ ID NO: 9) (see, e.g., WO 2014 / 165825, which is incorporated herein by reference in its entirety for all purposes). The guanine at the 5' end can facilitate transcription by RNA polymerase in cells. Other examples of CRISPR RNA recognition sequences may include two guanine nucleotides at the 5' end (e.g., GGN 20NGG; SEQ ID NO: 10), to facilitate efficient transcription by T7 polymerase in vitro. See, for example, WO2014 / 065596, which is incorporated herein by reference in its entirety for all purposes. Other CRISPR RNA recognition sequences may have SEQ ID NOs: 8-10 that are 4-22 nucleotides long, including 5'G or GG and 3'GG or NGG. Other CRISPR RNA recognition sequences may have SEQ ID NOs: 8-10 that are 14 to 20 nucleotides long. Specific examples of CRISPR RNA recognition sequences include DNA sequences that are complementary to nucleic acids comprising any one of SEQ ID NOS: 11-38.

[0152] The CRISPR RNA recognition sequence may be any nucleic acid sequence that is endogenous or exogenous to the cell. The CRISPR RNA recognition sequence may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory sequence) or may include both. In some cases, the CRISPR RNA recognition sequence may be within a gene or nucleic acid associated with the disease and / or within a gene or nucleic acid associated with a signaling pathway. Disease-associated genes or nucleic acids include any genes or nucleic acids that produce abnormal levels or abnormal forms of transcription or translation products in cells derived from disease-infected tissues compared to tissues or cells of non-disease controls. For example, a disease-associated gene may have one or more mutations or genetic variations that are directly related to the etiology of the disease or are in linkage disequilibrium with one or more genes associated with the etiology of the disease. The transcription or translation product may be known or unknown and may be at normal or abnormal levels. Examples of disease-associated genes and nucleic acids are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) at the U.S. National Library of Medicine in Bethesda, Maryland, and specific information can be found on the World Wide Web. For additional examples of disease-associated genes and nucleic acids, see U.S. Pat. No. 8,697,359, which is incorporated herein by reference in its entirety for all purposes.

[0153] The mutation in the pathogenic gene can be a recessive mutation or a dominant mutation. A diploid organism (i.e., an organism with two copies of each chromosome) usually carries two copies of each gene. If the two copies in an individual are identical, the individual is homozygous for the gene. If the two copies are different alleles, the individual is heterozygous for the gene. The term "genotype" includes whether an individual carries a mutation in a single gene (or multiple genes), and the term "phenotype" includes the physical or functional results of the genotype. Recessive mutations include such mutations: wherein both alleles must mutate to observe a mutant phenotype (i.e., the organism must be homozygous for the mutant allele to show a mutant phenotype). Recessive mutations can, for example, inactivate infected genes and cause loss of function. For example, recessive mutations can remove all or part of the genes in the chromosome, destroy the expression of a gene, or change the structure of the encoded protein, thereby changing its function. In contrast, dominant mutations include mutations in which a mutant phenotype can also be observed in an organism that is heterozygous for the mutation (i.e., the organism carries a mutant allele and a wild-type allele). Dominant mutations can, for example, lead to gain of function. For example, a dominant mutation can increase the activity of a given gene product, confer new activity to a gene product, or lead to its inappropriate temporal or spatial expression. Dominant mutations can also be associated with loss of function. In some cases, if two copies of a gene are required to function normally, removing a single copy may cause a mutant phenotype. Such genes are haploinsufficient. In other cases, a mutation in one allele may result in changes in protein structure that interfere with the function of the wild-type protein encoded by the other allele. Such mutations are dominant negative mutations. Some alleles may be associated with both recessive and dominant phenotypes.

[0154] Some CRISPR RNA recognition sequences are within genes or nucleic acids that contain mutations. Such mutations may, for example, be dominant mutations or recessive mutations. In some cases, dominant mutations are within cells that are heterozygous for dominant mutations (that is, cells contain wild-type alleles and mutant alleles with dominant mutations). In some such cases, the CRISPR RNA recognition sequence may be within the mutant allele rather than the wild-type allele. Alternatively, the CRISPR RNA recognition sequence may be within the wild-type allele rather than the mutant allele.

[0155] III. Targeting Vectors and Nucleic Acid Inserts

[0156] Methods and compositions disclosed herein can also utilize targeting vectors comprising nucleic acid inserts and homology arms to modify the genome in cells. In such methods, nucleic acid inserts are integrated into the genome target locus determined by homology arms through homologous recombination events. The methods provided herein can utilize nuclease reagents (e.g., Cas proteins) to be combined with homologous recombination events. Such methods use nicks or double-strand breaks formed by nuclease reagents at nuclease cleavage sites and combine homologous recombination to facilitate the integration of nucleic acid insert targeting into the genome target locus.

[0157] A. Targeting Vectors and Nucleic Acid Inserts for Cells Other Than 1-Cell Embryos

[0158] (1) Nucleic acid insert

[0159] One or more separate nucleic acid inserts can be used in the methods disclosed herein, and can be introduced into cells by separate targeting vectors or on the same targeting vectors. Nucleic acid inserts include DNA segments to be integrated into the genomic target locus. Integrating nucleic acid inserts at the target locus can result in adding a nucleic acid sequence of interest to the target locus, the disappearance of the nucleic acid sequence of interest at the target locus, and / or the replacement (i.e., deletion and insertion) of the nucleic acid sequence of interest at the target locus.

[0160] The nucleic acid inset or the corresponding nucleic acid replaced at the target locus can be a coding region, an intron, an exon, a non-translated region, a regulatory region, a promoter, an enhancer, or any combination thereof. In addition, the nucleic acid inset or the corresponding nucleic acid replaced at the target locus can be any desired length, including, for example, a length between 10 to 100 nucleotides, a length of 100 to 500 nucleotides, a length of 500 nucleotides to 1 kb, a length of 1 kb to 1.5 kb nucleotides, a length of 1.5 kb to 2 kb nucleotides, a length of 2 kb to 2.5 kb nucleotides, a length of 2.5 kb to 3 kb nucleotides, a length of 3 kb to 5 kb nucleotides, a length of 5 kb to 8 kb nucleotides, a length of 8 kb to 10 kb nucleotides or a longer length. In other cases, the length can be about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 250 kb, about 250 kb to about 300 kb, about 300 kb to about 350 kb, about 350 kb to about 400 kb, about 400 kb to about 800 kb, about 800 kb to 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, about 2.5 Mb to about 2.8 Mb, about 2.8 Mb to about 3 Mb. In other cases, the length can be at least 100, 200, 300, 400, 500, 600, 700, 800 or 900 nucleotides or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or larger. Some nucleic acid inserts may be even smaller. For example, an insert of about 4 nucleotides to about 12 nucleotides in length can be inserted to form a restriction endonuclease site.

[0161] In some targeting vectors, the nucleic acid insert can be about 5 kb to about 200 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 60 kb to about 70 kb, about 80 kb to about 90 kb, about 90 kb to about 100 kb, about 100 kb to about 110 kb, about 120 kb to about 130 kb, about 130 kb to about 140 kb, about 140 kb to about 150 kb, about 150 kb to about 160 kb, about 160 kb to about 170 kb, about 170 kb to about 180 kb, about 180 kb to about 190 kb, about 190 kb to about 200 kb. Alternatively, the nucleic acid insert can be from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to about 300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400 kb.

[0162] In some cases, replacing the nucleic acid at the target locus results in a nucleic acid sequence deletion ranging from about 1 kb to about 200 kb, about 2 kb to about 20 kb, or about 0.5 kb to about 3 Mb. In some cases, the extent of the deletion is greater than the total length of the 5' homology arm and the 3' homology arm.

[0163] In some cases, the extent of deletion of a nucleic acid sequence is in the range of about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 50 kb to about 70 kb, about 50 kb to about 80 kb, about 50 kb to about 90 kb, about 50 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, about 90 kb to about 100 kb, about 100 kb to about 110 kb, about 110 kb to about 120 kb, about 120 kb to about 130 kb, about 130 kb to about 140 kb, about 140 kb to about 150 kb, about 150 kb to about 160 kb. kb, about 160 kb to about 170 kb, about 170 kb to about 180 kb, about 180 kb to about 190 kb, about 190 kb to about 200 kb, about 200 kb to about 250 kb, about 250 kb to about 300 kb, about 300 kb to about 350 kb, about 350 kb to about 400 kb, about 400 kb to about 800 kb, about 800 kb to 1 Mb, about 1 Mb to about 1.5 Mb, From about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, from about 2.5 Mb to about 2.8 Mb, from about 2.8 Mb to about 3 Mb, from about 200 kb to about 300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb.

[0164] In other cases, the nucleic acid insert or the corresponding nucleic acid replaced at the target locus can be at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more.

[0165] The nucleic acid insert can comprise genomic DNA or any other type of DNA. For example, the nucleic acid insert can be derived from: a prokaryote, a eukaryote, a yeast, a bird (e.g., a chicken), a non-human mammal, a rodent, a human, a rat, a mouse, a hamster, a rabbit, a pig, a cow, a deer, a sheep, a goat, a cat, a dog, a ferret, a primate (e.g., a marmoset, a rhesus monkey), a domestic mammal or an agricultural mammal, or any other organism of interest.

[0166] The nucleic acid insert and / or the nucleic acid at the target locus may comprise a coding sequence or a non-coding sequence, such as a regulatory element (e.g., a promoter, an enhancer, or a transcriptional repressor binding element). For example, the nucleic acid insert may comprise a knock-in allele of at least one exon of an endogenous gene, or a knock-in allele of an entire endogenous gene (i.e., a "gene exchange knock-in").

[0167] For example, nucleic acid insert can be homologous or orthologous to the sequence of targeted deletion at genomic target locus.Homologous or orthologous nucleic acid insert can replace the sequence of targeted deletion at genomic target locus.Homologous sequence includes the same or substantially similar nucleic acid sequence as known reference sequence, so that it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with known reference sequence.Orthologous sequence includes the nucleic acid sequence functionally equivalent to the known reference sequence in another species from a species.If the insertion of nucleic acid insert causes non-human nucleic acid sequence to be replaced by homology or orthologous human nucleic acid sequence (that is, nucleic acid insert replaces corresponding non-human DNA sequence to be inserted into its endogenous genomic locus), then orthologous sequence can cause the humanization of locus.

[0168] The nucleic acid insert may also comprise a conditional allele. The conditional allele may be a multifunctional allele as described in US2011 / 0104799, which is incorporated herein by reference in its entirety for all purposes. For example, a conditional allele may comprise: (a) an actuator sequence in a sense orientation relative to transcription of a target gene; (b) a drug selection cassette (DSC) in a sense or antisense orientation; (c) a nucleotide sequence of interest (NSI) in an antisense orientation; and (d) an inverted conditional module (COIN) in an antisense orientation, which utilizes an exon-splitting intron and an invertible gene trap-like module. See, e.g., US2011 / 0104799, which is incorporated herein by reference in its entirety for all purposes. A conditional allele may also comprise a recombinable unit that recombines upon exposure to a first recombinase to form a conditional allele, wherein the conditional allele (i) lacks an actuator sequence and a DSC; and (ii) contains an NSI in a sense orientation and a COIN in an antisense orientation. See US2011 / 0104799, which is incorporated herein by reference in its entirety for all purposes.

[0169] Some nucleic acid inserts contain polynucleotides encoding selection markers. The selection marker can be contained in a selection cassette. Such selection markers include, but are not limited to, neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r), puromycin-N-acetyltransferase (puromycin r ), blasticidin S deaminase (bsr r ), xanthine / guanine phosphoribosyltransferase (gpt) or herpes simplex virus thymidine kinase (HSV-k), or a combination thereof. The polynucleotide encoding the selection marker can be operably linked to a promoter active in the targeted cell. Examples of promoters are described elsewhere herein.

[0170] In some targeting vectors, the nucleic acid insert comprises a reporter gene. Examples of reporter genes are genes encoding the following substances: luciferase, beta-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean, T-Sapphire, alkaline phosphatase, or a combination thereof. Such reporter genes can be effectively connected to a promoter active in the targeted cell. Examples of promoters are described elsewhere herein.

[0171] In some targeting vectors, the nucleic acid insert comprises one or more expression cassettes or deletion cassettes. A given expression cassette may comprise a nucleotide sequence of interest, a nucleic acid encoding a selection marker, and / or a reporter gene, as well as various regulatory components that affect expression. Examples of selectable markers and reporter genes that may be included are discussed in detail elsewhere herein.

[0172] In some targeting vectors, nucleic acid insets include nucleic acids flanked by site-specific recombinant target sequences. Although the entire nucleic acid inset can flank this site-specific recombinant target sequence, any region of interest in the nucleic acid inset or a single polynucleotide may also flank this type of site. The site-specific recombinant target sequence that can be located at any polynucleotide of interest on both sides in nucleic acid insets or nucleic acid insets may include, for example, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att, FRT, rox, and combinations thereof. In one example, the site-specific recombinant site is located at both sides of the polynucleotide encoding selection marker and / or reporter gene contained in the nucleic acid inset. After the nucleic acid inset is integrated at the targeting locus, the sequence between the site-specific recombinant site can be removed.

[0173] (2) Targeting vector

[0174] A targeting vector can be used to introduce a nucleic acid insert into a genomic target locus, and the targeting vector comprises a nucleic acid insert and homology arms located on both sides of the nucleic acid insert. The targeting vector can be a linear form or a circular form, and can be single-stranded or double-stranded. The targeting vector can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). For ease of reference, homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms. The term relates to the relative position of homology arms and nucleic acid inserts in the targeting vector. 5' and 3' homology arms correspond to the region in the targeting locus, which is referred to herein as "5' target sequence" and "3' target sequence", respectively. Some targeting vectors include 5' and 3' homology arms without nucleic acid inserts. Such targeting vectors can play the role of making the sequence between 5' and 3' target sequences deleted without inserting nucleic acid inserts.

[0175] When two regions have a sufficient level of sequence identity, homology arms and target sequences "correspond" to each other, thereby acting as substrates for homologous recombination reactions. The term "homology" includes DNA sequences that are identical or have a common sequence identity to corresponding sequences. The sequence identity between a given target sequence and the corresponding homology arms present on a targeting vector can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of the sequence identity shared by the homology arms (or its fragment) of the targeting vector and the target sequence (or its fragment) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, so that the sequence undergoes homologous recombination. Furthermore, the corresponding homology region between the homology arms and the corresponding target sequence can be of any length sufficient to promote homologous recombination at the recognition site of cleavage. For example, a given homology arm and / or corresponding target sequence can comprise a corresponding homology region that is at least about 5-10 kb, 5-15 kb, 5-20 kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb, 5-55 kb, 5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb, 5-95 kb, 5-100 kb, 100-200 kb, or 200-300 kb long or longer (as described in the LTVEC vectors described elsewhere herein), such that the homology arms have sufficient homology to the corresponding target sequence within the genome of the cell to undergo homologous recombination.

[0176] Homology arms may correspond to loci that are natural to the cell (e.g., target loci), or alternatively, they may correspond to regions of heterologous or exogenous DNA segments that are integrated into the cell genome, including, for example, transgenes, expression cassettes, or heterologous or exogenous DNA regions. Alternatively, the homology arms of the targeting vector may correspond to regions of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), human artificial chromosomes, or any other modified regions contained in appropriate host cells. Further, the homology arms of the targeting vector may correspond to regions of BAC libraries, cosmid libraries, or P1 phage libraries, or may be derived from regions of BAC libraries, cosmid libraries, or P1 phage libraries. In some cases, the homology arms of the targeting vector correspond to loci that are natural, heterologous or exogenous to the following organisms: prokaryotes, yeast, avian (e.g., chicken), non-human mammals, rodents, rats, mice, hamsters, rabbits, pigs, cattle, deer, sheep, goats, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys), domestic mammals or agricultural mammals, or any other target organisms. In some cases, the homology arms correspond to loci in cells, and in the absence of nicks or double-strand breaks induced by nuclease agents (e.g., Cas proteins), the loci cannot be targeted using conventional methods, or can only be incorrectly or only with significantly lower efficiency targeting. In some cases, the homology arms are derived from synthetic DNA.

[0177] In some targeting vectors, the 5' and 3' homology arms correspond to the targeted genome. Alternatively, the homology arms can be from related genomes. For example, the targeted genome is the mouse genome of a first cell line, and the targeting arms are from the mouse genome of a second cell line, wherein the first cell line is different from the second cell line. In some cases, the homology arms are from the genome of the same animal or from the genome of the same cell line, for example, the targeted genome is the mouse genome of a first cell line, and the targeting arms are from the mouse genome of the same mouse or from the mouse genome of the same cell line.

