RU2751237C1 - Methods and compositions for directed genome modification - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology, in particular to an in vitro method for modifying the genome at a genomic locus of interest in a human induced pluripotent stem cell. To implement the method, a Cas9 protein, an mRNA encoding a Cas9 protein, or a DNA encoding a Cas9 protein, a guide RNA containing CRISPR RNA that hybridizes with the target CRISPR sequence at the genomic locus of interest, and a tracrRNA, or a DNA encoding a guide RNA, are introduced into the specified cell. A large directional vector (LTVEC) is also introduced. In this case, the guide RNA is designed to avoid recognition of any sequence in the nucleotide insert, and after this introduction, the genome of the human induced pluripotent stem cell is modified so that it contains a directed genetic modification.

EFFECT: present invention makes it possible to expand the arsenal of tools for performing precise directed modifications of the genome to identify therapeutic agents.

63 cl, 55 dwg, 48 tbl, 6 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of US Provisional Patent Application No. 61/914768, filed December 11, 2013, US Provisional Patent Application No. 62/017416, filed June 26, 2014, US Provisional Patent Application No. 62/029261 filed July 25, 2014, U.S. Provisional Application No. 62/052906, filed September 19, 2014, U.S. Provisional Patent Application No. 62/059527, filed October 03, 2014, and U.S. Provisional Patent Application No. 62 / 064384, filed October 15, 2014, each of which is incorporated by reference in its entirety and in every sense.

LINK TO SEQUENCE LIST FILED AS A TEXT FILE THROUGH EFS WEB

[0002] An official copy of the sequence listing filed in

electronically via EFS-Web as an ASCII sequence listing file named 453460SEQLIST.TXT, created on October 15, 2014 and measuring 27.5 kilobytes, and filed concurrently with the disclosure. The sequence listing, which is in this document in ASCII encoding, is part of the description of the invention and is fully incorporated into this document by reference.

LEVEL OF TECHNOLOGY

[0003] Although rats are recognized as an important animal model system that can mimic the pathology of many human diseases, including, but not limited to, cardiovascular (eg, high blood pressure), metabolic (eg, obesity, diabetes), neurological (eg, pain pathologies ) diseases and various types of cancer, the use of rats in modeling human diseases has been limited compared to mice, in part due to the inaccessibility of pluripotent germline-transmitted rat cells that can retain their pluripotency after a series of in vitro genetic modifications, for example, one or more serial electroporations , and partly due to the lack of effective targeting methods that allow the introduction or removal of large genomic DNA sequences or replacement of large genomic DNA sequences with exogenous nucleotide sequences in pluripotent rat cells.

[0004] There is a need in the art for compositions and methods that allow precise targeted changes in the genome of an organism that can open or expand existing target detection areas and allow for faster and easier validation of therapeutic agents.

SUMMARY OF THE INVENTION

[0005] Methods are provided for modifying a genomic locus of interest in a eukaryotic cell by targeted genetic modification. Such a method comprises (a) introducing into a eukaryotic cell: (i) a large targeting vector (LTVEC) containing a first nucleic acid flanked by a 5 'homology arm and a 3' homology arm, wherein the LTVEC contains at least 10 kb. .; (ii) a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein, (iii) a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding a guide RNA (nRNA) containing a nucleotide sequence , which hybridizes with the target sequence, and trans-activating RNA CRISPR (tracrRNA), while the first and second promoters are active in a eukaryotic cell; and (b) identifying a modified eukaryotic cell containing targeted genetic modification at the genomic locus of interest.

[0006] In one embodiment, the targeted genetic modification is a biallelic genetic modification.

[0007] In one embodiment, the LTVEC is at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb .about., at least 50 kbp, at least 60 kbp, at least 70 kbp, at least 80 kbp. or at least 90 kb. In another embodiment, the LTVEC is at least 100 kb, at least 150 kb. or at least 200 kbp.

[0008] In one embodiment, the eukaryotic cell is a mammalian cell. In one embodiment, the mammalian cell is a fibroblast.

[0009] In one embodiment, the eukaryotic cell is a pluripotent cell. In one embodiment, the pluripotent cell is a human pluripotent cell. In one embodiment, the human pluripotent cell is a human embryonic stem (ES) cell or a human adult stem cell. In another embodiment, the human pluripotent cell is a developmentally restricted human progenitor cell. In another embodiment, the human pluripotent stem cell is a human induced pluripotent stem (iPS) cell.

[0010] In one embodiment, the Cas protein is Cas9.

[0011] In one embodiment, the target sequence is flanked by the sequence of a protospacer contiguous motif (PAM). In one embodiment, the target sequence is directly flanked at 3 (end by a protospacer adjacent motif (PAM) sequence).

[0012] In some embodiments, the 5 'and 3' arms of homology total between about 10 kbp. up to about 150 kbp In some embodiments, the 5 'and 3' arms of LTVEC homology total from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to 150 kbp

[0013] Additionally, there are provided methods in which targeted genetic modification comprises: (a) replacing an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) removal of the endogenous nucleotide sequence; (c) deletion of an endogenous nucleotide sequence, the deletion range being from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (d) insertion of an exogenous nucleotide sequence; (e) insertion of an exogenous nucleotide sequence in the range of about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp; (f) insertion of an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (g) insertion of a chimeric nucleotide sequence containing a human and non-human nucleotide sequence; (h) insertion of a conditioning allele flanked by target sequences of site-specific recombinase; (i) insertion of a selectable marker or reporter gene operably linked to a third promoter active in the pluripotent cell; or (j) a combination of both.

[0014] In one embodiment, the genomic locus of interest comprises (i) a 5 'target sequence that is homologous to the 5' arm of homology; and (ii) a 3 'target sequence that is homologous to the 3' homology arm.

[0015] In some embodiments, the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 3 ppm. In some embodiments, the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 10 kb, at least 10 kb, but less than 20 kb, at least 20 kb, but less than 40 kb, at least 40 kb, but less than 60 kb. v., at least 60 kb, but less than 80 kb, at least about 80 kb, but less than 100 kb, at least at least 100 kb but less than 150 kb, or at least 150 kb but less than 200 kb, at least about 200 kb bp, but less than about 300 kbp, at least about 300 kbp, but less than about 400 kbp, at least about 400 kbp. about., but less than about 500 kbp, at least about 500 kbp, but less than about 1 ppm, at least about 1 ppm. but less than about 1.5 ppm, at least about 1.5 ppm, but less than about 2 ppm, at least about 2 ppm. o., but less than about 2.5 m.p., or at least at least about 2.5 ppm, but less than about 3 ppm.

[0016] In one embodiment, the genomic locus of interest comprises an interleukin-2 gamma receptor locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or both Rag1 and Rag2.

[0017] In one embodiment, the first and second expression constructs are on the same nucleic acid molecule.

[0018] In addition, there is provided a method for modifying the genome, comprising exposing the genome to the Cas protein and CRISPR RNA in the presence of a large targeting vector (LTVEC) containing a nucleotide sequence of at least 10 kb, while after exposure to the Cas protein, RNA CRISPR and LTVEC modifies the genome so that it contains a nucleotide sequence of at least 10 kb.

[0019] In some such methods, the LTVEC comprises a nucleotide sequence of at least 20 kb, at least 30 kb, at least 40 kb, at least of 50 kb, at least 60 kb, at least 70 kb, at least 80 kb or at least 90 kb. In some such methods, the LTVEC contains a nucleotide sequence of at least 100 kb, at least 150 kb. or at least 200 kbp.

[0020] Additionally, there is provided a method for modifying the genome, including bringing the genome into contact with the Cas protein, CRISPR RNA that hybridizes to the target sequence, and tracrRNA in the presence of a large targeting vector (LTVEC), wherein the LTVEC consists of at least 10 kb .O. and contains a first nucleic acid flanked by a 5 'homology arm and a 3' homology arm, wherein after contact with Cas protein, CRISPR RNA and tracrRNA in the presence of LTVEC, the genome is modified at the genomic locus of interest so that it contains the first nucleic acid. The target sequence can be located within or near the genomic locus of interest.

[0021] In some such methods, the genome is in a eukaryotic cell and the Cas protein, CRISPR RNA, and tracrRNA and LTVEC are introduced into the eukaryotic cell. Some such methods further include identifying a modified eukaryotic cell containing targeted genetic modification at the genomic locus of interest.

[0022] In some such methods, CRISPR RNA and tracrRNA are introduced together in the form of a single guide RNA (nRNA). In other methods, CRISPR RNA and tracrRNA are added separately.

[0023] In some such methods (a) the Cas protein is introduced into a eukaryotic cell in the form of a protein, messenger RNA (mRNA) encoding the Cas protein, or DNA encoding the Cas protein; (b) CRISPR RNA is introduced into a eukaryotic cell in the form of RNA or DNA encoding CRISPR RNA; and (c) tracrRNA is introduced into a eukaryotic cell in the form of RNA or DNA encoding tracrRNA.

[0024] In some methods (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding the Cas protein; (b) the DNA encoding CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding CRISPR RNA; and (c) the DNA encoding tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a fourth nucleic acid encoding tracrRNA, the first, second and third promoters being active in a eukaryotic cell. Optionally, the first, second and / or third expression constructs are in the same nucleic acid molecule.

[0025] In some methods (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding the Cas protein; a (b) DNA encoding CRISPR RNA and DNA encoding tracrRNA are in the form of a second expression construct containing a second promoter operably linked to a third nucleic acid encoding an nRNA containing CRISPR RNA and tracrRNA; the first and second promoters are active in the eukaryotic cell. Optionally, the first and second expression constructs are on the same nucleic acid molecule.

[0026] In some methods, Cas protein, CRISPR RNA, and tracrRNA are introduced into a eukaryotic cell as a protein-RNA complex.

[0027] In some methods, targeted genetic modification includes the simultaneous removal of an endogenous nucleotide sequence at a genomic locus of interest and insertion of a first nucleic acid at a genomic locus of interest. In some methods, the endogenous nucleotide sequence deleted is from about 30 kb. to about 110 kb, and the inserted first nucleic acid is from about 40 kb. up to about 140 kbp In some methods, the endogenous nucleotide sequence deleted is from about 38 kb. to about 110 kb, and the inserted first nucleic acid is from about 43 kb. up to about 134 kbp

[0028] In some methods, the targeted genetic modification is a biallelic genetic modification. Optionally, the biallelic genetic modification includes the removal of an endogenous nucleotide sequence and the insertion of the first nucleic acid at the genomic locus of interest on two homologous chromosomes.

[0029] In some methods, the modified eukaryotic cell is compound heterozygous at the genomic locus of interest. In some methods, the modified eukaryotic cell is hemizygous at the genomic locus of interest. Optionally, targeted genetic modification at a genomic locus of interest on one chromosome includes deletion of an endogenous nucleotide sequence and insertion of a first nucleic acid. Optionally, targeted genetic modification includes: (1) deletion of an endogenous nucleotide sequence at a genomic locus of interest on two homologous chromosomes; and (2) inserting the first nucleic acid at the genomic locus of interest on the first chromosome and disrupting the genomic locus of interest on the second chromosome. The first chromosome can be one of two homologous chromosomes, and the second chromosome can be the other homologous chromosome.

[0030] In some methods, the LTVEC is at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb ., at least 50 kbp, at least 60 kbp, at least 70 kbp, at least 80 kbp. or at least 90 kb. Optionally, the LTVEC is at least 100 kb, at least 150 kb. or at least 200 kbp.

[0031] In some methods, the first 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 at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, or at least 300 kb .O. In some methods, the first nucleic acid is from about 40 kbp. up to about 140 kbp In some methods, the first nucleic acid contains from about 43 kb. up to about 134 kbp

[0032] In some methods, the eukaryotic cell is a mammalian cell, a fibroblast, a pluripotent cell, a non-human pluripotent cell, a pluripotent rodent cell, a mouse or rat embryonic stem (ES) cell, a human pluripotent cell, a human embryonic stem cell, a cell, a human restricted progenitor cell, or a human induced pluripotent stem (iPS) cell.

[0033] In some methods, the Cas protein is Cas9. In some methods, the target sequence is directly flanked by a protospacer adjacent motif (PAM) sequence.

[0034] In some methods, the 5 'and 3' arms of LTVEC homology total from about 10 kbp. up to about 150 kbp Optionally, the 5 'and 3' arms of LTVEC homology add up to about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to 150 kbp

[0035] In some methods, targeted genetic modification comprises: (a) replacing an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) removal of the endogenous nucleotide sequence; (c) deletion of an endogenous nucleotide sequence, the deletion range being from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (d) insertion of an exogenous nucleotide sequence; (e) insertion of an exogenous nucleotide sequence in the range of about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp; (f) insertion of an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (g) insertion of a chimeric nucleotide sequence containing a human and non-human nucleotide sequence; (h) insertion of a conditioning allele flanked by target sequences of site-specific recombinase; (i) insertion of a selectable marker or reporter gene operably linked to a third promoter active in the pluripotent cell; or (j) a combination of both.

[0036] In some methods, the genomic locus of interest comprises (i) a 5 'target sequence that is homologous to the 5' arm of homology; and (ii) a 3 'target sequence that is homologous to the 3' homology arm. Optionally, the 5 'target sequence and the 3' target sequence are separated by at least 5 kb, but less than 3 pb. Optionally, the 5 'target sequence and the 3' target sequence are separated by at least 5 kb but less than 10 kb, at least 10 kb but less than 20 kb kb, at least 20 kb but less than 40 kb, at least 40 kb, but less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb but less than 100 kb, at least 100 kb kbp but less than 150 kbp, or at least 150 kbp but less than 200 kbp, at least about 200 kbp ., but less than about 300 kbp, at least about 300 kbp, but less than about 400 kbp, at least about 400 kbp, but less than about 500 kbp, at least about 500 kbp, but less than about 1 ppm, at least about 1 ppm, but less than about 1.5 ppm, at least about 1.5 ppm, but less than about 2 ppm, at least about 2 ppm, but less than about 2.5 ppm, or at least about 2.5 ppm, but not its less than about 3 m.p. Optionally, the 5 'target sequence and the 3' target sequence are separated by at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kbp, at least 110 kbp, at least 120 kbp, at least 130 kbp, at least 140 kbp bp, at least 150 kbp, at least 160 kbp, at least 170 kbp, at least 180 kbp, by at least 190 kbp or at least 200 kbp. In some methods, the 5 'and 3' target sequences are separated from about 30 kb. up to about 110 kbp. In some methods, the 5 'and 3' target sequences are separated from about 38 kb. up to about 110 kbp.

[0037] In some methods, the genomic locus of interest comprises an interleukin-2 gamma receptor locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or both Rag1 and Rag2. In other methods, the genomic locus of interest comprises the Adamts5 locus, the Trpa1 locus, the Folh1 locus, or the Erbb4 locus. In other methods, the genomic locus of interest contains the Lrp5 locus. In other methods, the genomic locus of interest comprises a C5 (Hc) locus, a Ror1 locus, or a Dpp4 locus.

[0038] Additionally, there is provided a method for producing a non-human animal of the F0 generation, which contains a targeted genetic modification at a genomic locus of interest, comprising: (a) contacting a genome in a non-human ES cell with Cas protein, CRISPR RNA and tracrRNA in the presence of a large targeting vector (LTVEC) for the formation of a modified non-human ES cell, while the LTVEC consists of at least 10 kb. and contains a first nucleic acid flanked by a 5 'homology arm and a 3' homology arm; (b) identifying a modified non-human ES cell containing targeted genetic modification at the genomic locus of interest; (c) introducing a modified non-human ES cell into a non-human host embryo; and (d) bearing a non-human host embryo by a surrogate mother, wherein the surrogate mother produces a non-human F0 generation animal that contains a targeted genetic modification at the genomic locus of interest.

[0039] In some such methods, CRISPR RNA and tracrRNA are introduced together in the form of a single guide RNA (nRNA). In other such methods, CRISPR RNA and tracrRNA are added separately.

[0040] In some such methods (a) the Cas protein is introduced into a non-human ES cell in the form of a protein, messenger RNA (mRNA) encoding the Cas protein, or DNA encoding the Cas protein; (b) CRISPR RNA is introduced into a non-human ES cell in the form of RNA or DNA encoding CRISPR RNA; and (c) tracrRNA is introduced into a non-human ES cell in the form of RNA or DNA encoding tracrRNA.

[0041] In some such methods (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding the Cas protein; (b) the DNA encoding CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding CRISPR RNA; and (c) the DNA encoding tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a fourth nucleic acid encoding tracrRNA, the first, second and third promoters being active in a non-human ES cell. Optionally, the first, second and third expression constructs are on the same nucleic acid molecule.

[0042] In some such methods (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding the Cas protein; a (b) DNA encoding CRISPR RNA and DNA encoding tracrRNA are in the form of a second expression construct containing a second promoter operably linked to a third nucleic acid encoding an nRNA containing CRISPR RNA and tracrRNA; the first and second promoters are active in the non-human ES cell. Optionally, the first and second expression constructs are on the same nucleic acid molecule.