[0178] The homology arms of the targeting vector can have any length sufficient to promote homologous recombination events with the corresponding target sequence, including, for example, at least 5-10 kb, 5-15 kb, 5-20 kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb, 5-55 kb, 5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb, 5-95 kb, 5-100 kb, 100-200 kb, or 200-300 kb in length or longer. As described in further detail below, large targeting vectors can employ targeting arms of greater length.

[0179] Nuclease reagents (e.g., CRISPR / Cas systems) can be used in combination with targeting vectors to help modify the target locus. Such nuclease reagents can promote homologous recombination between the targeting vector and the target locus. When nuclease reagents are used in combination with targeting vectors, the targeting vector may include 5' and 3' homology arms corresponding to 5' and 3' target sequences positioned sufficiently close to the nuclease cleavage site, to promote homologous recombination events between the target sequence and the homology arms after nicks or double-strand breaks are produced at the nuclease cleavage site. The term "nuclease cleavage site" includes DNA sequences (e.g., Cas9 cleavage sites) that produce nicks or double-strand breaks with nuclease reagents therein. The target sequence corresponding to the 5' and 3' homology arms of the targeting vector in the targeting locus is "located sufficiently close to" the nuclease cleavage site, provided that the distance can promote nicks or double-strand breaks at the recognition site and homologous recombination events occur between the 5' and 3' target sequences and the homology arms. Thus, in certain cases, the target sequence corresponding to the 5' and / or 3' homology arm of the targeting vector is within at least 1 nucleotide of a given recognition site, or within at least 10 nucleotides to about 14 kb of a given recognition site. In some cases, the nuclease cleavage site is immediately adjacent to at least one or both of the target sequences.

[0180] The spatial relationship between the target sequence corresponding to the homology arm of the targeting vector and the nuclease cleavage site can vary. For example, the target sequence can be located at the 5' end of the nuclease cleavage site, the target sequence can be located at the 3' end of the nuclease cleavage site, or the target sequence can be located on both sides of the nuclease cleavage site.

[0181] The combined use of a targeting vector (including, for example, a large targeting vector) and a nuclease agent can improve the targeting efficiency compared to the use of the targeting vector alone. For example, when the targeting vector is used in combination with a nuclease agent, the targeting efficiency of the targeting vector can be improved by at least two times, at least three times, at least four times, or at least ten times compared to the use of the targeting vector alone.

[0182] (3) Large targeting vector

[0183] Some targeting vectors are "large targeting vectors" or "LTVECs," which include targeting vectors comprising homology arms corresponding to and derived from nucleic acid sequences larger than those typically used by other methods intended for homologous recombination in cells. LTVECs also include targeting vectors comprising nucleic acid inserts having nucleic acid sequences larger than those typically used by other methods intended for homologous recombination in cells. For example, LTVECs enable modification of large loci, which are not possible with conventional plasmid-based targeting vectors due to size limitations. For example, the targeted locus may be (i.e., the 5' and 3' homology arms may correspond to) a cellular locus that cannot be targeted using conventional methods, or can only be targeted incorrectly or only with significantly lower efficiency, in the absence of a nick or double-strand break induced by a nuclease agent (e.g., a Cas protein).

[0184] Examples of LTVECs include vectors derived from bacterial artificial chromosomes (BACs), human artificial chromosomes, or yeast artificial chromosomes (YACs). Non-limiting examples of LTVECs and methods for making them are described in, for example, U.S. Pat. No. 6,586,251; U.S. Pat. No. 6,596,541; U.S. Pat. No. 7,105,348; and WO 2002 / 036789 (PCT / US01 / 45375), each of which is incorporated herein by reference. LTVECs can be in linear or circular form.

[0185] The LTVEC can be of any length, including, for example, from about 50 kb to about 300 kb, from about 50 kb to about 75 kb, from about 75 kb to about 100 kb, from about 100 kb to about 125 kb, from about 125 kb to about 150 kb, from about 150 kb to about 175 kb, from about 175 kb to about 200 kb, from about 200 kb to about 225 kb, from about 225 kb to about 250 kb, from about 250 kb to about 275 kb, or from about 275 kb to about 300 kb. Alternatively, the LTVEC can be at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more. The size of the LTVEC may be too large to be screened for targeting events by conventional assays such as Southern blotting and long fragment (e.g., 1 kb-5 kb) PCR.

[0186] In some cases, the LTVEC comprises a nucleic acid insert ranging from about 5 kb to about 200 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 60 kb to about 70 kb, about 80 kb to about 90 kb, about 90 kb to about 100 kb, about 100 kb to about 110 kb, about 120 kb to about 130 kb, about 130 kb to about 140 kb, about 140 kb to about 150 kb, about 150 kb to about 160 kb, about 160 kb to about 170 kb, about 170 kb to about 180 kb, about 180 kb to about 190 kb, or about 190 kb to about 200 kb. In other cases, the nucleic acid insert can range from about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 250 kb, about 250 kb to about 300 kb, about 300 kb to about 350 kb, or about 350 kb to about 400 kb.

[0187] In some LTVECs, the sum of the 5' homology arm and the 3' homology arm is at least 10 kb. In other LTVECs, the 5' homology arm is in the range of about 5 kb to about 100 kb, and / or the 3' homology arm is in the range of about 5 kb to about 100 kb. Each homology arm can be, for example, from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 30 kb, from about 30 kb to about 40 kb, from about 40 kb to about 50 kb, from about 50 kb to about 60 kb, from about 60 kb to about 70 kb, from about 70 kb to about 80 kb, from about 80 kb to about 90 kb, from about 90 kb to about 100 kb, from about 100 kb to about 110 kb, from about 110 kb to about 120 kb, from about 120 kb to about 130 kb, from about 130 kb to 140 kb, from about 140 kb to about 150 kb, from about 150 kb to about 160 kb, from about 160 kb to about 170 kb, from 170 kb to about 180 kb, from about 180 kb to about 190 kb, or from about 190 kb to about 200 kb. The sum of the 5' and 3' homology arms can be, for example, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, about 90 kb to about 100 kb, about 100kb to about 110kb, about 110kb to about 120kb, about 120kb to about 130kb, about 130kb to 140kb, about 140kb to about 150kb, about 150kb to about 160kb, about 160kb to about 170kb, 170kb to about 180kb, about 180kb to about 190kb, or about 190kb to about 200kb. Alternatively, each homology arm can be at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, or at least 200 kb. Likewise, the sum of the 5' and 3' homology arms can be at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, or at least 200 kb.

[0188] In some cases, the LTVEC and nucleic acid insert are designed to allow for a deletion of about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb at the target locus. Alternatively, the deletion can be at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more.

[0189] In other cases, the LTVEC and the nucleic acid insert are designed to allow insertion of an exogenous nucleic acid sequence ranging from about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 250 kb, about 250 kb to about 300 kb, about 300 kb to about 350 kb, or about 350 kb to about 400 kb into the target locus. Alternatively, the insertion can be at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more.

[0190] In other cases, the region deleted in the nucleic acid insert and / or endogenous locus is at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or more.

[0191] B. Targeting Vectors and Nucleic Acid Inserts for One-Cell Embryos

[0192] The length of the targeting vector used in 1 cell stage embryo is no more than 5kb, and can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), they can be single-stranded or double-stranded, and they can be circular form or linear form.The length of the exemplary targeting vector used in 1 cell stage embryo is between about 50 nucleotides and about 5kb.For example, the length of the targeting vector used in 1 cell stage embryo can be between about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900 or about 900 to about 1,000 nucleotides. Alternatively, the length of the targeting vector used in the 1-cell stage embryo can be between about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 2.5 kb, about 2.5 kb to about 3 kb, about 3 kb to about 3.5 kb, about 3.5 kb to about 4 kb, about 4 kb to about 4.5 kb, or about 4.5 kb to about 5 kb. Alternatively, the length of the targeting vector used in the 1-cell stage embryo can be, for example, no more than 5 kb, 4.5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, or 50 nucleotides. With respect to a single-stranded DNA donor, an exemplary targeting vector can be between about 60 nucleotides and about 200 nucleotides (e.g., about 60 nucleotides to about 80 nucleotides, about 80 nucleotides to about 100 nucleotides, about 100 nucleotides to about 120 nucleotides, about 120 nucleotides to about 140 nucleotides, about 140 nucleotides to about 160 nucleotides, about 160 nucleotides to about 180 nucleotides, or about 180 nucleotides to about 200 nucleotides).

[0193] Such targeting vectors comprise 5' and 3' homology arms corresponding to regions within the targeted locus (5' target sequence and 3' target sequence, respectively). Optionally, the targeting vector comprises a nucleic acid insert (e.g., a DNA segment integrated at the genomic target locus) flanking the 5' and 3' homology arms. Integration of the nucleic acid insert at the target locus can result in the addition of a nucleic acid sequence of interest to the target locus, the deletion of a nucleic acid sequence of interest at the target locus, or the substitution (i.e., deletion and insertion) of a nucleic acid sequence of interest at the target locus.

[0194] The corresponding homology region between homology arm and corresponding target sequence can have any length that is enough to promote homologous recombination.The length of the exemplary homology arm used for 1 cell stage embryo is between about 20 nucleotide and about 2.5kb (for example, a length of about 30 nucleotide to about 100 nucleotide).For example, given homology arm and / or corresponding target sequence can comprise a length of about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 300 to about 350, about 350 to about 400, about 400 to about 450 or about 450 to about 500 nucleotide corresponding homology region, so that the corresponding target sequence in homology arm and cytogenomic group has the homology that is enough to homologous recombination. Alternatively, a given homology arm and / or corresponding target sequence may comprise a corresponding homology region having a length of about 0.5 kb to about 1 kb, about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, or about 2 kb to about 2.5 kb. For a single-stranded DNA donor, an exemplary homology arm may be between about 30 nucleotides and about 60 nucleotides (e.g., about 30 nucleotides to about 40 nucleotides, about 40 nucleotides to about 50 nucleotides, or about 50 nucleotides to about 60 nucleotides).

[0195] As described above, the homology arms may correspond to loci that are native to the cell (e.g., target loci), or they may correspond to regions of heterologous or exogenous DNA segments that are integrated into the cell's genome. As described above, the 5' and 3' target sequences are preferably located close enough to the Cas cleavage site to promote homologous recombination events between the target sequence and the homology arms after single-strand breaks (nicks) or double-strand breaks are generated at the Cas cleavage site.

[0196] Nucleic acid inserts or corresponding nucleic acids deleted and / or replaced at the target locus can have various lengths. Exemplary nucleic acid inserts or corresponding nucleic acids deleted and / or replaced at the target locus have a length between about 10 nucleotides and about 5 kb. For example, the length of nucleic acid inserts or corresponding nucleic acids deleted and / or replaced at the target locus can be about 1 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 1 The length of the restriction endonuclease site can be between about 40, about 140 to about 150, about 150 to about 160, about 160 to about 170, about 170 to about 180, about 180 to about 190, about 190 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, or about 900 to about 1,000 nucleotides. For example, an insert of about 4 nucleotides to about 12 nucleotides in length can be inserted to form a restriction endonuclease site. Likewise, the length of the nucleic acid insert or the corresponding nucleic acid deleted and / or replaced at the target locus can be between about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 2.5 kb, about 2.5 kb to about 3 kb, about 3 kb to about 3.5 kb, about 3.5 kb to about 4 kb, about 4 kb to about 4.5 kb, or about 4.5 kb to about 5 kb. The length of the nucleic acid deleted from the genomic target locus can also be between about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, about 90 kb to about 100 kb, about 100 kb to about 200 kb, about kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 600 kb, about 600 kb to about 700 kb, about 700 kb to about 800 kb, about 800 kb to about 900 kb, about 900 kb to about 1 Mb, or longer.Alternatively, the length of the nucleic acid deleted from the genomic target locus can be between about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, about 2.5 Mb to about 3 Mb, about 3 Mb to about 4 Mb, about 4 Mb to about 5 Mb, about 5 Mb to about 10 Mb, about 10 Mb to about 20 Mb, about 20 Mb to about 30 Mb, about 30 Mb to about 40 Mb, about 40 Mb to about 50 Mb, about 50 Mb to about 60 Mb, about 60 Mb to about 70 Mb, about 70 Mb to about 80 Mb, about 80 Mb to about 90 Mb, or about 90 Mb to about 100 Mb.

[0197] As described above, the nucleic acid insert may comprise genomic DNA or any other type of DNA, the nucleic acid insert or the corresponding nucleic acid deleted and / or replaced at the target locus may be a coding region or a non-coding region, and the nucleic acid insert may be homologous or orthologous to the sequence targeted for deletion at the target genomic locus. The nucleic acid insert may also comprise a conditional allele, a polynucleotide encoding a selection marker, a reporter gene, one or more expression cassettes, one or more deletion cassettes, or a nucleic acid insert comprising a nucleic acid flanked by a site-specific recombination target sequence as described above.

[0198] C. Promoter

[0199] Various nucleic acid sequences described herein can be effectively connected to a promoter. Such promoters can be, for example, active in rats, eukaryotes, mammals, non-human mammals, humans, rodents, mice or hamster pluripotent cells. Promoters active in 1-cell embryos can also be used. Promoters can be, for example, constitutively active promoters, conditional promoters, inducible promoters, temporally restricted promoters (for example, promoters regulated by development) or spatially restricted promoters (for example, cell-specific or tissue-specific promoters). Examples of promoters can be found in, for example, WO 2013 / 176772, which is incorporated herein by reference in its entirety.

[0200] Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol regulated promoters (e.g., alcohol dehydrogenase (alcA) gene promoters), tetracycline regulated promoters (e.g., tetracycline responsive promoters, tetracycline operator sequences (tetO), tet-On promoters or tet-Off promoters), steroid regulated promoters (e.g., rat glucocorticoid receptor, promoters of estrogen receptors or promoters of ecdysone receptors) or metal regulated promoters (e.g., metalloprotein promoters). Physically regulated promoters include, for example, temperature regulated promoters (e.g., heat shock promoters) and light regulated promoters (e.g., light-inducible promoters or light-repressible promoters).

[0201] The tissue-specific promoter can be, for example, a neuron-specific promoter, a glial-specific promoter, a muscle cell-specific promoter, a cardiac cell-specific promoter, a kidney cell-specific promoter, a bone cell-specific promoter, an endothelial cell-specific promoter, or an immune cell-specific promoter (e.g., a B cell promoter or a T cell promoter).

[0202] Developmentally regulated promoters include, for example, promoters that are active only during embryonic development or only in adult cells.

[0203] The promoter can also be selected according to the cell type. For example, various known promoters can be used for non-human mammalian cells, non-human pluripotent cells, human induced pluripotent stem cells directly derived from differentiated adult cells, non-pluripotent human cells, rodent cells, rat cells, mouse cells, hamster cells, fibroblasts or CHO cells.

[0204] IV. Methods of Modifying Genomes and Producing Genetically Modified Non-Human Animals

[0205] A. Methods for Modifying Genomes

[0206] Provided are various methods for modifying the genome in a cell by using two guide RNAs to target different regions in a single genome target locus. Also provided are methods for targeting different regions in a single genome target locus using two or more guide RNAs (e.g., three guide RNAs or four guide RNAs). These methods can be performed in vitro, in vitro, or in vivo. Such methods promote the formation of biallelic genetic modifications and may include genome shearing or other targeted modifications, such as the absence of a nucleotide sequence in the genome and being replaced by an exogenous nucleic acid sequence simultaneously.

[0207] Targeted gene modifications generated by homologous recombination between the targeting vector and the target locus can be very inefficient, especially in cell types other than rodent embryonic stem cells. The use of a targeting vector combined with nuclease-directed double-stranded DNA breaks at the target locus can greatly improve the efficiency of heterozygous targeting of simple modifications such as small deletions or insertions.

[0208] Combining the targeting vector with a CRISPR / Cas9 nuclease directed by a guide RNA (gRNA) can also improve the hybrid targeting efficiency of very large and very low-efficiency gene modifications, such as deletion of mouse genes and simultaneous replacement with human equivalents (humanization). Such modifications can involve very large (e.g., >50 kb) deletions and insertions (see Lrp5, C5 (Hc), Ror1, and Trpa1 targeting in Example 1).