[0043] In some such methods, Cas protein, CRISPR RNA, and tracrRNA are introduced into a non-human ES cell as a protein-RNA complex.

[0044] In some such methods, targeted genetic modification includes the simultaneous removal of an endogenous nucleotide sequence at a genomic locus of interest and insertion of a first nucleic acid at a genomic locus of interest.

[0045] In some such methods, the targeted genetic modification is a biallelic genetic modification. Optionally, the biallelic genetic modification includes the removal of an endogenous nucleotide sequence and the insertion of the first nucleic acid at the genomic locus of interest on two homologous chromosomes.

[0046] In some such methods, the modified non-human ES cell is compound heterozygous at the genomic locus of interest. In some such methods, the modified non-human ES cell is hemizygous at the genomic locus of interest. Optionally, targeted genetic modification at a genomic locus of interest on one chromosome includes deletion of an endogenous nucleotide sequence and insertion of a first nucleic acid. Optionally, targeted genetic modification includes: (1) deletion of an endogenous nucleotide sequence at a genomic locus of interest on two homologous chromosomes; and (2) inserting the first nucleic acid at the genomic locus of interest on the first chromosome and disrupting the genomic locus of interest on the second chromosome. The first chromosome can be one of two homologous chromosomes, and the second chromosome can be the other homologous chromosome.

[0047] In some such methods, the Cas protein is Cas9.

[0048] Additionally, there are provided methods for modifying the genome at a genomic locus of interest in a eukaryotic cell, mouse cell, or human cell, comprising bringing the genome in contact with Cas protein, CRISPR RNA that hybridizes to a target sequence at the genomic locus of interest, and tracrRNA in the presence of a large direction vector (LTVEC), while the LTVEC is at least 10 kb. and contains a first nucleic acid flanked by a 5 'homology arm that is homologous to the 5' target sequence at the genomic locus of interest, and a 3 'homology arm that is homologous to the 3' target sequence at the genomic locus of interest, the first nucleic acid being at least at least 30 kbp. and / or 5 'target sequence and 3' target sequence are separated by at least 30 kb, while after contact with Cas protein, CRISPR RNA and tracrRNA in the presence of LTVEC, the genome is modified so that it contains targeted genetic a modification involving the insertion of the first nucleic acid at the genomic locus of interest.

[0049] Any of the above methods may further comprise introducing Cas protein, CRISPR RNA, tracrRNA, and LTVEC into a eukaryotic cell, mouse cell, or human cell. Any of the above methods may further comprise identifying a modified eukaryotic cell, a modified mouse cell, or a modified human cell containing a targeted genetic modification at the genomic locus of interest.

[0050] In some of the above methods, CRISPR RNA and tracrRNA are introduced together in the form of a single transcript. In some of the above methods, CRISPR RNA and tracrRNA are introduced separately.

[0051] In some of the above methods (a) the Cas protein is introduced into a eukaryotic cell, a mouse cell, or a human cell in the form of a protein, messenger RNA (mRNA) encoding the Cas protein, or DNA encoding the Cas protein; (b) CRISPR RNA is introduced into eukaryotic cell, mouse cell or human cell in the form of RNA or DNA encoding CRISPR RNA; and (c) tracrRNA is introduced into a eukaryotic cell, mouse cell, or human cell in the form of RNA or DNA encoding tracrRNA. In some of the above methods, Cas protein, CRISPR RNA, and tracrRNA are introduced into a eukaryotic cell, mouse cell, or human cell as a protein-RNA complex.

[0052] In some of the above methods (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding the Cas protein; (b) the DNA encoding CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding CRISPR RNA; and (c) the DNA encoding tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a fourth nucleic acid encoding tracrRNA, wherein the first, second and third promoters are active in a eukaryotic cell, mouse cell, or human cell. In some of the above methods, the first, second and third expression constructs are on the same nucleic acid molecule.

[0053] In some of the above methods (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding the Cas protein; a (b) DNA encoding CRISPR RNA and DNA encoding tracrRNA are in the form of a second expression construct containing a second promoter operably linked to a third nucleic acid encoding an nRNA containing CRISPR RNA and tracrRNA; wherein the first and second promoters are active in a eukaryotic cell, a mouse cell, or a human cell. In some of the above methods, the first and second expression constructs are on the same nucleic acid molecule.

[0054] In some of the above methods, the LTVEC is at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb .about., at least 50 kbp, at least 60 kbp, at least 70 kbp, at least 80 kbp. or at least 90 kb. In some of the above methods, the LTVEC is at least 100 kb, at least 150 kb. or at least 200 kbp.

[0055] In some of the above methods, the first nucleic acid is at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb 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 or at least 300 kbp. In some of the above methods, the first nucleic acid is from about 40 kbp. up to about 140 kbp

[0056] In some of the above methods, the 5 'and 3' shoulders of LTVEC homology total from about 10 kbp. up to about 150 kbp In some of the above methods, the 5 'and 3' homology arms of LTVEC total from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to 150 kbp

[0057] In some of the above methods, the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 3 ppm. In some of the above methods, the 5 'target sequence and 3' target sequence are separated by at least 5 kb but less than 10 kb, at least 10 kb but less than 20 kb, at least 20 kb, but less than 40 kb, at least 40 kb, but less than 60 kb. v., at least 60 kb, but less than 80 kb, at least about 80 kb, but less than 100 kb, at least at least 100 kb but less than 150 kb, or at least 150 kb but less than 200 kb, at least about 200 kb bp, but less than about 300 kbp, at least about 300 kbp, but less than about 400 kbp, at least about 400 kbp. o., but less than about 500 kbp, at least about 500 kbp, but less than about 1 ppm, at least about 1 ppm. but less than about 1.5 ppm, at least about 1.5 ppm, but less than about 2 ppm, at least about 2 ppm. o., but less than about 2.5 p.o., or at least about 2.5 mpo, but less than about 3 mpo. In some of the above methods, the 5 'target sequence and 3' target sequence are separated by at least 20 kb, at least 30 kb, at least 40 kb, at least at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb so 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 kbp. or at least 200 kbp. In some of the above methods, the 5 'and 3' target sequences are separated from about 30 kb. up to about 110 kbp.

[0058] In some of the above methods, the eukaryotic cell is not a rat cell. In some of the above methods, the eukaryotic cell is a pluripotent cell, a non-pluripotent cell, a mammalian cell, a human cell, a non-human mammalian cell, a rodent cell, a mouse cell, a hamster cell, a non-human pluripotent cell, a human pluripotent cell, a pluripotent or a pluripotent or a pluripotent human cell. In some of the above methods, the eukaryotic cell is a primary cell or an immortalized cell. In some of the above methods, the rodent pluripotent cell is a mouse or rat embryonic stem (ES) cell.

[0059] In some of the above methods, the mouse cell or human cell is a primary cell or an immortalized cell. In some of the above methods, the mouse cell or human cell is a pluripotent cell. In some of the above methods, the mouse pluripotent cell is a mouse embryonic stem (ES) cell. In some of the above methods, the human pluripotent cell is a human embryonic stem (ES) cell, an adult human stem cell, a developmentally limited human progenitor cell, or a human induced pluripotent stem (iPS) cell. In some of the above methods, human iPS cells are maintained in a medium containing a basic medium and supplements, the medium comprising: (a) a leukemia inhibiting factor (LIF) e polypeptide; (b) a glycogen synthase kinase (GSK3) inhibitor; and (c) a MEK inhibitor; the osmolarity of the medium is from about 175 mOsm / kg to about 280 mOsm / kg.

[0060] In some of the above methods, the Cas protein is Cas9. In some of the above methods, the target sequence is directly flanked by a protospacer adjacent motif (PAM) sequence.

[0061] In some of the above methods, targeted genetic modification includes the simultaneous removal of an endogenous nucleotide sequence at a genomic locus of interest and the insertion of a first nucleic acid at a genomic locus of interest in one step. In some of the above methods, the endogenous nucleotide sequence deleted is from about 30 kb. to about 110 kb, and the inserted first nucleic acid is from about 40 kb. up to about 140 kbp

[0062] In some of the above methods, the targeted genetic modification is a biallelic genetic modification. In some of the above methods, biallelic genetic modification involves the removal of an endogenous nucleotide sequence and the insertion of a first nucleic acid at a genomic locus of interest on two homologous chromosomes. In some of the above methods, a modified eukaryotic cell, a modified mouse cell, or a modified human cell is compound heterozygous at the genomic locus of interest. In some of the above methods, the modified mouse cell or modified human cell is hemizygous at the genomic locus of interest. In some of the foregoing methods, directed genetic modification at a genomic locus of interest on one chromosome comprises removing an endogenous nucleotide sequence and inserting a first nucleic acid. In some of the above methods, targeted genetic modification comprises: (1) removing an endogenous nucleotide sequence at a genomic locus of interest on the first and second homologous chromosomes; and (2) inserting the first nucleic acid at the genomic locus of interest on the first chromosome and disrupting the genomic locus of interest on the second chromosome.