[0209] During homology-mediated repair of one or more double-strand breaks generated at the target genomic locus by a nuclease (such as Cas9), the one or more breaks are first processed by excision of the 5' end to form a 3' single-stranded overhang sequence. Rad51 then polymerizes on the single-stranded DNA to search for homologous sequences, strands invade into undamaged homologous template double-helix DNA (e.g., targeting vector), and form an intermediate D-loop structure to facilitate the use of undamaged homologous DNA (e.g., targeting vector) as a template to repair the one or more double-strand breaks. The chromosomal sequence is then replaced with a nucleic acid insert from the targeting vector through a double exchange event involving homologous regions on both sides. Whether this process can proceed smoothly is affected by several factors, such as the size of the nucleic acid insert, the length of the region homologous to the homologous arms of the targeting vector, and the position of the region homologous to the homologous arms of the targeting vector (e.g., relative to the position of the one or more double-strand breaks).

[0210] As the size of the nucleic acid insert or the sequence deleted at the target genomic locus increases, the excision process becomes more unpredictable, the stability of the intermediate D-loop structure decreases and becomes more unpredictable, and the success rate of the recombination process is generally reduced and becomes more unpredictable. For example, as the size of the targeted modification increases, the risk of internal recombination also increases, especially when the replaced sequence has a certain similarity to the inserted sequence. When such internal recombination occurs, homologous recombination exchanges occur within the expected target region, and the complete nucleic acid insert will not be incorporated into the genomic target locus. In addition, it is generally believed that as the distance between the double-strand break and the mutation or insertion site increases (e.g., more than 100 bp or 200 bp), the efficiency of HR-mediated insertion decreases. See Beumer et al. (2013) Genes\Genomes\Genetics 3:657-664; Elliott et al. (1998) Mol. Cell. Biol. 18:93-101; and Byrne et al. (2015) Nucleic Acids Research 43(3):e21, each of which is incorporated herein by reference in its entirety for all purposes.

[0211] In order to enable targeted gene modification to form a large deletion at the target genome locus and insert a large segment of foreign DNA at the same time, it is necessary to form a double ω structure as a recombination intermediate. The greater the degree of modification, the lower the structural stability. In cell types other than 1-cell embryos, LTVECs with a sum of 10 kb or greater total homology can be used. LTVECs with a sum of 10 kb or greater total homology for homology arms improve the stability of the double ω recombination intermediate, thereby facilitating large deletions mediated by nucleases and being replaced by large nucleic acid inserts at the same time, and further not only causing double-strand breaks adjacent to the homology region to occur to enhance the targeting efficiency, but also causing double-strand breaks away from the homology region to occur to enhance the targeting efficiency.

[0212] For gene modifications involving a great degree of humanization, combining the targeting vector with a CRISPR / Cas9 nuclease system guided by two gRNAs can further enhance the targeting efficiency, exceeding the efficiency achieved with one gRNA (see Lrp5, C5 (Hc), Ror1, and Trpa1 humanization in Example 1). In this regard, the use of two gRNAs can produce unexpected results. Compared with targeting with one gRNA (which produces low frequencies of biallelic modifications or does not produce them at all), targeting with two gRNAs results in a significantly increased rate of producing homozygous targeted cells, homozygous deleted cells, and compound heterozygous targeted cells (including hemizygous targeted cells).

[0213] The method for generating three allele types (i.e., homozygous targeting, homozygous deletion, and compound heterozygous targeting (especially hemizygous targeting)) in a single targeting experiment provides new possibilities and enhanced efficiency for targeted gene modification. For simple gene modifications, such as targeted deletion of a gene in mouse ES cells and its replacement by a sequence encoding a protein expressed by a reporter gene (e.g., β-galactosidase or a fluorescent protein), combining the targeting vector with the CRISPR / Cas9 system guided by two gRNAs facilitates the generation of heterozygous targeted ES cells, which can then be expressed by Methods These cells are used to produce completely ES cell-derived F0 generation mice. See Poueymirou et al. (2007) Nat. Biotech. 25: 91-99 (Poueymirou et al., 2007, "Nature Biotechnology", Vol. 25, pp. 91-99), which is incorporated herein by reference in its entirety for all purposes. These mice can be used to study tissue-specific gene expression using reporter gene knock-in alleles. Homozygous targeted ES cell clones produced in the same experiment can be converted into VelociMice with homozygous targeted gene deletions, and then the phenotypic results of their gene knockouts and gene expression in receptors can be studied. Producing VelociMice by ES cells with homozygous CRISPR inducible deletions of target genes enables verification of the knockout phenotype observed in homozygous targeted mice, and can reveal the phenotypic differences between pure gene deletions and deletions with receptor insertion and drug selection cassettes. Compound heterozygous (especially hemizygous) ES cell clones carrying both targeted deletion-insertion alleles and CRISPR-induced deletions allow the opportunity to generate VelociMice for research with the same opportunity to generate clones derived from homozygous targeting and homozygous deletions. In addition, these mice can be mated to simultaneously form targeted deletion and simple deletion mutant mouse strains from a single ES cell clone.

[0214] When extended to the humanized situation, these advantages are more valuable.An important use of humanized mouse genes is to form an animal model for testing human-specific therapeutic agents.In order to make humanization an effective model, mouse genes must be removed or inactivated to avoid interactions between mice and human gene products, which may damage biological functions or suitable interactions with drugs.At the same time, human genes must be able to replace the biological functions of their mouse counterparts.These requirements can be tested by combining the Cas9 nuclease guided by two gRNAs with a targeting vector, wherein the targeting vector is designed to make the mouse gene deleted and replaced by human genes at the same time.The VelociMice derived from ES cells with homozygous targeted humanization can be compared with the VelociMice derived from ES cells with homozygous CRISPR inducible deletions of mouse genes.If the knockout deletion causes an observable mutant phenotype and the humanized mouse does not express this phenotype but is normal, then the human gene can replace the biological function of the mouse gene. Homozygous humanized mice or humanized mice carrying a compound heterozygous (e.g., hemizygous) combination of humanized alleles and CRISPR-inducible deletion alleles can be used as animal models to study the mechanism of action and efficacy of human-specific therapeutic agents. Compound heterozygous (e.g., hemizygous) VelociMice can also be used to produce humanized and deleted knockout strains of mice by conventional mating. Therefore, from a single gene targeting experiment combining two gRNA CRISPR systems with a targeting vector, genetically modified mice were generated, thereby obtaining important mouse models for preclinical testing of therapeutic agents and knockout strains for studying the biological functions of mouse homologs of human drug targets.

[0215] (1) Methods for generating, improving or increasing the frequency of biallelic modifications

[0216] Provided herein is a method for carrying out biallelic modification to the intracellular genome, or a method for improving or increasing the frequency of carrying out biallelic modification to the intracellular genome. Such methods can result in, for example, genome shearing to remove most of the genomic DNA between two sequences of the genomic DNA subsequently reorganized. Such methods can also result in the insertion of a nucleic acid inset or the disappearance of most of the genomic DNA and its replacement by a nucleic acid inset.

[0217] The method for modifying the genome in a cell provided herein includes contacting the genome with a first Cas protein, a first CRISPR RNA hybridized with a first CRISPR RNA recognition sequence in a genomic target locus, a second CRISPR RNA hybridized with a second CRISPR RNA recognition sequence in a genomic target locus, and tracrRNA. Optionally, the genome can be further contacted with an additional CRISPR RNA hybridized with a CRISPR RNA recognition sequence in a genomic target locus, such as a third CRISPR RNA hybridized with a third CRISPR RNA recognition sequence in a genomic target locus and / or a fourth CRISPR RNA hybridized with a fourth CRISPR RNA recognition sequence in a genomic target locus. Bi-allelic modification can be produced by contacting the genome with a first Cas protein, a first CRISPR RNA hybridized with a first CRISPR RNA recognition sequence in a genomic target locus, a second CRISPR RNA hybridized with a second CRISPR RNA recognition sequence in a genomic target locus, and tracrRNA. As described in further detail below, Cas proteins, CRISPR RNA, and tracrRNA can be introduced into cells in any form and by any means. Likewise, all or some of the Cas proteins, CRISPR RNAs, and tracrRNAs may be introduced sequentially, simultaneously or in any combination. The contact of the genome may be direct (i.e., a component directly contacts the genome itself) or indirect (i.e., a component interacts with other components that directly contact the genome).

[0218] The genome may include a pair of first homologous chromosomes and a second homologous chromosome, which include a genomic target locus. The first Cas protein may cut one or both of these chromosomes in the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence (i.e., at the first cleavage site in the first CRISPR RNA recognition sequence and / or at the second cleavage site in the second CRISPR RNA recognition sequence). If the third CRISPR RNA and / or the fourth CRISPR RNA are also used, the first Cas protein may cut one or both of these chromosomes in the third CRISPR RNA recognition sequence and / or the fourth CRISPR RNA recognition sequence (i.e., at the third cleavage site in the third CRISPR RNA recognition sequence and / or at the fourth cleavage site in the fourth CRISPR RNA recognition sequence). The cleavage event may then produce at least one double-strand break in one or both of these chromosomes. The cleavage event may also produce at least two double-strand breaks in one or both of these chromosomes. If a Cas nickase is used, the cleavage event may produce at least one single-strand break in one or both of these chromosomes, or at least two single-strand breaks in one or both of these chromosomes. If a third CRISPR RNA and / or a fourth CRISPR RNA is used, the cleavage event may produce at least three or four single-strand or double-strand breaks in one or both of these chromosomes. The end sequences produced by the double-strand breaks may then be recombined, or the end sequences produced by the single-strand breaks may then be recombined. Cells with modified genomes comprising bi-allelic modifications may then be identified.

[0219] For example, a first Cas protein can cut the genome at a first cleavage site within a first CRISPR RNA recognition sequence in a first homologous chromosome and a second homologous chromosome and at a second cleavage site within a second CRISPR RNA recognition sequence in at least the first homologous chromosome, thereby generating terminal sequences in the first homologous chromosome and the second homologous chromosome. The terminal sequences can then be recombined to form a genome with a double allele modification comprising a targeted modification. The targeted modification can include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in at least the first chromosome.

[0220] The first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may be at any position within the genomic target locus. The first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may be on either side of any genomic region of interest. For example, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may be on either side of all or part of the coding sequence of a gene such as an Lrp5 locus, a C5 (Hc) locus, a Ror1 locus, or a Trpa1 locus. The first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may also be on either side of all or part of the coding sequence of the Cmah gene. Alternatively, the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may be on either side of a non-coding sequence (such as a regulatory element (e.g., a promoter), or both a coding sequence and a non-coding sequence). The third CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence may be, for example, at any position within the genomic region of interest flanking the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence.

[0221] For example, the third CRISPR RNA recognition sequence may be adjacent to the first CRISPR RNA recognition sequence, and the fourth CRISPR RNA recognition sequence may be adjacent to the second CRISPR RNA recognition sequence. Thus, the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence may be a first pair of CRISPR RNA recognition sequences, and the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence may be a second pair of CRISPR RNA recognition sequences. For example, the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence (and / or the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence) may be adjacent to the second CRISPR RNA recognition sequence. The RNA recognition sequences) may be spaced from about 25 bp to about 50 bp, from about 50 bp to about 100 bp, from about 100 bp to about 150 bp, from about 150 bp to about 200 bp, from about 200 bp to about 250 bp, from about 250 bp to about 300 bp, from about 300 bp to about 350 bp, from about 350 bp to about 400 bp, from about 400 bp to about 450 bp, from about 450 bp to about 500 bp, from about 500 bp to about 600 bp, from about 600 bp to about 700 bp, from about 700 bp to about 800 bp, or from about 800 bp to about 900 bp. In some embodiments, the first and third CRISPR RNA recognition sequences are from about 1 kb to about 800 bp, from about 800 bp to about 900 bp, from about 900 bp to about 1 kb, from about 1 kb to about 1.5 kb, from about 1.5 kb to about 2 kb, from about 2 kb to about 2.5 kb, from about 2.5 kb to about 3 kb, from about 3 kb to about 3.5 kb, from about 3.5 kb to about 4 kb, from about 4 kb to about 4.5 kb, from about 4.5 kb to about 5 kb, from about 5 kb to about 6 kb, from about 6 kb to about 7 kb, from about 7 kb to about 8 kb, from about 8 kb to about 9 kb, or from about 9 kb to about 10 kb. For example, the first and third CRISPR RNA recognition sequences (and / or the second and fourth CRISPR RNA recognition sequences) can be separated by about 100 bp to about 1 kb. Alternatively, the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence (and / or the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence) may overlap.

[0222] The first pair of CRISPR RNA recognition sequences may be located near the 5' end of the genomic target locus, and the second pair of CRISPR RNA recognition sequences may be located near the 3' end of the genomic target locus. Alternatively, the first pair and the second pair may be located near the 5' end of the genomic target locus or may be located near the 3' end of the target locus. Alternatively, one or two pairs may be located inside the genomic target locus. For example, the first CRISPR RNA recognition sequence or the first pair of CRISPR RNA recognition sequences may be less than 25 bp, less than 50 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, less than 500 bp, less than 600 bp, less than 700 bp, less than 800 bp, less than 900 bp, less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb or less than 10 kb from the 5' end of the genomic target locus. Likewise, the second CRISPR RNA recognition sequence or the first pair of CRISPR RNA recognition sequences may be less than 25 bp, less than 50 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, less than 500 bp, less than 600 bp, less than 700 bp, less than 800 bp, less than 900 bp, less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, or less than 10 kb from the 3' end of the genomic target locus.

[0223] Alternatively, the first CRISPR RNA recognition sequence or the first pair of CRISPR RNA recognition sequences may be, for example, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb from the 5' end of the genomic target locus. Similarly, the second CRISPR RNA recognition sequence or the first pair of CRISPR RNA recognition sequences may be, for example, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb from the 3' end of the genomic target locus.

[0224] Alternatively, the first CRISPR RNA recognition sequence or the first pair of CRISPR RNA recognition sequences can be, for example, about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, or about 900 bp to about 1000 bp. p, about 900 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 6 kb, about 6 kb to about 7 kb, about 7 kb to about 8 kb, about 8 kb to about 9 kb, about 9 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, or about 90 kb to about 100 kb. Similarly, the second CRISPR RNA recognition sequence or the first pair of CRISPR The RNA recognition sequence can be, for example, about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp. p, about 900 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 6 kb, about 6 kb to about 7 kb, about 7 kb to about 8 kb, about 8 kb to about 9 kb, about 9 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 60 kb, about 60 kb to about 70 kb, about 70 kb to about 80 kb, about 80 kb to about 90 kb, or about 90 kb to about 100 kb.

[0225] The first cleavage site and the second cleavage site or the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can be separated by, for example, about 1 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb. The first cleavage site and the second cleavage site or the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can also be separated by, for example, about 3 Mb to about 4 Mb, about 4 Mb to about 5 Mb, about 5 Mb to about 10 Mb, about 10 Mb to about 20 Mb, about 20 Mb to about 30 Mb, about 30 Mb to about 40 Mb, about 40 Mb to about 50 Mb, about 50 Mb to about 60 Mb, about 60 Mb to about 70 Mb, about 70 Mb to about 80 Mb, about 80 Mb to about 90 Mb, or about 90 Mb to about 100 Mb. The first cleavage site and the second cleavage site or the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can also be separated, for example, by about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, or about 900 bp to about 1 kb.Likewise, the first pair of CRISPR RNA recognition sequences can be separated from the second pair of CRISPR RNA recognition sequences by, for example, about 25 bp to about 50 bp, about 50 bp to about 100 bp, about 100 bp to about 150 bp, about 150 bp to about 200 bp, about 200 bp to about 250 bp, about 250 bp to about 300 bp, about 300 bp to about 350 bp, about 350 bp to about 400 bp, about 400 bp to about 450 bp, about 450 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1 kb, about 1 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, about 2.5 Mb to about 3 Mb, about 3 Mb to about 4 Mb, about 4 Mb to about 5 Mb, about 5 Mb to about 10 Mb, about 10 Mb to about 20 Mb, about 20 Mb to about 30 Mb, about 30 Mb to about 40 Mb, about 40 Mb to about 50 Mb, about 50 Mb to about 60 Mb, about 60 Mb to about 70 Mb, about 70 Mb to about 80 Mb, about 80 Mb to about 90 Mb, or about 90 Mb to about 100 Mb.

[0226] Alternatively, the first cleavage site and the second cleavage site or the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can be separated by, for example, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more. Likewise, the first pair of CRISPR RNA recognition sequences can be separated from the second pair of CRISPR RNA recognition sequences by, e.g., at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more.