[0063] In some of the above methods, targeted genetic modification comprises: (a) replacing an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) removal of the endogenous nucleotide sequence; (c) deletion of an endogenous nucleotide sequence, the deletion range being from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (d) insertion of an exogenous nucleotide sequence; (e) insertion of an exogenous nucleotide sequence in the range of about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp; (f) insertion of an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (g) insertion of a chimeric nucleotide sequence containing a human and non-human nucleotide sequence; (h) insertion of a conditioning allele flanked by target sequences of site-specific recombinase; (i) insertion of a selectable marker or reporter gene operably linked to a third promoter active in the pluripotent cell; or (j) a combination of both.

[0064] In some of the above methods, the genomic locus of interest comprises an interleukin-2 gamma receptor locus, ApoE locus, Rag1 locus, Rag2 locus, both Rag1 and Rag2 locus, Adamts5 locus, Trpa1 locus, Folh1 locus, Erbb4 locus, Lrp5 locus, locus C5 (Hc), locus Ror1 or locus Dpp4. In some of the above methods, the genomic locus of interest contains extrachromosomal DNA.

[0065] Also provided are methods for producing a non-human F0 generation animal or mouse that contains targeted genetic modification at a genomic locus of interest, comprising: (a) modifying a non-human or murine ES cell by any of the above methods; (b) identifying a modified non-human or murine ES cell containing targeted genetic modification at the genomic locus of interest; (c) introducing a modified non-human or mouse ES cell into a non-human or mouse embryo host; and (d) bearing a non-human or mouse host embryo by a surrogate mother, the surrogate mother producing a non-human animal or mouse of the F0 generation that contains a targeted genetic modification at the genomic locus of interest.

BRIEF DESCRIPTION OF THE GRAPHIC MATERIALS

[0066] Figure 1 illustrates rat ESCs that grow in compact spherical colonies that usually detach and float in the dish.

[0067] Figures 2A-D illustrate various pluripotency markers expressed by rat ESCs: A illustrates Oct-4 (green); B illustrates Sox-2 (red); C illustrates DAPI (blue); D illustrates the layering of pluripotency markers expressed by cESCs.

[0068] Figure 3 illustrates that rat ESCs express low levels of alkaline phosphatase (a marker of pluripotency).

[0069] Figure 4 illustrates the karyotype of line DA.2B, which is 42X, Y. Karyotyping was performed because rat ESCs often become tetrahaploid; therefore, lines were pre-screened by counting metaphase chromosomal preparations, and then lines with most normal results were formally karyotyped.

[0070] Figures 5A-B are photographs illustrating the chromosome number analysis of the rat ES cell line ACI.G1.

[0071] Figures 6A-B are photographs illustrating the chromosome number analysis of the DA.2B rat ES cell line.

[0072] Figures 7A-B are photographs illustrating the chromosome number analysis of the DA.2C rat ES cell line.

[0073] Figure 8 is a close-up of the rat ESCs of Figure 1.

[0074] Figure 9 illustrates the production of chimeras by injection into blastocysts and the transfer of the genome of rat ESCs through the germ line. Chimeras were obtained by injection into blastocysts using parental rat ESCs ACI.G1. Chimeras with a high percentage usually have white muzzles.

[0075] Figure 10 illustrates F1 agouti pups with littermate albino pups derived from the ACI / SD chimera marked with an asterisk (*) in Figure 9.

[0076] Figure 11 is a schematic representation of the rat ApoE locus, and the gray rectangles represent the zinc finger nuclease cutting sites (ZFN1 and ZFN2). The genomic regions corresponding to the 5 'and 3' homology arms (5 kb and 5.4 kb, respectively) are indicated by dark gray rectangles. Exon 1 in the ApoE gene is non-coding and is shown as an unstained rectangle closest to the 5 'arm of homology. The three introns in the ApoE gene are indicated as lines. Exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle.

[0077] Figure 12 illustrates the targeting of the rat Rosa26 lucus, which is located between the Setd5 and Thumpd3 genes, as in the mouse, at regular intervals. Panel A illustrates the structure of the murine Rosa26 locus. Rosa26 murine transcripts consist of 2 or 3 exons. Panel B illustrates the structure of the rat Rosa26 locus; the rat locus contains a second exon 1 (Ex1b) in addition to an exon homologous to murine exon 1 (Ex1a); the third exon was not identified in rats. Panel C illustrates the targeted rat Rosa26 allele; homology arms of 5 kbp. each were cloned by PCR using genomic DNA from DA cESCs; the targeted allele contains a splice acceptor (SA) cassette -lacZ-hUB-neo, replacing a 117 bp deletion. in nitrone of rat Rosa26.

[0078] Figure 13A illustrates a control brain of a 14 week old wild-type rat that was stained with X-gal. For the control brain, a low background LacZ staining was observed (top view).

[0079] Figure 13B illustrates the expression of LacZ in the brain of a heterozygous rRosa26 rat (14 weeks old). The lacZ reporter was expressed throughout the brain of the rRosa26 heterozygote.

[0080] Figure 13C illustrates a control heart and thymus (insert) of a 14 week old wild-type rat that was treated with X-gal. The control heart and thymus showed low background LacZ staining.

[0081] Figure 13D illustrates LacZ expression in the heart and thymus (insert) of a 14 week old heterozygous rRosa26 rat. The lacZ reporter was expressed throughout the heart and thymus of the rROSA26 heterozygote.

[0082] Figure 13E illustrates control lungs of a 14 week old wild-type rat that were treated with X-gal. Control lungs showed low background LacZ staining.

[0083] Figure 13F illustrates the expression of LacZ in the lungs of a 14 week old heterozygous rRosa26 rat. The lacZ reporter was expressed throughout the lung volume of the rRosa26 heterozygote.

[0084] Figures 13G and H illustrate the expression of LacZ in rat E12.5 embryos. In contrast to the wild-type control embryo (H), which showed low background LacZ staining, the heterozygous rRosa26 embryo showed LacZ reporter expression throughout the embryo.

[0085] Figures 13I and J illustrate LacZ expression in E14.5 rat embryos. In contrast to the wild-type control embryo (J), which showed low background LacZ staining, the heterozygous rRosa26 embryo showed LacZ reporter expression throughout the embryo.

[0086] Figure 14 illustrates homologous or non-homologous recombination that occurs within a rat ES cell after electroporation of a targeting vector containing a selection cassette (lacZ-neo cassette).

[0087] Figure 15 illustrates the mechanism by which genome-editing endonucleases (eg, ZFN and TALEN) create a double-stranded break (DSB) in the genomic target sequence and activate non-homologous end-joining (NHSC) in an ES cell.

[0088] Figure 16 illustrates a gene targeting method that uses ZFN / TALEN to improve homologous recombination efficiency of the targeting vector. DSB stands for double-strand break.

[0089] Figure 17 illustrates chimeras ApoE-ZFN-AB5, obtained by the method of obtaining chimeras and transmission of the germline of the modified locus of the rat ApoE. Targeted modification was carried out using zinc finger nucleases.

[0090] Figure 18 is a schematic illustration of targeting IL2r-γ in combination with zinc finger nucleases that target ZFN U and ZFN D. Shows a region of the rat IL2r-γ locus that is targeted by ZFN U and ZFN D (SEQ ID No. 93). The figure shows the cutting areas ZFN.

[0091] Figure 19 is a schematic representation of targeting IL2r-γ in combination with zinc finger nucleases that target ZFN U and ZFN D, or in combination with nRNA (nRNA1, nRNA2, nRNA3, nRNA4). Areas of the rat IL2r-γ locus that are targeted by ZFN U and ZFN D or nRNA1-4 are shown, and ZFN cut sites are indicated.

[0092] Figure 20 is a schematic representation of a rat ApoE locus and a targeting plasmid. The upper diagram shows the genomic structure of the rat ApoE locus and the genomic loci corresponding to the 5 'and 3' homology arms (5 kb and 5.4 kb, respectively; dark gray rectangles). Exon 1 in the ApoE gene is non-coding and is shown as an unstained rectangle closest to the 5 'homology arm. The three introns in the ApoE gene are indicated as lines. Exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle. The bottom panel illustrates the targeting plasmid. The 5 'and 3' homology arms (5 kb and 5.4 kb, respectively) are indicated by dark gray rectangles. The targeting vector contains a reporter gene (lacZ) and a self-destructive cassette flanked by loxP regions (unstained arrows). The self-destructive cassette contains a murine Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing a human ubiquitin promoter operably linked to a neomycin resistance gene.

[0093] Figure 21A is a schematic representation of targeting the ApoE locus in rat ES cells using zinc finger nucleases and a targeting vector containing a reporter gene (LacZ) and a self-destructive cassette containing the murine Prm1 promoter operably linked to the Crei gene and a selection cassette drug susceptibility containing a human ubiquitin promoter operably linked to a neomycin resistance gene. Figure 21B illustrates a homozygous targeted ApoE locus.

[0094] Figure 22 is a schematic representation of the rat ApoE locus and large targeting vector (LTVEC). The top panel shows the genomic structure of the rat ApoE locus and the genomic regions corresponding to the 5 'and 3' homology arms (45 kb and 23 kb, respectively; dark gray rectangles). Exon 1 ApoE is non-coding and is shown as an unstained rectangle closest to the 5 'homology arm. The three nitrons in the ApoE gene are indicated as lines, and exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle. The bottom panel shows LTVEC for modification of the rat ApoE locus. The 5 'and 3' homology arms (45 kb and 23 kb, respectively) are indicated by dark gray rectangles. LTVEC contains a reporter gene (lacZ) and a self-destructive cassette flanked by loxP regions (open arrows) that contains the murine Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter operably linked to the resistance gene to neomycin.

[0095] Figure 23 is a schematic representation of the rat APOE locus and the gray rectangles represent the cut sites with zinc finger nucleases (ZFN1 and ZFN2) used in conjunction with a large targeting vector (LTVEC) to enhance homologous recombination between the targeting vector and the target cognate region of the chromosome ...

[0096] Figure 24 illustrates the rat IL2r-γ locus in which a 3.2 kb deletion has been made. and a reporter gene insert (uZPB) and a self-destructive cassette containing a drug susceptibility selection cassette (hUb-neo) and a Crei gene operably linked to the murine Prm1 promoter were introduced.

[0097] Figure 25 is another image of the rat IL2r-γ locus in which a 3.2 kb deletion has been made. and a reporter gene insert (uZPB) and a self-destructive cassette containing the Crei gene operably linked to the murine Prm1 promoter and a drug susceptibility selection cassette (hUb-Neo) were introduced.

[0098] Figure 26 is a schematic representation of the rat Rag2 locus and large targeting vector (LTVEC) for modifying the rat Rag2 locus. The top panel shows the genomic structure of the rat Rag2 locus and cognate genomic regions corresponding to the 5 'and 3' homology arms (48 kb and 84 kb, respectively; dark gray rectangles). Rag2 contains one exon, indicated by dashed gray shading. The bottom panel is LTVEC. The 5 'and 3' homology arms (48 kb and 84 kb, respectively) are indicated by dark gray rectangles. LTVEC contains a reporter gene (lacZ) and a self-destructive cassette flanked by loxP regions (open arrows) that contains the rat Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter operably linked to the resistance gene to neomycin.

[0099] Figure 27 shows the genomic structure of the rat Rag1 / Rag2 locus and genomic regions removed by targeting Rag2 (Rag2 deletion) or dual targeting Rag2 / Rag1 (Rag2 / Rag1 deletion).

[00100] Figure 28 is a schematic representation of the rat Rag2 and Rag1 loci and the large targeting vector (LTVEC) used to modify the loci. The top panel shows the genomic structure of the Rag1 and Rag2 loci and the cognate genomic regions corresponding to the 5 'and 3' homology arms (48 kb and 15 kb, respectively; dark gray rectangles). Each of Rag2 and Rag] contains one exon, indicated by gray dotted shading. The bottom panel is LTVEC. The 5 'and 3' homology arms (48 kb and 15 kb, respectively) are indicated by dark gray rectangles. LTVEC contains a reporter gene (lacZ) and a self-destructive cassette flanked by loxP regions (open arrows) that contains the rat Prm 1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter operably linked to the gene neomycin resistance.

[00101] Figure 29 illustrates flow cytometric analysis for PBS expression and for the CD3 T cell marker (Panels A and D), the B220 B cell marker (Panels B and E), and the CD161a NK cell marker (Panels C and F ) in peripheral blood mononuclear cells (PBMCs) obtained from chimeric II2rg- / y rats (panels A-C) and DA DT rats (panels DF). Double positive cells are shown in the R8 quadrant. Figure 29 illustrates that II2rg- / y PBMCs do not express mature lymphocyte markers.

[00102] Figure 30 illustrates that PBS-positive lymphocytes were detected in the peripheral blood of 2 of 3 chimeras II2rg- / y.

[00103] Figure 31 is a schematic representation of the rat Il2rg locus and a targeting plasmid for complete humanization of the rat Il2rg locus. The top panel shows the genomic structure of the rat Il2rg locus and cognate genomic regions corresponding to the 5 'and 3' homology arms (4.3 kb and 4.0 kb, respectively; dark gray rectangles) ... The bottom panel is the targeting plasmid. The 5 'and 3' homology arms (4.3 kb and 4.0 kb, respectively) are indicated by dark gray rectangles. The targeting plasmid contains the human IL-2rg genomic region and a deletion cassette flanked by loxP regions (uncolored arrows) that contains a drug susceptibility selection cassette containing a human ubiquitin promoter operably linked to a neomycin resistance gene.

[00104] Figure 32 is a schematic representation of the rat Il2rg locus and a targeting plasmid for humanizing the ectodomain of the rat Il2rg locus. The top panel shows the genomic structure of the rat Il2rg locus and cognate genomic regions corresponding to the 5 'and 3' homology arms (4.3 kb and 4.0 kb, respectively; dark gray rectangles) ... The bottom panel is the targeting plasmid. The 5 'and 3' homology arms (4.3 kb and 4.0 kb, respectively) are indicated by dark gray rectangles. The targeting plasmid contains the human ectodomain genomic region of IL-2rg and a self-destructive cassette flanked by loxP regions (unstained arrows) that contains the rat Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter functionally with a neomycin resistance gene.

[00105] Figure 33 shows the sequence alignment of the human IL-2rg protein (SEQ ID # 20; NP 000197.1); rat IL-2rg protein (SEQ ID No. 21; NP 543165.1); and a chimeric IL-2rg protein (SEQ ID NO: 22) containing the human IL-2rg ectodomain fused to the rat IL-2rg protein residue. The junction of human and rat IL-2rg is indicated by a vertical line.

[00106] Figure 34 is a schematic representation of CRISPR / Cas9 humanization of the murine Lrp5 gene; LTVEC is shown in the top panel and the mouse Lrp5 locus is shown in the bottom panel. The humanized area is an ectodomain. Arrows indicate target sites for each nRNA (gA, gB, gB2, gC, gD, gE2, gE, gF) and ZFN (a-d).

[00107] Figure 35 illustrates the percent targeting efficiency of LTVECs targeting genes with increasing deletion size (Figure 35A), and LTVECs with increasing size human gene inserts (Figure 35B). LTVECs were used alone (gray squares or triangles) or in combination with ZFN (black squares or triangles).

[00108] Figure 36 is a schematic representation of CRISPR / Cas9 humanization of the entire coding region of the murine Trpa1 gene; LTVEC is shown in the top panel and the mouse Trpa1 locus is shown in the bottom panel. Arrows indicate target sites for each nRNA (gA, gA2, gB, gC, gD, gE2, gE, gF).

[00109] Figure 37 is a schematic representation of CRISPR / Cas9 humanization of the ectodomain (exon 2 to stop codon) of the mouse Folh1 gene; LTVEC is shown in the top panel and the mouse Folh1 locus is shown in the bottom panel. Arrows indicate target sites for each nRNA (gA, gA2, gB, gC, gD, gE, gE2, gF).

[00110] Figure 38 is a schematic representation of CRISPR / Cas9 humanization of the region from exon 2 to the stop codon of the mouse C5 gene (Hc); LTVEC is shown in the top panel and the mouse C5 (Hc) locus is shown in the bottom panel. Arrows indicate target sites for each nRNA (gA, gB, gB2, gC, gD, gE2, gE, gF).