[0227] Alternatively, the first cleavage site and the second cleavage site or the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may be separated by less than 25 bp, less than 50 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, less than 500 bp, less than 600 bp, less than 700 bp, less than 800 bp, less than 900 bp, less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb or less than 10 kb. Likewise, the first pair of CRISPR RNA recognition sequences may be separated from the second pair of CRISPR RNA recognition sequences by, e.g., less than 25 bp, less than 50 bp, less than 100 bp, less than 150 bp, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, less than 500 bp, less than 600 bp, less than 700 bp, less than 800 bp, less than 900 bp, less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, or less than 10 kb.

[0228] The end sequence produced by cutting the genome at the first cleavage site and / or the second cleavage site may be a blunt end or a staggered end, and the deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may include all or part of the nucleic acid sequence between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence (including the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence). Similarly, the end sequence produced by cutting the genome at the third cleavage site and / or the fourth cleavage site may be a blunt end or a staggered end. For example, the deletion may include only a portion of the nucleic acid sequence between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence and / or only a portion of the first CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence. Alternatively, the deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence may include all nucleic acid sequences between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence. Similarly, the deletion may include the first CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence or a portion thereof. In some cases, the deletion also includes sequence located outside the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence (i.e., sequence that does not include and is not between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence).

[0229] The deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can be of any length. For example, the deleted nucleic acid can be about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb.

[0230] Alternatively, the deleted nucleic acid can be, e.g., at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb or more. In some cases, the deleted nucleic acid may be at least 550 kb, at least 600 kb, at least 650 kb, at least 700 kb, at least 750 kb, at least 800 kb, at least 850 kb, at least 900 kb, at least 950 kb, at least 1 Mb, at least 1.5 Mb, at least 2 Mb, at least 2.5 Mb, at least 3 Mb, at least 4 Mb, at least 5 Mb, at least 10 Mb, at least 20 Mb, at least 30 Mb, at least 40 Mb, at least 50 Mb, at least 60 Mb, at least 70 Mb, at least 80 Mb, at least 90 Mb, or at least 100 Mb (e.g., most of a chromosome).

[0231] The deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can be a precise deletion, wherein the deleted nucleic acid consists of a nucleic acid sequence only between the first Cas protein cleavage site and the second Cas protein cleavage site, so that there is no additional deletion or insertion at the modified genomic target locus. The deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can also be an imprecise deletion extending beyond the first Cas protein cleavage site and the second Cas protein cleavage site (consistent with imprecise repair by non-homologous end joining (NHEJ)), resulting in additional deletions and / or insertions at the modified genomic locus. For example, the deletion can extend beyond the first Cas protein cleavage site and the second Cas protein cleavage site by about 1 bp, about 2 bp, about 3 bp, about 4 bp, about 5 bp, about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp or more. Likewise, the modified genomic locus may comprise additional insertions consistent with imprecise repair by NHEJ, such as insertions of about 1 bp, about 2 bp, about 3 bp, about 4 bp, about 5 bp, about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, or more.

[0232] The contact process can be carried out under the situation that there is no exogenous donor sequence or there is exogenous donor sequence, and prerequisite is that if cell is 1 cell stage embryo, the length of exogenous donor sequence is no more than 5kb.Exogenous molecule or sequence comprise the molecule or sequence that is not normally present in the cell.Normal existence comprises existence with respect to the specific development stage and environmental conditions of cell.Exogenous molecule or sequence for example can comprise the mutant form of corresponding endogenous sequence in cell, such as the humanized form of endogenous sequence.By contrast, endogenous molecule or sequence comprise the molecule or sequence that is normally present in the specific cell of specific development stage under specific environmental conditions.

[0233] The exogenous donor sequence can be in the targeting vector and can include the nucleic acid insert of 5' and 3' homology arms corresponding to the 5' and 3' target sequences in the genome, provided that if the cell is a 1-cell stage embryo, the length of the targeting vector does not exceed 5kb. In cell types other than the 1-cell stage embryo, the targeting vector may be longer. In cell types other than the 1-cell stage embryo, the targeting vector may be a large targeting vector (LTVEC) such as described herein, and may be at least 10kb. Therefore, in some methods, the genome is further contacted with the targeting vector, and the nucleic acid insert is inserted between the 5' and 3' target sequences.

[0234] Alternatively, the exogenous donor sequence can include 5' and 3' homology arms that do not contain nucleic acid inserts. Such targeting vectors that do not contain nucleic acid inserts can facilitate precise deletions between the 5' and 3' target sequences within the genome. Such precise deletions can be, for example, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb , at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, at least 500 kb, at least 550 kb, at least 600 kb, at least 650 kb, at least 700 kb, at least 750 kb, at least 800 kb, at least 850 kb, at least 900 kb, at least 950 kb, at least 1 Mb, at least 1.5 Mb or at least 2 Mb or longer.

[0235] In some such methods, the 5' and 3' homology arms correspond to the 5' and 3' target sequences at the genomic target locus comprising the first CRISPR RNA recognition sequence of the first CRISPR RNA and / or the second CRISPR RNA recognition sequence of the second CRISPR RNA. The first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence or the first cleavage site and the second cleavage site may be adjacent to the 5' target sequence, adjacent to the 3' target sequence, or neither adjacent to the 5' target sequence nor adjacent to the 3' target sequence. Alternatively, the first CRISPR RNA recognition sequence or the first cleavage site may be adjacent to the 5' target sequence, and the second CRISPR RNA recognition sequence or the second cleavage site may be adjacent to the 3' target sequence. Alternatively, the first CRISPR RNA recognition sequence or the first cleavage site may be adjacent to the 5' target sequence or the 3' target sequence, and the second CRISPR RNA recognition sequence or the second cleavage site may be adjacent to the 5' target sequence nor adjacent to the 3' target sequence.

[0236] For example, the first CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence may be located between the 5' and 3' target sequence, or may be adjacent to or near the 5' target sequence and / or the 3' target sequence, such as within 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, 200 kb, 250 kb, 300 kb, 350 kb, 400 kb, 450 kb, or 500 kb of the 5' and / or 3' target sequence. Likewise, the first cleavage site and / or the second cleavage site may be located between the 5' and 3' target sequences or may be adjacent to or proximal to the 5' target sequence and / or the 3' target sequence, such as within 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, 200 kb, 250 kb, 300 kb, 350 kb, 400 kb, 450 kb, or 500 kb of the 5' and / or 3' target sequence. The RNA recognition sequence or the first cleavage site may be 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, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 110kb, 120kb, 130kb, 140kb, 150kb, 160kb, 170kb, 180kb, 190kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb or 500kb.Likewise, the second CRISPR RNA recognition sequence or the second cleavage site may be 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, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb ... 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, 100kb, 110kb, 120kb, 130kb, 140kb, 150kb, 160kb, 170kb, 180kb, 190kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb or 500kb.

[0237] Alternatively, the first CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence may be located at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 800 bp, at least 900 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb or at least 500 kb. Likewise, the first cleavage site and / or the second cleavage site may be located at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 20 ...3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, at least 3 kb, kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb.For example, the first CRISPR RNA recognition sequence or the first cleavage site can be located at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 20 kb, at least 25 kb, at least 26 kb, at least 27 kb, at least 28 kb, at least 29 kb, at least 30 kb, at least 31 kb, at least 32 kb, at least 33 kb, at least 34 kb, at least 35 kb, at least 36 kb, at least 37 kb, at least 38 kb, at least 39 kb, at least 40 kb, at least 41 kb, at least 42 kb, at least 43 kb, at least 44 kb At least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb. Similarly, the second CRISPR / Cas9 at the second cleavage site The RNA recognition sequence may be located at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 15 kb, at least 16 kb, at least 17 kb, at least 18 kb, at least 19 kb, at least 20 kb, at least 21 kb, at least 22 kb, at least 23 kb, at least 24 kb, at least 25 kb, at least 26 kb, at least 27 kb, at least 28 kb, at least 29 kb, at least 30 kb, at least 31 kb, at least 32 kb, at least 33 kb kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb.

[0238] For example, the first CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence may be located about 50 bp to about 100 bp, about 200 bp to about 300 bp, about 300 bp to about 400 bp, about 400 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb from the 5' and / or 3' target sequence. , about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, or about 400 kb to about 500 kb. Likewise, the first cleavage site and / or the second cleavage site may be located about 50 bp to about 100 bp, about 200 bp to about 300 bp, about 300 bp to about 400 bp, about 400 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, or about 1 kb to about 2 kb from the 5' and / or 3' target sequence. to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb or about 400 kb to about 500 kb.For example, the first CRISPR RNA recognition sequence or the first cleavage site can be located about 50 bp to about 100 bp, about 200 bp to about 300 bp, about 300 bp to about 400 bp, about 400 bp to about 500 bp, about 500 bp to about 600 bp, about 600 bp to about 700 bp, about 700 bp to about 800 bp, about 800 bp to about 900 bp, about 900 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, or about 2 kb to about 5 kb from the 5' target sequence or from both the 5' and 3' target sequences. kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, or about 400 kb to about 500 kb. Similarly, the second CRISPR The RNA recognition sequence or second cleavage site can be located from about 50 bp to about 100 bp, from about 200 bp to about 300 bp, from about 300 bp to about 400 bp, from about 400 bp to about 500 bp, from about 500 bp to about 600 bp, from about 600 bp to about 700 bp, from about 700 bp to about 800 bp, from about 800 bp to about 900 bp, from about 900 bp to about 1 kb, from about 1 kb to about 2 kb, from about 2 kb to about 3 .... kb to about 3 kb, about 3 kb to about 4 kb, about 4 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 30 kb, about 30 kb to about 40 kb, about 40 kb to about 50 kb, about 50 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, or about 400 kb to about 500 kb.

[0239] Alternatively, the first CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence or the first cleavage site and / or the second cleavage site may be located greater than 50 bp, greater than 100 bp, greater than 200 bp, greater than 300 bp, greater than 400 bp, greater than 500 bp, greater than 600 bp, greater than 700 bp, greater than 800 bp, greater than 900 bp, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, greater than 10 kb, greater than 20 kb, greater than 30 kb, greater than 40 kb, greater than 50 kb, greater than 60 kb, greater than 70 kb, greater than 80 kb, greater than 90 kb or greater than 100 kb from the 5' and / or 3' target sequence. For example, the first CRISPR RNA recognition sequence or the first cleavage site can be located greater than 50 bp, greater than 100 bp, greater than 200 bp, greater than 300 bp, greater than 400 bp, greater than 500 bp, greater than 600 bp, greater than 700 bp, greater than 800 bp, greater than 900 bp, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, greater than 10 kb, greater than 20 kb, greater than 30 kb, greater than 40 kb, greater than 50 kb, greater than 60 kb, greater than 70 kb, greater than 80 kb, greater than 90 kb, or greater than 100 kb from the 5' target sequence or from both the 5' and 3' target sequences. Likewise, the second CRISPR RNA recognition sequence or the second cleavage site may be located greater than 50 bp, greater than 100 bp, greater than 200 bp, greater than 300 bp, greater than 400 bp, greater than 500 bp, greater than 600 bp, greater than 700 bp, greater than 800 bp, greater than 900 bp, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater than 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, greater than 10 kb, greater than 20 kb, greater than 30 kb, greater than 40 kb, greater than 50 kb, greater than 60 kb, greater than 70 kb, greater than 80 kb, greater than 90 kb, or greater than 100 kb from the 3' target sequence or from both the 5' and 3' target sequences.

[0240] The methods described herein improve and increase the frequency of biallelic modifications. Specifically, by contacting the genome with the first CRISPR RNA and the second CRISPR RNA simultaneously, the efficiency of producing biallelic modifications can be increased compared to contacting the genome with the first CRISPR RNA or the second CRISPR RNA alone. The efficiency of producing biallelic modifications can also be increased by contacting the genome with the first CRISPR RNA, the second CRISPR RNA and the third CRISPR RNA or the first CRISPR RNA, the second CRISPR RNA, the third CRISPR RNA and the fourth CRISPR RNA. Bi-allelic modifications include such events: wherein the same locus on the corresponding homologous chromosomes (e.g., in diploid cells) is modified identically, or wherein the same locus on the corresponding homologous chromosomes is modified differently. Homologous chromosomes include chromosomes (e.g., chromosomes paired during meiosis) where the genes at the same locus are identical but the alleles may be different. The term "allele" includes a gene sequence in any form of one or more alternative forms. In a diploid cell or organism, two alleles of a given sequence typically occupy a corresponding locus on a pair of homologous chromosomes.

[0241] Bi-allelic modification can result in homozygosity of targeted modification or compound heterozygosity (e.g., hemizygosity) of targeted modification. Performing a single targeted experiment on a group of cells can produce cells that are homozygous for targeted modification (e.g., humanization of a locus), cells that are compound heterozygous for the targeted modification (including cells that are hemizygous for the targeted modification), and cells that are homozygous sheared between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence (i.e., a large nucleic acid sequence is deleted between the two CRISPR RNA recognition sequences). Homozygosity includes a situation where both alleles of the target locus (i.e., the corresponding alleles on two homologous chromosomes) have targeted modifications. Compound heterozygosity includes a situation where both alleles of the target locus (i.e., alleles on two homologous chromosomes) have been modified, but they are modified in different ways (e.g., one allele is targeted and the other allele is inactivated or destroyed). The destruction of endogenous nucleic acid sequences can be, for example, when repairing the double-strand breaks produced by Cas proteins with non-homologous end joining (NHEJ)-mediated DNA repair, causing the repair to produce mutant alleles comprising insertions or deletions of nucleic acid sequences, thereby causing destruction of the genomic locus. Examples of destruction include changes in regulatory elements (such as promoters or enhancers), missense mutations, nonsense mutations, frameshift mutations, truncated mutations, null mutations, or insertions or deletions of a small amount of nucleotides (for example, causing frameshift mutations). Destruction can result in inactivation (i.e., loss of function) or loss of alleles.

[0242] For example, if a cell has one allele modified by targeting and has another allele that cannot be expressed or otherwise function, then biallelic modification can result in compound heterozygosity. Compound heterozygosity includes hemizygosity. Hemizygosity includes such a situation: there is only one allele of the target locus (i.e., the allele on one of the two homologous chromosomes). For example, if the targeted modification occurs in one allele, and the other allele has a corresponding loss or deletion, then biallelic modification can result in hemizygosity of the targeted modification.

[0243] In a specific example, the biallelic modification may include a deletion between a first CRISPR RNA recognition sequence and a second CRISPR RNA recognition sequence in a first homologous chromosome and a second homologous chromosome pair. This deletion may occur simultaneously, or this deletion may occur initially in the first homologous chromosome, wherein homozygosity is achieved by the cell using the first homologous chromosome as a donor sequence to repair one or more double-strand breaks in the second homologous chromosome via homologous recombination (such as by gene conversion). The deleted nucleic acid sequences in the first homologous chromosome and the second homologous chromosome may be identical, partially overlapping, or different. Alternatively, the biallelic modification may include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in the first homologous chromosome and a loss of the corresponding allele or locus in the second homologous chromosome. Alternatively, the biallelic modification may include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in the first homologous chromosome and an inactivation or destruction of the corresponding allele or locus between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in the second homologous chromosome.

[0244] If a donor sequence is used, the biallelic modification may include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence and an insertion of a nucleic acid insert between the 5' and 3' target sequences in the first and second homologous chromosome pairs, resulting in a homozygous modified genome. The deletion and insertion may occur simultaneously in both chromosomes, or the deletion and insertion may occur initially in the first homologous chromosome, where homozygosity is achieved by the cell using the first homologous chromosome as a donor sequence to repair the double-strand break in the second homologous chromosome via homologous recombination (such as by gene conversion). For example, without wishing to be bound by any particular theory, the insertion of the nucleic acid insert may occur in the first homologous chromosome (with or without cleavage by the Cas protein), and the second homologous chromosome may subsequently be modified by a gene conversion event stimulated by cleavage by the Cas protein on the second homologous chromosome.

[0245] Alternatively, biallelic modifications may result in a modified genome of a compound heterozygous. For example, targeted modifications may include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence in both the first homologous chromosome and the second homologous chromosome and an insertion of a nucleic acid insert in the first homologous chromosome but not in the second homologous chromosome. Alternatively, targeted modifications may include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence, and an insertion of a nucleic acid insert in the first homologous chromosome and an inactivation or destruction of the corresponding allele or locus in the second homologous chromosome. Alternatively, biallelic modifications may result in a modified genome of a hemizygous, wherein targeted modifications may include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence and an insertion of a nucleic acid insert in the first homologous chromosome and a loss or deletion of the corresponding allele or locus in the second homologous chromosome.