[00111] Figure 39 is a schematic representation of CRISPR / Cas9 humanization of the entire coding region of the murine Adamts5 gene; LTVEC is shown in the top panel and the mouse Adamts5 locus is shown in the bottom panel. Arrows indicate target sites for each nRNA (gA, gA2, gB, gC, gD, gE2, gE, gF).

[00112] Figure 40 is a schematic representation of CRISPR / Cas9 humanization of exons 4-15 of the mouse Erbb4 gene; LTVEC is shown in the top panel and the mouse Erb4 locus is shown in the bottom panel. Arrows indicate target sites for each nRNA (gA, gB, gB2, gC, gD, gE2, gE, gF).

[00113] Figure 41 is a schematic representation of CRISPR / Cas9 humanization of exons 2-7 of the murine Ror1 gene; LTVEC is shown in the top panel and the mouse Ror1 locus is shown in the bottom panel. Arrows indicate target sites for each nRNA (gA, gB, gC, gD, gE, gF).

[00114] Figure 42 is a schematic representation of a CRISPR / Cas9 humanization region from exon 2 to the stop codon of the murine Dpp4 gene; LTVEC is shown in the top panel and the mouse Dpp4 locus is shown in the bottom panel. Arrows indicate target sites for each nRNA (gA, gB, gB2, gC, gD, gE2, gE, gF).

[00115] Figure 43 illustrates X-gal stained brains of 12 month old female rats. Figures 43A-C illustrate the brain of a wild-type rat, and Figures 43D-F illustrate the brain of the rat ApoE ^+/- . Figures 43A and D illustrate a top view, Figures 43B and E illustrate a bottom view, and Figures 43C and F illustrate a close-up view.

[00116] Figure 44 illustrates the heart of 12 month old female rats (A and C) and the corresponding blood vessels in close-up (B and D), stained with X-gal. Figures 44A and B illustrate the heart and blood vessels, respectively, obtained from a wild-type rat, and Figures 44C and D illustrate the heart and blood vessels, respectively, obtained from an ApoE ^+/− rat. Staining was observed in the heart cavity and some vessels (eg, vena cava).

[00117] Figure 45 illustrates the liver of 12 month old female rats stained with X-gal. Figures 45A and B illustrate the liver obtained from a wild-type rat and Figures 45C and D illustrate the liver of an ApoE ^+/- rat. Figures 45B and D show a close-up view of the liver.

[00118] Figure 46 illustrates the determination of cholesterol, LDL, HDL and triglyceride levels (Figures 46A-D, respectively) in homozygous ApoE-targeted rats, heterozygous ApoE-targeted rats and wild-type rats at 6 weeks of age, 9 weeks, 12 weeks, and 15 weeks.

[00119] Figure 47 is a schematic representation of the rat ApoE locus (top panel) and a large targeting vector (LTVEC) that targets the rat ApoE locus (bottom panel). The top panel shows the genomic structure of the rat ApoE locus and the genomic regions corresponding to the 5 'and 3' homology arms (45 kb and 23 kb, respectively; dark gray rectangles). Exon 1 ApoE is non-coding and is shown as an unstained rectangle closest to the 5 'homology arm. The three introns in the ApoE gene are indicated as lines, and exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle. Target regions for nRNA2 (SEQ ID NO. 87) and nRNA3 (SEQ ID NO. 88) ApoE are indicated. The bottom panel shows LTVEC for modification of the rat ApoE locus. The 5 'and 3' homology arms (45 kb and 23 kb, respectively) are indicated by dark gray rectangles. LTVEC contains a reporter gene (lacZ) and a self-destructive cassette flanked by loxP regions (open arrows) that contains the murine Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter operably linked to the resistance gene to neomycin.

[00120] Figure 48 is a schematic representation of the rat Rag2 locus (top panel) and a large targeting vector (LTVEC) that targets the rat Rag2 locus (bottom panel). The top panel shows the genomic structure of the rat Rag2 locus and cognate genomic regions corresponding to the 5 'and 3' homology arms (48 kb and 84 kb, respectively; dark gray rectangles). Rag2 contains one exon, indicated by dashed gray shading. Rag2 contains one exon, indicated by the gray dotted line. Target regions are indicated for nRNA1 (SEQ ID No. 89) and nRNA4 (SEQ ID No. 90) Rag2. The bottom panel is LTVEC. The 5 'and 3' homology arms (48 kb and 84 kb, respectively) are indicated by dark gray rectangles. LTVEC contains a reporter gene (lacZ) and a self-destructive cassette flanked by loxP regions (open arrows) that contains the rat Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter operably linked to the resistance gene to neomycin.

[00121] Figure 49 is a schematic representation of the rat Il2rg locus (upper panel) and a targeting plasmid for humanizing the ectodomain of the rat Il2rg locus (lower panel). The top panel shows the genomic structure of the rat Il2rg locus and cognate genomic regions corresponding to the 5 'and 3' homology arms (4.3 kb and 4.0 kb, respectively; dark gray rectangles) ... Target regions for nRNA2 (SEQ ID NO. 891) and nRNA4 (SEQ ID NO. 92) Il2rg are indicated. The bottom panel is the targeting plasmid. The 5 'and 3' homology arms (4.3 kb and 4.0 kb, respectively) are indicated by dark gray rectangles. The targeting plasmid contains the human ectodomain genomic region of IL-2rg and a self-destructive cassette flanked by loxP regions (unstained arrows) that contains the rat Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter functionally with a neomycin resistance gene.

[00122] Figure 50 is a schematic representation of the rat Rag2 and Rag1 loci and a large targeting vector (LTVEC) used to modify loci in Il2rg-targeted rat ES cells (clone Il2rg-CG12). The top panel shows the genomic structure of the Rag1 and Rag2 loci and the cognate genomic regions corresponding to the 5 'and 3' arms of homology (48 kb and 15 kb, respectively; gray rectangles). Each of Rag2 and Rag1 contains one exon, indicated by unstained arrows. The bottom panel is LTVEC. The 5 'and 3' homology arms (48 kb and 15 kb, respectively) are indicated by gray rectangles. LTVEC contains a reporter gene (uPPB) and a puromycin resistance gene separated by an internal ribosome entry site (IRES) and functionally linked to the actin promoter. The LTVEC further comprises a self-destructive cassette flanked by loxP regions (unstained arrows) that contains the rat Prm1 promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing the human ubiquitin promoter operably linked to the neomycin resistance gene.

[00123] Figure 51 illustrates a diagram of the replacement of a portion of the human ADAM6 locus with nucleic acid containing the murine Adam6a and Adam6b loci using LTVEC and guide RNA in human iPS cells. The target site for the guide RNA is indicated by an arrow.

[00124] Figure 52A illustrates the morphology observed for human iPS cells cultured for 8 days in medium 2i. Figure 52B illustrates the morphology observed for human iPS cells cultured for 12 days in medium 2i.

[00125] Figures 53A-53D illustrate the morphology observed for human iPS cells cultured in mTeSR ™ -hLIF medium or low osmolarity VG2i medium for 6 days. Figures 53A and 53B illustrate the morphology observed for human iPS cells cultured in mTeSR ™ -hLIF medium (Figure 53A) or VG2i medium (Figure 53B) for 6 days. Figures 53C and 53D illustrate the morphology observed for human iPS cells cultured on Newborn Human Foreskin Fibroblast Feeding Cells (NuFF) in mTeSR ™ -hLIF medium (Figure 53C) or VG2i medium (Figure 53D) for 6 days.

[00126] Figure 54A illustrates reprogrammed human iPS cells cultured in VG2i medium that have been stained for alkaline phosphatase. Figures 54B and 54C illustrate reprogrammed human iPS cells cultured in VG2i medium that have been immunostained for NANOG expression.

[00127] Figures 55A-55C illustrate enzymatic dissociation and subculture of reprogrammed human iPS cells cultured in VG2i medium. Figure 55A illustrates reprogrammed human iPS cells cultured in VG2i medium prior to enzymatic trypsin dissociation in the absence of a ROCK inhibitor. Figure 55B illustrates human iPS cells cultured in VG2i medium for 1 day after subculture. Figure 55C illustrates human iPS cells cultured in VG2i medium for 4 days after subculture.

DETAILED DESCRIPTION OF THE INVENTION

[00128] Compositions and methods are provided for modifying a genomic locus of interest from a rat, eukaryotic organism, non-rat eukaryotic organism, non-human mammal, human, non-rat rodent, mouse or hamster by bacterial homologous recombination (BHR) in a prokaryotic cell. Also provided are compositions and methods for genetically modifying a genomic locus of interest, for example, a genomic locus of interest, a rat, a eukaryotic organism, a non-rat eukaryotic organism, a mammal, a non-human mammal, a human, a rodent, a non-rat rodent, or a mouse, using a large targeting vector (LTVEC) in combination with endonucleases. Also provided are compositions and methods for producing a genetically modified non-human animal, eg, a rat, a mouse, a non-rat rodent, containing one or more targeted genetic modifications. Also provided are isolated human and non-human totipotent or pluripotent stem cells, in particular rat embryonic stem cells, which are capable of maintaining pluripotency after one or more serial genetic modifications in vitro, and which are capable of transmitting targeted genetic modifications to subsequent generations through the germ line.

Glossary

[00129] As used herein, the term "embryonic stem cell" or "ES cell" includes an embryo-derived totipotent or pluripotent cell that is capable of developing into any tissue of the developing embryo after being introduced into the embryo. As used herein, the term "pluripotent cell" includes an undifferentiated cell that has the ability to develop into more than one type of differentiated cell. The term "non-pluripotent cell" includes cells that are not pluripotent cells.

[00130] As used herein, the term "homologous nucleic acid" includes a nucleotide sequence that is identical or substantially similar to a known control sequence. In one embodiment, the term "homologous nucleic acid" is used to characterize a sequence having an amino acid sequence that is 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 even 100% identical with a known control sequence.

[00131] As used herein, the term "orthologous nucleic acid" includes a nucleotide sequence of one species that is functionally equivalent to a known control sequence of another species.

[00132] As used herein, the term "large targeting vector" or "LTVEC" (from the English. "Large targeting vector") includes large targeting vectors for eukaryotic cells that are obtained from fragments of cloned genomic DNA, larger than those commonly used in other approaches to implement homologous targeting of genes in eukaryotic cells. Examples of LTVECs include, but are not limited to, bacterial artificial chromosome (IIR) and yeast artificial chromosome (DIR).

[00133] As used herein, the term "allele modification" (MOA) includes the modification of a specific DNA sequence from one allele of a gene (s) or chromosomal locus (s) in a genome. As described herein, examples of "allele modification (MOA)" include, but are not limited to, deletions, substitutions, or insertions of at least one nucleotide, or deletions of many thousands of base pairs constituting the gene (s) or chromosomal locus (s) of interest, and also any and all possible variations between these two extremes.

[00134] As used herein, the term "recombination site" includes a nucleotide sequence that is recognized by a site-specific recombinase and that can serve as a substrate for the act of recombination.

[00135] "Serial" genetic modifications include two or more modifications independently performed on a cell (e.g., eukaryotic cell, non-rat eukaryotic cell, mammalian cell, human cell, non-human mammalian cell, pluripotent cell, non-pluripotent cell, non-human, pluripotent cell human pluripotent cell, human ES cell, human adult stem cell, human restricted progenitor cell, human iPS cell, human cell, rodent cell, non-rat cell, rat cell, mouse cell, hamster cell, fibroblast or ovarian cell Chinese hamster (CHO)). The first modification can be carried out by electroporation or any other method known in the art. A second modification of the same cellular genome is then carried out using a suitable second nucleotide construct. The second modification can be accomplished by a second electroporation or any other method known in the art. In various embodiments of the invention, after the first and second genetic modifications of the same cell, the third, fourth, fifth, sixth, etc. serial genetic modifications (one after the other) can be carried out using, for example, serial electroporation or any other suitable method known in the art (serial).

[00136] As used herein, the term "site-specific recombinase" includes a group of enzymes that can facilitate recombination between "recombination sites" when two recombination sites are physically separated within the same nucleic acid molecule or are on separate nucleic acid molecules. Examples of "site-specific recombinases" include, but are not limited to, Cre, Flp, and Dre recombinases.

[00137] The term "germline" in relation to a nucleotide sequence includes a nucleotide sequence that can be passed on to offspring.

[00138] The expression "heavy chain" or "heavy chain of an immunoglobulin" includes an immunoglobulin heavy chain sequence, including an immunoglobulin heavy chain constant region sequence, from any organism. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise indicated. Heavy chain fragments include CDRs, CDRs, and FRs, and combinations thereof. Typical heavy chain comprises the following elements: variable domain (from N-terminus to C-terminus) domain C _H 1, hinge, C _H 2 domain and C _H 3 domain is functional fragment comprises a heavy chain fragment, which is capable of specifically recognizing an epitope (for example, recognize an epitope with K _D in the micromolar, nanomolar or picomolar range) that is capable of being expressed and secreted from the cell and that contains at least one CDR. The heavy chain variable domains are encoded by the variable region nucleotide sequence, which generally contains V _H , D _H and J _H segments derived from a set of V _H , D _H and J _H segments present in the germline. The sequences, location and nomenclature for the V, D and J heavy chain segments for various organisms can be found in the IMGT database, which is accessible via the Internet at (www) with the URL "imgt.org".

[00139] The expression "light chain" includes an immunoglobulin light chain sequence of any organism and, unless otherwise indicated, includes human kappa (κ) and lambda (λ) light chains and VpreB, as well as surrogate light chains. Light chain variable domains typically include three light chain CDRs and four framework regions (FRs), unless otherwise indicated. In general, a full-length light chain comprises, from the amino terminus to the carboxy terminus, a variable domain that contains FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the amino acid sequence of the constant region of the light chain. Light chain variable domains are encoded by the light chain variable region nucleotide sequence, which generally contains light chain V _L and J _L gene segments derived from a set of germline V and J light chain gene segments. The sequences, location and nomenclature for the V and J light chain gene segments for various organisms can be found in the IMGT database, which is available online at (www) with the URL "imgt.org". Light chains include those that, for example, do not selectively bind to the first or second epitope that is selectively bound by the epitope-binding protein in which they are located. Light chains also include those that bind and recognize or help the heavy chain to bind and recognize one or more epitopes that are selectively bound by the epitope-binding protein in which they are located.

[00140] The expression "operatively linked" represents the relationship in which the functionally linked components function as intended. In one case, the nucleotide sequence encoding the protein can be operably linked to regulatory sequences (eg, promoter, enhancer, silencer sequence, etc.) so as to maintain proper transcriptional regulation. In one instance, an immunoglobulin variable region nucleotide sequence (or V (D) J segments) may be operably linked to an immunoglobulin constant region nucleotide sequence so as to allow proper recombination between sequences to form an immunoglobulin heavy or light chain sequence.

1. Target locus containing nucleic acid

[00141] Various methods and compositions have been proposed that allow for the integration of at least one nucleotide insert into a target locus. As used herein, a "genomic locus of interest" comprises any segment or region of DNA within a genome into which a nucleotide insert is to be integrated. The terms "genomic locus of interest" and "genomic target of interest" may be used interchangeably. The genomic locus of interest may be native to the cell or, alternatively, may contain a heterologous or exogenous DNA segment that has been integrated into the genome of the cell. Such heterologous or exogenous DNA segments may include transgenes, expression cassettes, polynucleotide encoding selectable markers, or heterologous or exogenous regions of genomic DNA. The term "locus" is defined herein as a segment of DNA within genomic DNA. Genetic modifications described herein may include one or more deletions from a locus of interest, additions to a locus of interest, substitution of a locus of interest, and / or any combination thereof. The locus of interest may contain coding regions or non-coding regulatory regions.

[00142] The genomic locus of interest may further comprise any component of a targeted integration system, including, for example, a recognition site, a selectable marker, a previously integrated nucleotide insert, polynucleotides encoding nuclease agents, promoters, etc. Alternatively, the genomic locus of interest may be located in extrachromosomal DNA within a cell, such as a yeast artificial chromosome (DIR), a bacterial artificial chromosome (IIR), a human artificial chromosome, or any other engineered genomic region found in a suitable host cell. In various embodiments, the target locus may comprise a native, heterologous, or exogenous nucleotide sequence of a prokaryote, eukaryote, non-rat eukaryote, yeast, bacteria, non-human mammal, non-human cell, rodent, non-rat rodent, human, rat, mouse, a hamster, rabbit, pig, bull, deer, sheep, goat, chicken, cat, dog, ferret, primate (eg, marmoset, rhesus monkey), domestic mammal or agricultural mammal, or any other organism of interest, or combinations thereof. In some embodiments, the genomic locus of interest comprises a nucleotide sequence from a human, a mouse, or a combination thereof.

[00143] In certain embodiments, the target locus is derived from, for example, eukaryotic cell, non-rat eukaryotic cell, mammalian cell, human cell, non-human mammalian cell, pluripotent cell, non-pluripotent cell, non-human pluripotent cell, human pluripotent cell, human ES cell, human adult stem cell, human restricted progenitor cell, human iPS cell, human cell, rodent cell, non-rat rodent cell, rat cell, mouse cell, hamster cell, fibroblast or CHO cell.