[0246] Homozygous and compound heterozygous (especially hemizygous) targeted gene modification is advantageous, because the process (described in more detail below) of utilizing these modifications to produce genetically modified animals may be more efficient and less time-consuming. In many cases (such as removing genes to study the impact of their deletion), the pure heterozygosity (that is, one allele is modified but another allele is not changed) of targeted genetic modification is not enough. Adopting conventional targeting strategies, it is possible to obtain heterozygous F0 generation animals for large targeted genome deletions, but it is necessary for these heterozygous animals to carry out subsequent mutual mating to produce homozygous F1 generation animals for the deletion. These extra mating steps are not only costly, but also time-consuming. It is possible to form homozygous or compound heterozygous (especially hemizygous) F0 generations of genetically modified animals for targeted gene modification, significantly improving efficiency and saving time, because the mating steps needed are less.

[0247] (2) Gene conversion or loss of heterozygosity

[0248] In some methods, the genome to be modified is in a cell that is heterozygous for the first allele, and the gene is modified to become homozygous for the first allele. The term "heterozygous" includes such a situation: wherein the genome comprises different alleles at one or more corresponding chromosome loci (for example, the corresponding loci on homologous chromosomes include different alleles). The term "homozygous" includes such a situation: wherein the genome comprises the same allele at the corresponding chromosome loci (for example, on the corresponding homologous chromosomes). In some such methods, homozygosity can be achieved by using the first allele as a donor sequence by cells to repair the double-strand breaks in the corresponding second allele via homologous recombination (such as gene conversion). Typically, the extent of gene conversion is limited to hundreds of base pairs. See, e.g., Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22:886-897, which is incorporated herein by reference in its entirety for all purposes. However, the use of paired guide RNAs that direct cleavage at different cleavage sites within a single locus can improve and enhance gene conversion capacity over longer stretches.

[0249] Such methods can be used for a variety of situations. The first allele may include a mutation. In some methods, for example, the first allele includes a desired targeted gene modification. If, for example, the goal is to form a non-human animal that is homozygous for targeted genetic modification, then the homozygosity of the modification can be saved in a large amount of time and cost. In other methods, the first allele is a wild-type allele of a gene, which corresponds to the second pathogenic allele of the gene. Or, the second allele may include any mutation. These methods can then be used to achieve the ultimate gene therapy goal of replacing the pathogenic allele at its natural chromosome locus using a wild-type allele.

[0250] In some such methods for modifying a genome that is heterozygous for a first allele to become homozygous for the first allele, the genome is contacted with a Cas protein, a tracrRNA, a first CRISPR RNA that hybridizes to a first CRISPR RNA recognition sequence within a second allele, and a second CRISPR RNA that hybridizes to a second CRISPR RNA recognition sequence within the second allele, wherein the first allele is on a first homologous chromosome and the second allele is at a corresponding locus on the second homologous chromosome (i.e., the first allele and the second allele can be corresponding alleles in the first homologous chromosome and the second homologous chromosome pair). Optionally, the genome can be contacted with an additional CRISPR RNA (e.g., a third CRISPR RNA, or a third CRISPR RNA and a fourth CRISPR RNA) that hybridizes to a CRISPR RNA recognition sequence within the second allele. The Cas protein can cleave one or both of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence (i.e., cleavage at a first cleavage site within the first CRISPR RNA recognition sequence and / or at a second cleavage site within the second CRISPR RNA recognition sequence). Genome cutting at the first cleavage site and / or the second cleavage site can form blunt ends or staggered ends in the genomic DNA. The cleavage site can then be repaired by the recombination between the first allele and the second allele, resulting in a modified genome that is homozygous for the first allele. Cells with the modified genome can then be identified.

[0251] In some methods, the first CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence is located in the second allele rather than the first allele. The first allele and / or the second allele may be a wild-type allele or may contain a targeted modification or other deviation from the wild-type allele. For example, the first allele may contain a desired targeted modification, and the second allele may be a wild-type allele. Alternatively, the first allele may be a wild-type allele, and the second allele may contain an undesirable modification, such as a pathogenic mutation. In some such methods, targeted gene repair or targeted gene correction occurs, so that the pathogenic mutation in the second allele is corrected by recombination using the first allele as a donor sequence.

[0252] The first cleavage site and the second cleavage site or the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can be separated by, for example, about 1 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb.

[0253] Alternatively, the first cleavage site and the second cleavage site or the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence can be separated by, for example, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb.

[0254] In some methods, the sequence difference between the first allele and the second allele spans about 100 bp to about 200 bp, about 200 bp to about 400 bp, about 400 bp to about 600 bp, about 600 bp to about 800 bp, about 800 bp to about 1 kb, about 1 kb to about 2 kb, about 2 kb to about 3 kb, about 4 kb to about 5 kb, about 5 kb to about 10 kb, about 10 kb to about 20 kb, about 20 kb to about 40 kb, about 4 kb, about 0 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb to about 150 kb, about 150 kb to about 200 kb, about 200 kb to about 300 kb, about 300 kb to about 400 kb, about 400 kb to about 500 kb, about 500 kb to about 1 Mb, about 1 Mb to about 1.5 Mb, about 1.5 Mb to about 2 Mb, about 2 Mb to about 2.5 Mb, or about 2.5 Mb to about 3 Mb.

[0255] In other methods, the difference between the first allele and the second allele spans at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 800 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, 20 kb, at least 30 kb, at least 4 kb, at least 190 kb, at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb.

[0256] In other methods for modifying the genome in a cell that is heterozygous for the first allele to make it homozygous for the first allele, the genome is contacted with a Cas protein, tracrRNA, and a first non-allele-specific CRISPR RNA that hybridizes with a first CRISPR RNA recognition sequence. The first allele is on the first homologous chromosome, and the CRISPR RNA recognition sequence is located at the near centromere side (i.e., closer to the centromere) of the locus corresponding to the first allele on the second homologous chromosome. The Cas protein can cut the first CRISPR RNA recognition sequence to produce a double-strand break. Recombination can then occur to modify the cell so that it becomes homozygous for the first allele.

[0257] Optionally, the cell is heterozygous for the one or more additional alleles, the first CRISPR RNA recognition sequence is located near the centromere of the locus corresponding to the one or more additional alleles on the second homologous chromosome, and the recombination modifies the cell to become homozygous for the one or more additional alleles.

[0258] Optionally, the method may further include contacting the genome with a second non-allele-specific CRISPR RNA that hybridizes to a second CRISPR RNA recognition sequence, the second CRISPR RNA recognition sequence being located near the centromere side of the locus corresponding to the first allele in the second homologous chromosome, wherein the Cas protein cuts at least one of the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence to produce at least one double-strand break. Optionally, the method may further include contacting the genome with an additional non-allele-specific CRISPR RNA (e.g., a third CRISPR RNA, or a third CRISPR RNA and a fourth CRISPR RNA) that hybridizes to a CRISPR RNA recognition sequence, the CRISPR RNA recognition sequence being located near the centromere side of the locus corresponding to the first allele on the second homologous chromosome. Cells with modified genomes may then be identified.

[0259] In some methods, the first CRISPR RNA recognition sequence (or the second CRISPR RNA recognition sequence, the third CRISPR RNA recognition sequence, or the fourth CRISPR RNA recognition sequence) is located on the second homologous chromosome instead of the first homologous chromosome. The first CRISPR RNA recognition site (or the second CRISPR RNA recognition site, the third CRISPR RNA recognition site, or the fourth CRISPR RNA recognition site) can be about 100bp to about 1kb, about 1kb to about 10kb, about 10kb to about 100kb, about 100kb to about 1Mb, about 1Mb to about 10Mb, about 10Mb to about 20Mb, about 20Mb to about 30Mb, about 30Mb to about 40Mb, about 40Mb to about 50Mb, about 50Mb to about 60Mb, about 60Mb to about 70Mb, about 70Mb to about 80Mb, about 80Mb to about 90Mb, or about 90Mb to about 100Mb from the centromere.

[0260] The first allele and / or one or more additional alleles may include mutations, such as targeted modifications. Alternatively, the first allele and / or one or more additional alleles may be wild-type alleles, and the corresponding locus on the second homologous chromosome may include mutations, such as pathogenic mutations. The first allele may be about 100bp to about 1kb, about 1kb to about 10kb, about 10kb to about 100kb, about 100kb to about 1Mb, about 1Mb to about 10Mb, about 10Mb to about 20Mb, about 20Mb to about 30Mb, about 30Mb to about 40Mb, about 40Mb to about 50Mb, about 50Mb to about 60Mb, about 60Mb to about 70Mb, about 70Mb to about 80Mb, about 80Mb to about 90Mb, or about 90Mb to about 100Mb from the first CRISPR RNA recognition site. Alternatively, the first allele may be at least 100 bp, at least 1 kb, at least 10 kb, at least 100 kb, at least 1 Mb, at least 10 Mb, at least 20 Mb, at least 30 Mb, at least 40 Mb, at least 50 Mb, at least 60 Mb, at least 70 Mb, at least 80 Mb, at least 90 Mb, or at least 100 Mb or more from the first CRISPR RNA recognition site.

[0261] The Cas protein can be Cas9. It can have nuclease activity on both strands of double-stranded DNA, or it can be a nickase. In some methods, the Cas protein and the first CRISPR RNA do not exist at the same time in nature.

[0262] The recombination may include loss of heterozygosity (e.g., polar or directional gene conversion or loss of heterozygosity) on the telomeric side of the double-strand break (i.e., toward the telomere). The second homologous chromosome region replaced by loss of heterozygosity may be about 100 bp to about 1 kb, about 1 kb to about 10 kb, about 10 kb to about 100 kb, about 100 kb to about 1 Mb, about 1 Mb to about 10 Mb, about 10 Mb to about 20 Mb, about 20 Mb to about 30 Mb, about 30 Mb to about 40 Mb, about 40 Mb to about 50 Mb, about 50 Mb to about 60 Mb, about 60 Mb to about 70 Mb, about 70 Mb to about 80 Mb, about 80 Mb to about 90 Mb, or about 90 Mb to about 100 Mb. Alternatively, the second homologous chromosome region that is replaced can be at least 100bp, at least 1kb, at least 10kb, at least 100kb, at least 1Mb, at least 10Mb, at least 20Mb, at least 30Mb, at least 40Mb, at least 50Mb, at least 60Mb, at least 70Mb, at least 80Mb, at least 90Mb or at least 100Mb or larger. For example, a majority of the chromosome can be replaced.

[0263] B. Methods for Producing Genetically Modified Non-Human Animals

[0264] Genetically modified non-human animals can be produced using various methods disclosed herein. In some cases, the method for producing genetically modified non-human animals includes: (1) using the above method to modify the genome of pluripotent cells; (2) selecting genetically modified pluripotent cells; (3) introducing genetically modified pluripotent cells into host embryos; and (4) implanting host embryos containing genetically modified pluripotent cells into surrogate mothers. Produce offspring derived from genetically modified pluripotent cells. Donor cells can be introduced into host embryos at any stage such as blastocyst stage or pre-morula stage (i.e., 4-cell stage or 8-cell stage). Produce offspring that can pass genetic modifications through the germline. Pluripotent cells can be non-human mammalian ES cells (e.g., mouse ES cells or rat ES cells) as discussed elsewhere herein. See, for example, U.S. Patent No. 7,294,754, which is incorporated herein by reference in its entirety for all purposes.

[0265] Alternatively, the method of producing a genetically modified non-human animal may include: (1) modifying the genome of a 1-cell stage embryo using the above method; (2) selecting the genetically modified embryo; and (3) implanting the genetically modified embryo into a surrogate mother, thereby producing offspring that are capable of transmitting the genetic modification through the germline.

[0266] Non-human mammals can also be generated using nuclear transfer techniques. Briefly, methods for nuclear transfer include the following steps: (1) enucleating an oocyte, or providing an enucleated oocyte; (2) isolating or providing a donor cell or nucleus to be mixed with the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstructed cell; (4) implanting the reconstructed cell into the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are generally removed from a sacrificed animal, but oocytes can also be isolated from the oviduct and / or ovary of a living animal. Oocytes can be matured in a variety of culture media known to those of ordinary skill in the art prior to enucleation. Enucleation of oocytes can be performed in a variety of ways known to those of ordinary skill in the art. Donor cells or nuclei can be inserted into enucleated oocytes to form reconstructed cells by microinjection of donor cells under the zona pellucida prior to fusion. Fusion can be induced by applying direct current pulse (electric fusion) across the contact / fusion plane, by exposing the cell to chemicals such as polyethylene glycol that promote fusion, or by inactivating viruses such as Sendai virus. Reconstruction cells can be activated by electricity and / or non-electricity before, during and / or after the fusion of nuclear donor and recipient oocyte. Activation methods include electric pulses, chemically induced shocks, sperm penetration, increasing divalent cation levels in oocytes, and reducing cell protein phosphorylation in oocytes (such as by kinase inhibitors). Activated reconstruction cells or embryos can be cultured in a culture medium known to those of ordinary skill in the art and then transplanted into the uterus of an animal. See, for example, US20080092249, WO / 1999 / 005266A2, US20040177390, WO / 2008 / 017234A1, and U.S. Patent No. 7,612,250, each of which is incorporated herein by reference in its entirety for all purposes.

[0267] Some methods for preparing genetically modified non-human animals include methods for producing F0 generation non-human animals. Such methods may include contacting the genome in non-human ES cells with Cas proteins, a first CRISPR RNA hybridized with a first CRISPR RNA recognition sequence, a second CRISPR RNA hybridized with a second CRISPR RNA recognition sequence, and tracrRNA. The Cas protein can cut the genome in the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence to produce an end sequence. The end sequence can undergo recombination to form a genome with a targeted modification, and the targeted modification may include a deletion between the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence.

[0268] The method may also include: (1) identifying non-human ES cells comprising targeted modifications; (2) introducing non-human ES cells comprising targeted modifications into non-human host embryos; and (3) gestating non-human host embryos in surrogate mothers. The surrogate mothers can then produce F0 generation non-human animals comprising targeted modifications. Host embryos comprising genetically modified pluripotent or totipotent cells (e.g., non-human ES cells) can be incubated until the blastocyst stage and then implanted into surrogate mothers to produce F0 animals. Animals with genetically modified genomic loci can be identified via modification of alleles (MOA) assays as described herein.

[0269] The various methods provided herein allow for the generation of genetically modified non-human F0 animals, wherein the cells of the genetically modified F0 animals comprise targeted modifications. It is recognized that, depending on the method used to generate the F0 animal, the number of cells in the F0 animal having the nucleotide sequence of interest and not containing the recombinase cassette and / or selection cassette will vary. By, for example, Method is introduced into the cell group of F0 animal of greater percentage from corresponding organism (for example 8-cell stage mouse embryo) to allow in morula pre-embryo to comprise the cell with the nucleotide sequence paid close attention to, and the nucleotide sequence paid close attention to comprises targeted genetic modification.In particular cases, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cell ratio of non-human F0 animal comprise the cell group with targeted modification.In other cases, at least one or more in the germ cell of F0 animal has targeted modification.

[0270] In some cases, the cells of the genetically modified F0 animal are heterozygous or compound heterozygous for the targeted modification. For example, the cells of the genetically modified F0 animal can be hemizygous for the targeted modification. In other cases, the cells of the genetically modified F0 animal are homozygous for the targeted modification.

[0271] In some cases, the F0 animals produced by the methods and compositions disclosed herein can be bred into wild-type animals to produce F1 generations that are heterozygous for the targeted modification. Subsequently, animals from the F1 generation can be mated with each other to produce F2 animals that are homozygous for the targeted modification. Specific primers and / or probes can be used to genotype the F1 progeny to determine whether the targeted genetic modification is present.

[0272] C. Genomes and Target Genomic Loci

[0273] The genome or target genomic locus modified by the methods disclosed herein may comprise any segment or region of DNA within the cell. The genome or target genomic locus may be native to the cell, may be a heterologous or exogenous segment of DNA integrated into the cell genome, or may be a combination thereof. Such heterologous or exogenous DNA segments may include transgenes, expression cassettes, polynucleotides encoding selectable markers, or heterologous or exogenous regions of genomic DNA.

[0274] The genome or target genomic locus may also comprise extrachromosomal DNA in the cell, such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a human artificial chromosome, or any other engineered genomic region contained in an appropriate host cell.

[0275] D. Forms of Cas9 and guide RNA

[0276] In some methods, genome contact includes introducing one or more Cas proteins, one or more CRISPR RNAs, and one or more tracrRNAs into the cell. The introduction can be accomplished by any means, and one or more of the components (e.g., two of the components, or all of the components) can be introduced into the cell simultaneously or sequentially in any combination.

[0277] CRISPR RNA and tracrRNA can be fused together as a guide RNA (gRNA) to be introduced into a cell. Alternatively, CRISPR RNA and tracrRNA can be different RNA molecules. CRISPR RNA can be introduced into a cell in the form of RNA or in the form of DNA encoding CRISPR RNA. Similarly, tracrRNA can be introduced into a cell in the form of RNA or in the form of DNA encoding tracrRNA, and gRNA can be introduced into a cell in the form of RNA or in the form of DNA encoding gRNA.

[0278] Cas protein can be introduced into cells in the form of protein, messenger RNA (mRNA) encoding Cas protein or DNA encoding Cas protein. In some methods, Cas protein, CRISPR RNA and tracrRNA can be introduced into cells as protein-RNA complexes. Similarly, Cas protein and gRNA can be introduced into cells as protein-RNA complexes. Cas protein can be cell permeable Cas protein (e.g., Cas protein with cell penetration domain).

[0279] The DNA encoding the Cas protein, CRISPR RNA or tracrRNA can be effectively connected to an active promoter in the cell. Such DNA can be one or more expression constructs. In some methods, one or more of such expression constructs can be components of a single nucleic acid molecule. For example, DNA encoding one or more Cas proteins, DNA encoding one or more CRISPR RNAs, and DNA encoding one or more tracrRNAs can all be components of a single nucleic acid molecule. Alternatively, between two, three, four or more nucleic acid molecules, they can be separated in any combination.

[0280] Similarly, the DNA encoding the Cas protein or the DNA encoding the gRNA can be effectively connected to an active promoter in the cell. Such DNA can also be one or more expression constructs. In some methods, one or more of such expression constructs can be components of a single nucleic acid molecule. For example, the DNA encoding one or more Cas proteins and the DNA encoding one or more gRNAs can both be components of a single nucleic acid molecule. Alternatively, between two, three, four or more nucleic acid molecules, they can be separated in any combination.

[0281] In some methods, the Cas protein and CRISPR RNA and / or tracrRNA do not exist together in nature. In some methods, for example, the Cas protein and the first CRISPR RNA do not exist together in nature, the Cas protein and the second CRISPR RNA do not exist together in nature, and / or the Cas protein and tracrRNA do not exist together in nature.

[0282] In some methods, the Cas protein is a Cas9 protein. The Cas protein can be fused to a heterologous polypeptide, such as a nuclear localization signal (NLS). The Cas protein can have full cleavage activity and can form double-strand breaks (e.g., double-strand breaks with blunt ends) in genomic DNA, or it can be a nickase that can only cut the strands of genomic DNA.

[0283] In some methods, paired nickases are used. For example, the genome can be contacted with the first nickase and the second nickase that cut the relative strand of DNA, thereby, the genome is modified by double nicking. The first nickase can cut the first strand (that is, complementary strand) of genomic DNA, and the second nickase can cut the second strand (that is, non-complementary strand) of genomic DNA. Alternatively, the two nickases can cut the same strand. The first nickase and the second nickase can be formed, for example, by mutating the catalytic residues in the RuvC domain of the first nickase (for example, the D10A mutation described elsewhere herein) and mutating the catalytic residues in the HNH domain of the second nickase (for example, the H840A mutation described elsewhere herein). Alternatively, the first nickase can be used to form two nicks.

[0284] In some such methods, double nicking can be used to form one or more double-strand breaks with staggered ends. For example, double nicking is used to form staggered ends at the first cleavage site and the second cleavage site. The first nicking enzyme can cut the first strand of DNA in the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence hybridized with the first CRISPR RNA and the second CRISPR RNA, and the second nicking enzyme can cut the second strand of DNA in the third CRISPR RNA target recognition sequence and the fourth CRISPR RNA target recognition sequence hybridized with the third CRISPR RNA and the fourth CRISPR RNA. Alternatively, the first nicking enzyme can be used to make the first CRISPR RNA recognition sequence, the second CRISPR RNA recognition sequence, the third CRISPR RNA recognition sequence, and the fourth CRISPR RNA recognition sequence produce nicks. The first CRISPR RNA target recognition sequence and the third CRISPR RNA target recognition sequence can be positioned to form a first nicking site, so that the nicks formed on the first and second strands of DNA by the first nicking enzyme and the second nicking enzyme form double-strand breaks (that is, the first nicking site includes the nicks in the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence). Similarly, the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence can be positioned to form a second cleavage site so that the nicks formed by the first nickase and the second nickase on the first and second strands of the DNA form a double-stranded break (i.e., the second cleavage site comprises the nicks in the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence). In some cases, the nicks in the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence and / or the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence can be offset nicks. The offset window can be, for example, at least about 5 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp or more.See Ran et al. (2013) Cell 154: 1380-1389; Mali et al. (2013) Nat. Biotech. 31: 833-838; and Shen et al. (2014) Nat. Methods 11: 399-404, each of which is incorporated herein by reference in its entirety for all purposes.

[0285] E. Methods for Introducing Nucleic Acids and Proteins into Cells

[0286] Various methods and compositions are provided herein to allow nucleic acid to be introduced into cells. In some cases, the system for introducing nucleic acid allows targeted integration at a specific genomic locus. Such systems use multiple components, and for ease of reference, the term "targeted genome integration system" generally includes all components required for the integration event (e.g., nuclease reagents, nuclease cleavage sites, insertion DNA polynucleotides, targeting vectors, target genome loci and one or more of the polynucleotides of interest).

[0287] The method provided herein may include introducing one or more polynucleotides or polypeptide constructs comprising one or more components of a targeted genome integration system into a cell."Introducing" includes presenting a sequence to a cell in a manner that enables a sequence (polypeptide or polynucleotide) to enter the interior of a cell. The method provided herein does not depend on a particular method for introducing nucleic acid or protein into a cell, as long as nucleic acid or protein is enabled to enter at least one interior of a cell. Methods for introducing nucleic acid and protein into various cell types are known in the art, and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[0288] In some cases, the cells used in the methods and compositions have a DNA construct stably incorporated into their genome. For example, the cells used in the methods disclosed herein may have native Cas-encoding genes stably incorporated into their genome (i.e., Cas-ready cells). "Stably incorporated" or "stably introduced" includes introducing a polynucleotide into a cell so that the nucleotide sequence is integrated into the genome of the cell and can be inherited to its progeny. Any protocol can be used to stably incorporate the various components of a DNA construct or a targeted genome integration system.

[0289] Transfection protocols and protocols for introducing polypeptide or polynucleotide sequences into cells can vary. Non-limiting transfection methods include chemical-based transfection methods using: liposomes; nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc Natl Acad Sci USA 74 (4): 1590-4, and Kriegler, M (1991). Transfer and Expression: A Laboratory Manual. New York: WH Freeman and Company.pp.96-97 (Kriegler, M, 1991, "Gene Transfection and Expression Laboratory Manual", New York WH Freeman Company, pp. 96-97); dendrimers; or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, ultrasound poration and optical transfection. Particle-based transfection includes the use of gene guns or magnet-assisted transfection (Bertram, J. (2006) Current Pharmaceutical Biotechnology 7, 277-28 (Bertram, J., 2006, "Current Pharmaceutical Biotechnology", Vol. 7, pp. 277-28)). Viral methods can also be used for transfection.

[0290] In some cases, the cells are expressed by electroporation, intracytoplasmic injection, viral infection, adenovirus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or Nucleofection. TM Mediates the introduction of nucleic acids or proteins into cells.

[0291] It is also possible to complete the introduction of nucleic acid or protein into cells (e.g., 1-cell stage embryos) by microinjection. In 1-cell stage embryos, microinjection can enter into female pronuclei and / or male pronuclei or into cytoplasm. If microinjection only enters into one pronucleus, male pronucleus is preferred because it has a larger size. MRNA microinjection preferably enters into cytoplasm (e.g., to directly deliver mRNA to the translation machinery), while Cas protein or microinjection of nucleic acid molecules encoding Cas protein or encoding RNA preferably enters into the nucleus / pronucleus. Alternatively, microinjection can be performed by injecting into the nucleus / pronucleus and the cytoplasm: a needle can be first introduced into the nucleus / pronucleus, and a first amount can be injected, and then when the needle is removed from the 1-cell stage embryo, a second amount can be injected into the cytoplasm. If the Cas protein is injected into the cytoplasm, the Cas protein preferably includes a nuclear localization signal to ensure delivery to the nucleus / pronucleus. The method for performing microinjection is well known. See, e.g., Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), Meyer et al. (2010) Pros Natl Acad Sci USA 107: 15022-15026 and Meyer et al. (2012) Proc Natl Acad Sci USA 107: 15022-15026. 109:9354-9359 (Meyer et al., 2012, Proc. Natl. Acad. Sci. USA, Vol. 109, pp. 9354-9359), each of which is incorporated herein by reference in its entirety for all purposes.

[0292] The introduction of nucleic acid or protein into cells can be performed one or more times over a period of time. For example, the introduction step can be performed at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, at least thirteen times, at least fourteen times, at least fifteen times, at least sixteen times, at least seventeen times, at least eighteen times, at least nineteen times, or at least twenty times over a period of time.

[0293] When both the nuclease agent and the targeting vector (e.g., LTVEC for cells other than 1-cell stage embryos) are introduced into the cells, they can be introduced simultaneously. Alternatively, the nuclease agent can be introduced separately from the targeting vector. For example, the nuclease agent can be introduced before the targeting vector is introduced, or the nuclease agent can be introduced after the targeting vector is introduced.

[0294] F. Recombination Mechanisms and Methods for Altering the Incidence of Non-homologous End Joining, Gene Conversion, or Homologous Recombination

[0295] Recombination includes any process of genetic information exchange between two polynucleotides and can occur by any mechanism. Recombination in response to double-strand breaks (DSBs) occurs mainly through two conservative DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22: 886-897 (Kasparek and Humphrey, 2011, "Cell and Developmental Biology Research Collection", Vol. 22, pp. 886-897), which is incorporated herein by reference in its entirety for all purposes. NHEJ includes repairing double-strand breaks in nucleic acids by directly connecting the broken ends to each other without the need for homologous templates. Connection of discontinuous sequences by NHEJ can generally lead to deletions, insertions or translocations near the double-strand break site. Recombination can also occur by homology-mediated repair (HDR) or homologous recombination (HR). HDR or HR includes a form of nucleic acid repair that may require nucleotide sequence homology, use a "donor" molecule as a template to repair a "target" molecule (i.e., a molecule that has undergone a double-strand break), and result in the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve mismatch correction of heteroduplex DNA formed between the broken target and the donor, and / or synthesis-dependent strand annealing in which the donor is used to resynthesize genetic information that will become part of the target, and / or related processes. In some cases, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into the target DNA.

[0296] In the context of modifying the cell genome that is heterozygous for alleles to be homozygous for alleles, recombination may include any means of deriving homozygous cells from heterozygous cells. Such means may include, for example, loss of heterozygosity (LOH), gene conversion, or exchange events that occur by any known recombination mechanism. Without wishing to be bound by theory, LOH may, for example, occur via mitotic recombination with or without gene conversion, or via chromosome loss and replication. See, for example, Lefebvre et al. (2001) Nat. Genet. 27: 257-258 (Lefebvre et al., 2001, "Nature-Genetics", Vol. 27, pp. 257-258), which is incorporated herein by reference in its entirety for all purposes. Gene conversion in this context may include unidirectional transfer of genetic material from a donor sequence to a highly homologous receptor (i.e., non-reciprocal exchange of genetic information from one molecule to its homolog). Gene conversion includes any means for copying alleles by any known recombination mechanism. For example, gene conversion can involve the non-reciprocal exchange of genetic information from a complete sequence to a homologous region containing a double-strand break, and it can occur between sister chromatids on either the same chromatid or different chromosomes, between homologous chromosomes, or between homologous sequences. See, for example, Chen et al. (2007) Nat. Rev. Genet. 8: 762-775 (Chen et al., 2007, "Nature Review-Genetics", Vol. 8, pp. 762-775), which is incorporated herein by reference in its entirety for all purposes. In certain cases, gene conversion is produced directly by homologous recombination due to copying genetic information from homologous chromosomes. When homologous sequences are different, this can lead to local loss of heterozygosity (LOH).

[0297] For example, LOH can occur through reciprocal chromatid exchange by mitotic crossover, or through chromatid duplication by breakage-induced replication. In either case, heterozygous modification can occur, where one chromosome is targeted prior to genome duplication. Alternatively, a single chromatid can be targeted after genome duplication, followed by interchromatid gene conversion.

[0298] In any one of the methods disclosed herein, the cell may be a cell modified to increase or decrease NHEJ activity. Similarly, the cell may be a cell modified to increase gene conversion or NHEJ activity. Such modifications may include modification of the expression or activity of genes involved in regulating NHEJ, gene conversion and / or HDR. For example, reducing NHEJ activity and / or increasing HDR activity may promote biallelic shearing of the genomic region between the CRISPR RNA recognition sequences corresponding to two gRNAs. Without being bound by any particular theory, a mechanism by which biallelic genome shearing may occur is to undergo NHEJ-mediated repair or HDR-mediated repair in the first allele, and to form the same second allele (see Example 1) via an HDR mechanism (such as gene conversion). Therefore, promoting HDR-mediated pathways (e.g., by reducing NHEJ activity or by increasing HDR activity) may also promote biallelic shearing of genomic regions. Similarly, without being bound by any particular theory, conversion from heterozygous cells to homozygous cells using pairs of guide RNAs targeting a single locus can be facilitated if NHEJ activity is reduced and HDR activity (e.g., gene conversion activity) is correspondingly increased.

[0299] Inhibitors can be used to increase or decrease NHEJ activity, or increase or decrease HDR activity. Such inhibitors can be, for example, small molecules or inhibitory nucleic acids such as short interfering nucleic acids (e.g., short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA)) or specific antisense oligonucleotides for gene transcripts. Inhibitors can be directed to enzymes that participate in NHEJ or HDR, or upstream regulation via post-translational modifications such as phosphorylation, ubiquitination and sumoylation.

[0300] In mammalian cells, NHEJ is the main DSB repair mechanism and is active throughout the cell cycle. In vertebrates, "standard" or "typical" NHEJ pathways (C-NHEJ) require several core factors, including DNA-PK, Ku70-80, Artemis, ligase IV (Lig4), XRCC4, CLF and Polμ to repair DSB. See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22: 886-897 (Kasparek and Humphrey, 2011, "Cell and Developmental Biology Research Collection", Vol. 22, pp. 886-897), which is incorporated herein by reference in its entirety for all purposes. During NHEJ, the high abundance end protection Ku protein binds to the DNA end as a mooring site for loading other NHEJ components.

[0301] Therefore, in some of the methods disclosed herein, the cell has been modified to reduce or eliminate or to increase the expression or activity of factors involved in C-NHEJ. For example, in some methods, the cell has been modified to reduce or eliminate the expression or activity of DNA-PK, Ku70-80, Artemis, ligase IV (Lig4), XRCC4, CLF and / or Polμ. In a specific method, the cell has been modified to reduce or eliminate the expression or activity of DNA-PK, or to increase the expression or activity of DNA-PK (e.g., the expression or activity of DNA-PKcs; an exemplary UniProt sequence designated as P97313). Examples of DNA-PKcs inhibitors include, for example, NU7026 and NU7441. See, for example, U.S. Patent No. 6,974,867, which is incorporated herein by reference in its entirety for all purposes. In a specific method, the cell has been modified to reduce or eliminate the expression or activity of ligase IV or to increase the expression or activity of ligase IV. An example of a ligase IV inhibitor is SCR7.

[0302] Inhibitors targeting cell cycle checkpoint proteins such as ATM (e.g., KU55933), CHK1 / CHK2 (e.g., KLD1162 or CHIR-124), and ATR (e.g., VE 821) can also be used to synergistically enhance the effects of specific DNA repair inhibitors or prevent unintended adverse effects such as cell cycle arrest and / or apoptosis (see Ciccia et al. (2010) Mol Cell 40:179, which is incorporated herein by reference in its entirety for all purposes.

[0303] Disruption of C-NHEJ can increase the level of aberrant ligation mediated by the "alternative" NHEJ (A-NHEJ) pathway and can also increase HR repair. The A-NHEJ pathway shows a bias toward microhomology-mediated ligation and follows slower kinetics than C-NHEJ. Several factors, including the MRN complex (MRE11, RAD50, NBS1), CtIP, XRCC1, PARP, Lig1, and Lig3, have been proposed to be involved. See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22: 886-897 and Claybon et al. (2010) Nucleic Acids Res. 38(21): 7538-7545, each of which is incorporated herein by reference in its entirety for all purposes.

[0304] Thus, in some of the methods disclosed herein, the cell has been modified to reduce or eliminate or to increase the expression or activity of a factor involved in A-NHEJ. For example, in some methods, the cell has been modified to reduce or eliminate the expression or activity of MRE11, RAD50, NBS1, CtIP, XRCC1, PARP (e.g., PARP1), Lig1, and / or Lig3. In other methods, the cell has been modified to increase the expression or activity of MRE11, RAD50, NBS1, CtIP, XRCC1, PARP (e.g., PARP1), Lig1, and / or Lig3. In a specific method, the cell has been modified to reduce or eliminate the expression or activity of PARP1 or to increase the expression or activity of PARP1 (exemplary UniProt sequence designated as P11103). Examples of PARP inhibitors (e.g., NU1025, Iniparib, Olaparib) include nicotinamide; isoquinolinone and dihydroisoquinolinone; benzimidazole and indole; phthalazin-1 (2H) -one and quinazolinone; isoindolinone and its analogs and derivatives; phenanthridine and phenanthridone; benzopyrone and its analogs and derivatives; unsaturated hydroxamic acid derivatives and its analogs and derivatives; pyridazines, including fused pyridazines and its analogs and derivatives; and / or other compounds such as caffeine, theophylline and thymidine and its analogs and derivatives. See, e.g., U.S. Pat. No. 8,071,579, which is incorporated herein by reference in its entirety for all purposes.

[0305] C-NHEJ also exhibits a competitive relationship with HR, such that disruption of C-NHEJ can also lead to increased HR repair. This competition between NHEJ and HR can be exploited because disruption of NHEJ can enhance gene targeting by reducing random integration and potentially increasing target integration through homologous recombination.

[0306] There are several forms of homologous recombination repair, including single-strand annealing, gene conversion, exchange and break-induced replication. Single-strand annealing is a minor form of HR repair, in which homologous single-stranded sequences are located on either side of the excised DSB annealing strand, resulting in chromosome reconstruction. Single-strand annealing produces deletions of varying sizes, depending on the separation distance of the two regions of sequence homology. Gene conversion includes the non-reciprocal exchange of genetic information from one molecule to its homolog, which is directly derived from the HR caused by copying genetic information from homologous chromosomes. When homologous sequences are not identical, this can lead to local LOH. Typically, the degree of gene conversion is limited to hundreds of base pairs. However, long-segment gene conversions in some genetic backgrounds have been reported, including RAD51C deletions. See Nagaraju et al. (2006) Mol. Cell. Biol. 26: 8075-8086, which is incorporated herein by reference in its entirety for all purposes. Exchanges can occur, for example, between homologous chromosomes, and if they occur in G1, the exchange has the potential for a reciprocal translocation, or if they occur in G2, the exchange has the potential for a non-reciprocal translocation and LOH extending from the break site to the distal telomere. Break-induced replication is a variant of HR in which DNA replication continues to the end of the chromosome with strand invasion. Thus, there are many mechanisms by which HR can promote LOH.

[0307] Thus, in some of the methods disclosed herein, the cells have been modified to reduce or eliminate or to increase the expression or activity of factors involved in HR. For example, in some methods, the cells have been modified to increase the expression or activity of RAD51, RAD52, RAD54, RAD55, RAD51C, BRCA1 and / or BRCA2. In other methods, the cells have been modified to reduce or eliminate the expression or activity of RAD51, RAD52, RAD54, RAD55, RAD51C, BRCA1 and / or BRCA2.

[0308] In some methods, the expression or activity of other proteins involved in regulating NHEJ and / or HR can be altered. For example, in some methods, the cells have been modified to reduce or eliminate the expression or activity of Chk2, to reduce or eliminate the expression or activity of Clspn, to reduce or eliminate the expression or activity of Setd2, to increase the expression or activity of Kat2a, and / or to increase the expression or activity of Rad51. In other methods, the cells have been modified to increase the expression or activity of Chk2, to increase the expression or activity of Clspn, to increase the expression or activity of Setd2, to reduce or eliminate the expression or activity of Kat2a, and / or to reduce or eliminate the expression or activity of Rad51.

[0309] Chk2 (also known as Chek2 and Rad53; the S. pombe homolog is Cds1) is a serine / threonine protein kinase required for checkpoint-mediated cell cycle arrest, DNA repair activation, and apoptosis in response to the presence of DNA double-strand breaks. See Blaikley et al. (2014) Nucleic Acids Research 42:5644-5656, which is incorporated herein by reference in its entirety for all purposes. Clspn (also known as Claspin; the S. pombe homolog is Mrc1) is a protein required for checkpoint-mediated cell cycle arrest in response to DNA damage. Deletion of homologs of Chk2 or Clspn in S. pombe has been reported to produce a hyper-recombination phenotype that exhibits a significantly increased level of break-induced gene conversion compared to the wild type. Specifically, gene conversion levels have been reported to be significantly increased, whereas nonhomologous end joining (NHEJ), sister chromatid conversion (SCC), and loss of heterozygosity (LOH) levels have been reported to be decreased. See Blaikley et al. (2014) Nucleic Acids Research 42: 5644-5656.

[0310] Kat2a (also known as Gcn5 and Gcn5l2) is a ubiquitous histone acetyltransferase that promotes transcriptional activation and has been reported to be associated with double-strand break repair. When non-homologous end joining is inhibited, the acetylation of histone H3 lysine 36 (H3K36) that Kat2a relies on increases chromatin accessibility, improves excision rate, and promotes homologous recombination. See Pai et al. (2014) Nat. Commun. 5: 4091 (Pai et al., 2014, "Nature Communications", Vol. 5, p. 4091), which is incorporated herein by reference in its entirety for all purposes. Setd2 (also known as Kiaa1732, Kmt3a and Set2) is a histone methyltransferase that specifically trimethylates lysine 36 (H3K36me3) of histone H3 using demethylated lysine 36 (H3K36me2) as a substrate. Setd2-dependent H3K36 methylation reduces chromatin accessibility, increases resection rate, and promotes NHEJ. See Pai et al. (2014) Nat. Commun. 5:4091.

[0311] Rad 51 (also known as Reca, Rad51A, and DNA repair protein Rad51 homolog 1) is a protein that acts with Rad52 and other proteins to achieve strand exchange during homologous recombination, thereby forming heteroduplex DNA that is resolved by mismatch repair to produce gene conversion segments. In mammalian cells, overexpression of Rad51 and Rad52 has been reported to increase the frequency of homologous recombination and gene conversion. See Yanez & Porter (1999) Gene Ther. 6: 1282-1290 and Lambert & Lopez (2000) EMBO J. 19: 3090-3099, which is incorporated herein by reference in its entirety for all purposes.

[0312] The modification in the expression or activity of the gene involved in regulating and controlling NHEJ, gene conversion and / or homology-mediated repair can be space-specific or timing-specific, and can also be inducible or temporary and reversible.For example, various forms of boxes can be constructed so that they can be missing in specific cells or tissue types, at specific developmental stages or when induced.Such boxes can adopt recombinase systems, wherein the boxes are flanked by recombinase recognition sites on both sides, and can be removed using recombinases expressed in the desired cell type, expressed at the desired developmental stage, or expressed or activated when induced.Such boxes can be further constructed to include a series of paired different recombinase recognition sites, which are arranged so that invalid alleles, conditional alleles or conditional / invalid combination alleles can be generated, as described in US 2011 / 0104799, which is incorporated herein by reference in its entirety for all purposes. The regulation of the recombinase gene can be controlled in various ways, such as by effectively linking the recombinase gene to a cell-specific, tissue-specific, or developmentally regulated promoter (or other regulatory element), or by effectively linking the recombinase gene to a 3'-UTR that contains a recognition site for a miRNA that is active only in a specific cell type, tissue type, or developmental stage. The recombinase can also be regulated, for example, by using a fusion protein that places the recombinase under the control of an effector or metabolite (e.g., a CreER whose activity is controlled by tamoxifen). T2 ), or the recombinase gene is placed under the control of an inducible promoter (e.g., an inducible promoter whose activity is controlled by doxycycline and TetR or a TetR variant). Examples of various forms of cassettes and ways to regulate recombinase genes are provided, for example, in US 8,518,392, US 8,354,389, and US 8,697,851, each of which is incorporated by reference in its entirety.

[0313] G. Cells and Animals

[0314] Various compositions and methods provided herein use cells, such as cells from animals.Such cells can be from non-human animals.Such cells can be eukaryotic cells, including, for example, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, and non-pluripotent human cells.Mammalian cells can be, for example, non-human mammalian cells, non-pluripotent human cells, rodent cells, rat cells, mouse cells, hamster cells, fibroblasts, or CHO cells.Eukaryotic cells can be non-human omnipotent cells, non-human pluripotent cells (e.g., mouse embryonic stem (ES) cells or rat ES cells) or non-pluripotent cells.Non-human omnipotent cells include undifferentiated cells, which can produce any cell type, and pluripotent cells include undifferentiated cells, which have the ability to develop into more than one differentiated cell type.Such pluripotent and / or omnipotent cells can be, for example, non-human embryonic stem (ES) cells or ES-like cells such as non-human induced pluripotent stem (iPS) cells.Non-human embryonic stem cells include non-human omnipotent or pluripotent cells of embryonic origin. Non-human ES cells can be derived from the inner cell mass of a blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0315] Eukaryotic cells can also be cells that are not primary somatic cells. Somatic cells can include any cell that is not a gamete, a germ cell, a gametocyte, or an undifferentiated stem cell.

[0316] Eukaryotic cells also include non-human primary cells. Non-human primary cells include cells or cell cultures directly separated from non-human organisms, organs or tissues. Primary cells include cells that are neither transformed cells nor immortal cells. They include any cells obtained from non-human organisms, organs or tissues, which were not previously transmitted with tissue culture, or previously transmitted with tissue culture but could not be transmitted with tissue culture indefinitely. Such cells can be separated by conventional techniques, and include, for example, somatic cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal stem cells, keratinocytes, melanocytes, mononuclear leukocytes, monocytes, adipocytes, preadipocytes, neurons, glial cells, hepatocytes, skeletal muscle myoblasts and smooth muscle cells. For example, primary cells can derive from connective tissue, muscle tissue, nervous system tissue or epithelial tissue.

[0317] Eukaryotic cells also include immortalized cells. Immortalized cells include cells from non-human multicellular organisms, which are usually not infinitely proliferated, but have escaped normal cell aging and can continue to divide on the contrary due to mutation or change. Such mutation or change can occur spontaneously or be deliberately induced. The example of immortalized cells includes Chinese hamster ovary (CHO) cells, human embryonic kidney cells (such as, HEK 293 cells) and mouse embryonic fibroblasts (such as, 3T3 cells). Many types of immortalized cells are well known in the art.

[0318] Immortalized cells or primary cells include cells typically used for culture or expression of recombinant genes or proteins.

[0319] Eukaryotic cells can also include 1-cell stage embryos (i.e., fertilized oocytes or zygotes) of non-human mammals. Such 1-cell stage embryos can be from any genetic background (e.g., BALB / c, C57BL / 6, 129, or a combination thereof), can be fresh or frozen, and can be derived from in vitro fertilization.

[0320] About cell, pluripotent and / or totipotent cell, ES cell, donor cell and / or host embryo, term " animal " comprises mammal, fish and bird.Mammal comprises, for example, non-human primate, monkey, orangutan, cat, dog, horse, bull, deer, bison, sheep, rodent (such as, mouse, rat, hamster, guinea pig), livestock (such as, cattle species such as cow, steer, etc.; Sheep species such as sheep, goat, etc.; With pig species such as piglet and boar).Bird comprises, for example, chicken, turkey, ostrich, goose, duck, etc..Also comprise domesticated animal and agricultural animal.Term " non-human animal " does not comprise people.

[0321] Mouse pluripotent and / or totipotent cells can be derived from 129 cell lines, C57BL / 6 cell lines, a mixture of 129 and C57BL / 6, BALB / c cell lines or Swiss Webster cell lines. Examples of 129 cell lines include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1 and 129T2. See, e.g., Festing et al. (1999) Mammalian Genome 10:836, which is incorporated herein by reference in its entirety for all purposes. Examples of C57BL cell lines include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal-wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. Mouse pluripotent and / or totipotent cells may also be derived from a mixed strain of the above-mentioned 129 cell line and the above-mentioned C57BL / 6 cell line (e.g., 50% 129 and 50% C57BL / 6). Similarly, mouse pluripotent and / or totipotent cells can be derived from a mixture of the above-mentioned 129 cell lines or a mixture of the above-mentioned BL / 6 cell lines (e.g., 129S6 (129 / SvEvTac) cell lines). A specific example of mouse ES cells is VGF1 mouse ES cells. See, for example, Auerbach et al. (2000) Biotechniques 29, 1024-1028, 1030, 1032 (Auerbach et al., 2000, Biotechnology, Vol. 29, pp. 1024-1028, 1030, 1032), which is incorporated herein by reference in its entirety for all purposes.

[0322] Rat pluripotent and / or totipotent cells can be derived from any rat cell line, including, for example, ACI rat cell line, Blackthorn (DA) rat cell line, Wistar rat cell line, LEA rat cell line, Sprague Dawley (SD) rat cell line, or Fischer rat cell lines such as Fisher F344 or Fisher F6. Rat pluripotent and / or totipotent cells can also be obtained from a cell line derived from a mixture of two or more of the cell lines listed above. For example, rat pluripotent and / or totipotent cells can be derived from a DA cell line or an ACI cell line. The ACI rat cell line is characterized by having black spines, a white abdomen and feet, and is RT1av1 Haplotype. Such cell lines are available from a variety of sources, including the Harlan Laboratory. An example of a rat ES cell line from an ACI rat is the ACI.G1 rat ES cell. The Blackthorn (DA) rat cell line is characterized by having a patterned integument and being RT1 av1 Haplotype. Such rats are available from a variety of sources, including Charles River and Harlan Laboratories. Examples of rat ES cell lines from DA rats are the DA.2B rat ES cell line and the DA.2C rat ES cell line. In some cases, rat pluripotent and / or totipotent cells are from inbred rat cell lines. See, e.g., US2014 / 0235933A1, filed February 20, 2014, which is incorporated herein by reference in its entirety for all purposes.

[0323] The example of human pluripotent cells includes the human induced pluripotent stem cells (human iPS cells) directly derived from the adult cells of differentiation. Human iPS cells can be produced by introducing the reprogramming factors of a specific group into cells, and the reprogramming factors can include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, l-Myc, n-Myc), Krüppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5) and / or related transcription factors (such as NANOG, LIN28 and / or Glis1). Human iPS cells can also be produced, for example, by using miRNA, small molecules or lineage-specific molecules that simulate the effect of transcription factors. Human iPS cells are characterized in that they can differentiate into any cell of three vertebrate germ layers (e.g., endoderm, ectoderm or mesoderm). Human iPS cells are also characterized in that they are able to propagate indefinitely under suitable in vitro culture conditions. See, for example, Takahashi and Yamanaka (2006) Cell 126: 663-676 (Takahashi and Yamanaka, 2006, "Cell", Vol. 126, pp. 663-676), which is incorporated herein by reference in its entirety for all purposes. Initial human iPS cells include cells expressing features similar to those of ectoderm cells after implantation, and cells that participate in lineage specialization and differentiation. Primitive human iPS cells include cells expressing features similar to those of ES cells of the inner cell mass of preimplantation embryos, and cells that do not participate in lineage specialization. See, for example, Nichols and Smith (2009) Cell Stem Cell 4: 487-492 (Nichols and Smith, 2009, "Cell Stem Cell", Vol. 4, pp. 487-492), which is incorporated herein by reference in its entirety for all purposes.

[0324] The cells that have been implanted into the host embryo may be referred to as "donor cells". The genetically modified pluripotent and / or totipotent cells may be from the same cell strain as the host embryo or from a different cell strain. Similarly, the surrogate mother may be from the same cell strain as the genetically modified pluripotent and / or totipotent cells and / or the host embryo, or the surrogate mother may be from a cell strain different from the genetically modified pluripotent and / or totipotent cells and / or the host embryo.

[0325] A variety of non-human host embryos can be used in the methods and compositions disclosed herein.For example, multipotent and / or omnipotent cells with targeted genetic modification can be introduced into morula pre-stage embryos (for example 8-cell stage embryos) from corresponding organisms.See, for example, US 7,576,259, US 7,659,442, US 7,294,754 and US 2008 / 0078000 A1, each of these patents is incorporated herein by reference in full for all purposes.In other cases, non-human donor ES cells can be implanted in non-human host embryos at 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage or 64-cell stage.Host embryo can also be blastocyst or can be blastocyst pre-stage embryos, morula pre-stage embryos, morula stage embryos, not dense morula stage embryos or dense morula stage embryos. When adopting mouse embryo, host embryo stage can be Theiler stage 1 (TS1), TS2, TS3, TS4, TS5 and TS6, with reference to Theiler (1989) " The House Mouse:Atlas of Mouse Development, " Springer-Verlag, New York (Theiler, 1989, " House Mouse: Mouse Development Atlas, " Springer Press, New York) described in Theiler stage, this document is incorporated herein by reference in its entirety for all purposes. For example, Theiler stage can be selected from TS1, TS2, TS3 and TS4. In some cases, host embryo comprises zona pellucida, and donor cell is the ES cell introduced into host embryo via the hole in zona pellucida. In other cases, host embryo is the embryo with less zona pellucida. In other cases, morula stage host embryo gathers.

[0326] H. Methods of Identifying Cells Having a Modified Genome

[0327] Some of the above methods also include a step of identifying cells with modified genomes. Various methods can be used to identify cells with targeted modifications such as deletions or insertions. Such methods can include identifying a cell with targeted modifications at the target locus (e.g., between a first CRISPR RNA recognition sequence and a second CRISPR RNA recognition sequence). Screening can be completed to identify such cells with modified genomic loci.

[0328] The screening step may include a quantitative determination of allele (MOA) modifications for evaluating the parental chromosome. For example, a quantitative determination may be performed via quantitative PCR such as real-time PCR (qPCR). Real-time PCR may utilize a first primer set identifying the target locus and a second primer set identifying a non-targeted reference locus. Primer sets may include a fluorescent probe identifying an amplified sequence.

[0329] The screening step may also include a retention assay, which is an assay for distinguishing between correctly targeting the insertion of a nucleic acid insert into a target genomic locus and randomly transgenic insertion of a nucleic acid insert into a genomic position outside the target genomic locus. Conventional assays such as long fragment PCR or Southern blotting for screening targeted modifications connect the inserted targeting vector to the targeted locus. However, LTVECs are not allowed to be screened by such conventional assays due to their larger homology arm size. In order to screen LTVEC targeting, allele modification (MOA) assays including allele loss (LOA) and allele gain (GOA) assays can be used (see, for example, US 2014 / 0178879 and Frendewey et al. (2010) Methods Enzymol. 476: 295-307 (Frendewey et al., 2010, "Enzymology Methods", Vol. 476, pp. 295-307, the document is incorporated herein by reference in its entirety for all purposes). Allele loss (LOA) assay reverses conventional screening logic and quantifies the number of copies of the natural locus to which the mutation is directed. In correctly targeted cell clones, the LOA assay detects one of the two natural alleles (for genes not on chromosome X or Y), and the other allele is destroyed by targeted modification. The same principle can be reversely applied as allele gain (GOA) assay, quantifying the number of copies of the inserted targeting vector. For example, the combined use of the GOA assay and the LOA assay shows that the correct targeted heterozygous clone has lost a copy of the natural target gene and gained a copy of a drug resistance gene or other insertion marker.

[0330] For example, quantitative polymerase chain reaction (qPCR) can be used as a method for allele quantification, but any method that can reliably distinguish between zero, one, and two copies of a target gene or between zero, one, and two copies of a nucleic acid insert can be used to develop a MOA assay. For example, especially compared to a reference gene (see, e.g., US 6,596,541, which is incorporated herein by reference in its entirety for all purposes), Can be used to quantify the copy number of a DNA template in a genomic DNA sample. Quantify a reference gene in the same genomic DNA as one or more target genes or one or more loci. Therefore, two Amplification (each using its corresponding probe). The probe determines the "Ct" (threshold cycle) of the reference gene, while other probes determine the Ct of the region of one or more target genes or one or more loci that is displaced by successful targeting (i.e., LOA assay). Ct is a measure of the amount of activity that is targeted to a target gene. The amount of initial DNA in each of the probes is proportional to the amount of DNA present, i.e. less abundant sequences require more PCR cycles to reach the threshold cycle. Reducing the number of copies of the template sequence by half will increase the Ct by approximately one unit. When compared to DNA from non-targeted cells, the Ct in cells where one allele of one or more target genes or one or more loci has been replaced by homologous recombination is increased by approximately one unit. The reaction will be directed to the target The reaction increases by one Ct, but does not increase the Ct for the reference gene. For the GOA assay, another The probe is used to determine the Ct of a nucleic acid insert that replaces one or more target genes or one or more loci through successful targeting.

[0331] Because paired gRNA can form a large Cas-mediated deletion at the target genomic locus, it can help to enhance the standard LOA assay and GOA assay to confirm the correct targeting performed by LTVEC (i.e., in cells rather than 1-cell stage embryos). For example, specifically, if the GOA assay uses a probe for the selection box in the LTVEC insert, then only the LOA assay and the GOA assay may not be able to distinguish the correctly targeted cell clones from the clones in which the large Cas-induced deletion of the target genomic locus and the random integration of LTVEC elsewhere in the genome occur simultaneously. Because the selection pressure in the targeted cell is based on the selection box, the random transgenic integration of LTVEC elsewhere in the genome generally includes the adjacent regions of the selection box and LTVEC, but does not include the more distal regions of LTVEC. For example, if a portion of LTVEC is randomly integrated into the genome, and LTVEC includes a nucleic acid insert of about 5kb or longer in length, with a selection box adjacent to the 3' homology arm, then generally speaking, the 3' homology arm, rather than the 5' homology arm, will be transgenicly integrated with the selection box. Alternatively, if the selection cassette is adjacent to the 5' homology arm, then generally the 5' homology arm, rather than the 3' homology arm, will be transgenicly integrated with the selection cassette. For example, if the LOA assay and the GOA assay are used to assess LTVEC targeted integration, and the GOA assay utilizes a probe for the selection cassette, then a heterozygous deletion at the target genomic locus combined with random transgenic integration of the LTVEC will give the same readout as a heterozygous targeted integration of the LTVEC at the target genomic locus. To verify correct targeting by the LTVEC, the retention assay may be used alone or in combination with the LOA assay and / or the GOA assay.

[0332] The retention assay determines the copy number of the DNA template in the 5' target sequence (corresponding to the 5' homology arm of the LTVEC) and / or the 3' target sequence (corresponding to the 3' homology arm of the LTVEC). Specifically, it is useful to determine the copy number of the DNA template in the target sequence corresponding to the homology arm of the adjacent selection box. In diploid cells, a copy number greater than 2 generally indicates random transgenic integration of LTVEC outside the target genomic locus rather than at the target genomic locus, which is undesirable. Correctly targeted clones will maintain a copy number of 2. In addition, a copy number less than 2 in such a retention assay generally indicates a larger Cas-mediated deletion extending beyond the region of the targeted deletion, which is also undesirable.

[0333] In an exemplary retention assay for identifying targeted insertion of a nucleic acid insert at a target genomic locus in a diploid cell, DNA is initially obtained from a cell that has been contacted with a large targeting vector (LTVEC) comprising a nucleic acid insert flanked by a first homology arm that hybridizes to a first target sequence and a second homology arm that hybridizes to a second target sequence, wherein the nucleic acid insert comprises a selection box adjacent to the first homology arm. Optionally, the selection box may comprise a drug resistance gene. The DNA is then exposed to a probe bound to the first target sequence, a probe bound to the nucleic acid insert, and a probe bound to a reference gene with a known copy number, wherein each probe produces a detectable signal after binding. Subsequently, the signal from each of the probes is detected. The signal from the reference gene probe is compared with the signal from the first target sequence probe to determine the copy number of the first target sequence, and the signal from the reference gene probe is compared with the signal from the nucleic acid insert probe to determine the copy number of the nucleic acid insert. A nucleic acid insert copy number of one or two and a first target sequence copy number of two typically indicates targeted insertion of the nucleic acid insert at the target genomic locus, while a nucleic acid insert copy number of one or more and a first target sequence copy number of three or more typically indicates random insertion of the nucleic acid insert at a genomic locus other than the target genomic locus.

[0334] The signal from the first target sequence probe binding can be used to determine the threshold cycle (Ct) value of the first target sequence, the signal from the reference gene probe binding can be used to determine the threshold cycle (Ct) value of the reference gene, and the copy number of the first target sequence can be determined by comparing the first target sequence C...

Claims

1. A method for modifying a genome in a mammalian cell for non-therapeutic purposes, wherein the genome is heterozygous for a first allele, comprising: (I) contacting the genome with: (a) the first Cas protein; as well as (b) a first guide RNA, wherein the first guide RNA comprises a first tracrRNA and a first CRISPR RNA, the first CRISPR RNA hybridizing to a first non-allele-specific CRISPR RNA recognition sequence; wherein the first allele is on a first homologous chromosome and the CRISPR RNA recognition sequence is located on the pericentromeric side of the locus corresponding to the first allele on a second homologous chromosome; and (c) a second guide RNA, wherein the second guide RNA comprises a second tracrRNA and a second CRISPR RNA, the second CRISPR RNA hybridizes to a second non-allele-specific CRISPR RNA recognition sequence, the second non-allele-specific CRISPR RNA recognition sequence being located near the centromere of the locus corresponding to the first allele on the second homologous chromosome, wherein the first allele is 100 kb to 60 Mb away from the first CRISPR RNA recognition sequence, wherein the Cas protein and the first guide RNA do not exist simultaneously in nature, wherein the Cas protein has nuclease activity on both strands of double-stranded DNA; and wherein the Cas protein cleaves the first CRISPR RNA recognition sequence to generate a double-stranded break, and the cell is modified to become homozygous for the first allele; and (II) identifying modified cells that are homozygous for said first allele.

2. The method according to claim 1, wherein step (I) comprises introducing into the cell: (a) the Cas protein or a nucleic acid encoding the Cas protein; and (b) a first guide RNA or a DNA encoding the first guide RNA.

3. The method of claim 2, wherein step (I) comprises introducing a nucleic acid encoding the Cas protein into the cell and introducing the first guide RNA into the cell, wherein the nucleic acid encoding the Cas protein is RNA.

4. The method of claim 2, wherein step (I) comprises introducing a nucleic acid encoding the Cas protein and a DNA encoding the first guide RNA into the cell, wherein the nucleic acid encoding the Cas protein is DNA.

5. The method of claim 2, wherein the Cas protein and the first guide RNA are introduced into the cell as a first protein-RNA complex.

6. The method of claim 4, wherein the first CRISPR RNA and the first tracrRNA are separate RNA molecules, and wherein: (1) DNA encoding the Cas protein is operably linked to a first promoter in a first expression construct; and / or (2) DNA encoding the first CRISPR RNA is operably linked to a second promoter in a second expression construct; and / or (3) DNA encoding the first tracrRNA is operably linked to a third promoter in a third expression construct; Wherein the first promoter, the second promoter and the third promoter are active in the cell.

7. The method of claim 6, wherein the first expression construct, the second expression construct, and the third expression construct are components of a single nucleic acid molecule.

8. The method according to claim 4, wherein: (1) DNA encoding the Cas protein is operably linked to a first promoter in a first expression construct; and / or (2) DNA encoding the first guide RNA is operably linked to a second promoter in a second expression construct, Wherein the first promoter and the second promoter are active in the cell.

9. The method of claim 8, wherein the first expression construct and the second expression construct are components of a single nucleic acid molecule.

10. The method according to any one of the preceding claims, wherein loss of heterozygosity occurs at the telomeric side of the double strand break.

11. The method according to any one of claims 1 to 9, wherein step (I) further comprises introducing the second guide RNA or a DNA encoding the second guide RNA into the cell.

12. The method of any one of claims 1-9, wherein the Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence.

13. The method of any one of claims 1-9, wherein the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence are located on the second homologous chromosome but not on the first homologous chromosome.

14. The method of any one of claims 1-9, wherein the first CRISPR RNA recognition sequence is 100 bp to 1 kb, 1 kb to 10 kb, 10 kb to 100 kb, 100 kb to 1 Mb, 1 Mb to 10 Mb, 10 Mb to 20 Mb, 20 Mb to 30 Mb, 30 Mb to 40 Mb, 40 Mb to 50 Mb, 50 Mb to 60 Mb, 60 Mb to 70 Mb, 70 Mb to 80 Mb, 80 Mb to 90 Mb, or 90 Mb to 100 Mb from the centromere.

15. The method of any one of claims 1-9, wherein the first allele is 1 Mb to 10 Mb, 10 Mb to 20 Mb, 20 Mb to 30 Mb, 30 Mb to 40 Mb, 40 Mb to 50 Mb, or 50 Mb to 60 Mb away from the first CRISPR RNA recognition sequence.

16. The method of any one of claims 1-9, wherein the first allele is at least 1 Mb, at least 10 Mb, at least 20 Mb, at least 30 Mb, at least 40 Mb, or at least 50 Mb away from the first CRISPR RNA recognition sequence.

17. The method of any one of claims 1-9, wherein the region in the second homologous chromosome replaced by loss of heterozygosity is 100 bp to 1 kb, 1 kb to 10 kb, 10 kb to 100 kb, 100 kb to 1 Mb, 1 Mb to 10 Mb, 10 Mb to 20 Mb, 20 Mb to 30 Mb, 30 Mb to 40 Mb, 40 Mb to 50 Mb, or 50 Mb to 60 Mb.

18. The method of any one of claims 1-9, wherein the region in the second homologous chromosome replaced by loss of heterozygosity is at least 100 bp, at least 1 kb, at least 10 kb, at least 100 kb, at least 1 Mb, at least 10 Mb, at least 20 Mb, at least 30 Mb, at least 40 Mb, or at least 50 Mb.

19. The method according to any one of claims 1 to 9, wherein (1) the first allele comprises a mutation; or (2) The first allele is a wild-type allele, and the corresponding site on the second homologous chromosome contains a mutation.

20. The method of claim 19, wherein the first allele comprises a mutation, wherein the mutation is a targeted modification.

21. The method of any one of claims 1-9, wherein the mammalian cell is a human cell or a rodent cell.

22. The method of claim 21, wherein the mammalian cell is the human iPS cell.

23. The method of claim 21, wherein the mammalian cell is a mouse embryonic stem (ES) cell or a rat ES cell.

24. The method of claim 23, wherein the mammalian cell is the mouse ES cell.

25. The method of any one of claims 1-9, wherein the Cas protein is Cas9.

26. The method of any one of claims 1-9, wherein the first CRISPR RNA and the first tracrRNA in the first guide RNA are fused together.

27. The method of any one of claims 1-9, wherein the first CRISPR RNA and the first tracrRNA are separate RNA molecules.

28. The method of any one of claims 1-9, wherein the cell has been modified to reduce non-homologous end joining (NHEJ) and / or to increase gene conversion or homology-directed repair (HDR).

29. The method of claim 28, wherein the cells have been modified to reduce the expression or activity of one or more of the following: DNA-PK, PARP1 and Ligase IV.

30. The method of claim 29, wherein the reduction in expression or activity is inducible, reversible, time-specific and / or spatially specific.

31. The method according to any one of claims 1-9, wherein the identification in step (II) comprises an allele modification assay.

32. The method according to any one of claims 1 to 9, wherein the identification in step (II) comprises screening for loss of heterozygosity.

33. The method of claim 32, wherein the screening comprises performing a structural variant polymorphism PCR assay and / or a single nucleotide variation allele typing assay on the telomeric side of the first CRISPR RNA recognition sequence.

34. The method of claim 33, wherein the screening comprises performing multiple structural variant polymorphism PCR assays and / or multiple single nucleotide variation allele typing assays at different positions on the telomeric side of the first CRISPR RNA recognition sequence.

35. The method of any one of claims 1-9, wherein the cell is heterozygous for one or more additional alleles, the first CRISPR RNA recognition sequence is located near the centromere of the locus corresponding to the one or more additional alleles on the second homologous chromosome, and homologous recombination modifies the cell to become homozygous for the one or more additional alleles, and step (II) comprises identifying a modified cell that is homozygous for the one or more additional alleles.

Images