[00144] In certain embodiments, the genomic locus of interest comprises a target rat nucleic acid locus. This region contains rat nucleic acid, which is integrated into the genome of the cell. Non-limiting examples of target loci include a genomic locus that encodes a protein expressed in a B cell, a genomic locus that expresses a polypeptide in an immature B cell, a genomic locus that expresses a polypeptide in a mature B cell, immunoglobulin (Ig) loci or receptor loci T cells, including, for example, the alpha T cell receptor locus. Additional examples of target genomic loci include Fcer1a locus, Tlr4 locus, Prlr locus, Notch4 locus, Accn2 locus, Adamts5 locus, Trpa1 locus, Folh1 locus, Lrp5 locus, IL2 receptor locus including, for example, IL2 gamma receptor locus (Il2rg), locus ApoE, Rag1 locus, Rag2 locus, Rag1 / Rag2 locus and Erbb4 locus. Any such target locus may be from a rat, or may be from a eukaryotic cell, a non-rat eukaryotic cell, a mammalian cell, a human cell, or a non-human mammalian cell.

[00145] In one embodiment, the target locus encodes the amino acid sequence of the heavy chain variable region of a mammalian immunoglobulin. In one embodiment, the target locus encodes the amino acid sequence of a rat immunoglobulin heavy chain variable region. In one embodiment of the invention, the target locus comprises a genomic DNA sequence containing an unrearranged nucleotide sequence of the variable region of the heavy chain of a rat, mouse, or human immunoglobulin operably linked to the nucleotide sequence of the constant region of the heavy chain of an immunoglobulin. In one embodiment, the immunoglobulin heavy chain constant region nucleotide sequence is a rat, mouse, or human immunoglobulin heavy chain constant region nucleotide sequence selected from CH1, hinge, CH2, CH3, and combinations thereof. In one embodiment, the heavy chain constant region nucleotide sequence comprises a CH1 hinge region-CH2-CH3. In one embodiment, the target locus comprises a rearranged rat, mouse, or human immunoglobulin heavy chain variable region nucleotide sequence operably linked to the immunoglobulin heavy chain constant region nucleotide sequence. In one embodiment, the immunoglobulin heavy chain constant region nucleotide sequence is a rat, mouse, or human immunoglobulin heavy chain constant region nucleotide sequence selected from CH1, hinge, CH2, CH3, and combinations thereof. In one embodiment, the heavy chain constant region nucleotide sequence comprises a CH1 hinge region-CH2-CH3.

[00146] In one embodiment, the target locus comprises a genomic DNA sequence that encodes the amino acid sequence of the variable region of the light chain of a mammalian immunoglobulin. In one embodiment of the invention, the genomic DNA sequence comprises a non-rearranged nucleotide sequence of the λ and / or κ light chain variable region of a mammal.

[00147] In one embodiment, the genomic DNA sequence comprises a rearranged nucleotide sequence of the λ and / or κ light chain variable region of a mammal. In one embodiment, the rearranged λ or κ light chain variable region nucleotide sequence is operably linked to a mammalian immunoglobulin light chain constant region nucleotide sequence selected from a λ light chain constant region nucleotide sequence and a κ light chain constant region nucleotide sequence. In one embodiment, the nucleotide sequence of a mammalian immunoglobulin light chain constant region is the nucleotide sequence of a rat immunoglobulin light chain constant region. In one embodiment, the mammalian immunoglobulin light chain constant region nucleotide sequence is the mouse immunoglobulin light chain constant region nucleotide sequence. In one embodiment, the nucleotide sequence of a mammalian immunoglobulin light chain constant region is the nucleotide sequence of a human immunoglobulin light chain constant region.

[00148] In the context of this document, the ApoE locus, the interleukin-2 gamma receptor locus (Il2rg), the Rag2 locus, the Rag1 locus and / or the Rag2 / Rag1 locus contains the corresponding regions of the genome (i.e., the mammalian genome, human genome or genome other than human mammal) in which each of these genes or gene combinations is located. Modification of any of the ApoE locus, interleukin-2 gamma receptor locus (Il2rg), Rag2 locus, Rag1 locus and / or Rag2 / Rag1 locus (i.e., ApoE locus, interleukin-2 gamma receptor locus, Rag2 locus, Rag1 locus and / or a combined mammalian, human, or non-human mammalian Rag2 / Rag1 locus) may include any desired change at a given locus. Non-limiting examples of modifications at a given locus (ie, mammalian, human, or non-human mammalian locus) are discussed in more detail below.

[00149] For example, in certain embodiments, one or more of the ApoE locus, the interleukin-2 gamma receptor locus (Il2rg), the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1 locus (the ApoE locus of a mammal, human or non-human mammal , locus of interleukin-2 gamma receptor of mammalian, human or non-human mammal, locus of Rag2 of mammalian, human or non-human mammal and / or locus of Rag2 / Rag1) is modified so that the activity and / or level of encoded ApoE protein or interleukin receptor protein -2 gamma, or Rag1 protein, or Rag2 protein, or a combination of Rag1 and Rag2 proteins decreased. In other embodiments, the ApoE protein, interleukin-2 gamma receptor protein, Rag1 protein or Rag2 protein, or a combination of Rag1 and Rag2 proteins is absent.

[00150] By "decreasing" is meant any decrease in the level or activity of a gene / protein encoded at the locus of interest. For example, a decrease in activity can include (1) a statistically significant decrease in the total level or activity of a given protein (i.e., ApoE, interleukin-2 gamma receptor, Rag2, Rag2, or a combination of Rag1 and Rag2), including, for example, a decrease in the level or activity by 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120% or more compared to the corresponding control ... Methods for assaying the decrease in concentration and / or activity of any of ApoE, interleukin-2 gamma receptor, Rag1 and Rag2 are known in the art.

[00151] In other embodiments, one or more of a mammalian, human or non-human mammalian ApoE locus, a mammalian, human or non-human mammalian interleukin-2 gamma receptor locus, a mammalian, human or non-human mammalian Rag2 locus, a locus Mammalian, human or non-human mammalian Rag1 and / or the mammalian, human or non-human mammalian Rag2 / Rag1 locus contain a modification that results in the activity and / or level of the encoded ApoE polypeptide, interleukin-2 gamma receptor polypeptide, polypeptide Rag2, Rag1 polypeptide or Rag1 polypeptide and Rag2 is increased. By "increasing" is meant any increase in the level or activity of a gene / protein encoded at a locus of interest. For example, an increase in activity may include (1) a statistically significant increase in the overall level or activity of a given protein (i.e., ApoE, interleukin-2 gamma receptor, Rag1, Rag2, or Rag1 and Rag2), including, for example, increasing the level or activity by 0 , 5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120% or more compared to the corresponding control. Methods for assaying the increase in concentration and / or activity of any of ApoE, Rag1, Rag2, and the interleukin-2 gamma receptor are known in the art.

[00152] Genetic modification of the mammalian, human or non-human mammalian ApoE locus, the mammalian, human or non-human mammalian interleukin-2 gamma receptor locus, the mammalian, human or non-human mammalian Rag2 locus, the mammalian, human or non-human Rag1 locus a human mammalian and / or a mammalian Rag2 / Rag1 locus, a human, or a non-human mammal may comprise deletion of an endogenous nucleotide sequence at a genomic locus, insertion of an exogenous nucleic acid at a genomic locus, or a combination thereof. The deletion and / or insertion can be anywhere on a given locus, as discussed elsewhere in this document.

[00153] Additional embodiments provided herein include modifying one or more of the ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1 locus of a mammal, human, or non-human mammal by replacing part locus ApoE, locus of receptor interleukin-2 gamma (Il2rg), locus Rag2, locus Rag1 and / or locus Rag2 / Rag1 corresponding homologous or orthologous part of locus ApoE, locus of receptor interleukin-2 gamma, locus Rag2, locus Rag1 and / or locus Rag2 / Rag1 of another organism.

[00154] In other embodiments, modification of one or more of the ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1 locus is performed by replacing part of the ApoE locus, the interleukin-2 gamma receptor locus (Il2rg ), the Rag2 locus, the Rag1 locus and / or the Rag2 / Rag1 locus, a polynucleotide insert having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% similarity along its entire length , 96%, 97%, 98%, 99% with a portion of the ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1 locus that it replaces.

[00155] A given polynucleotide insert and / or corresponding deletion region of a locus can be a coding region, an intron, an exon, an untranslated region, a regulatory region, a promoter or enhancer, or any combination or portion thereof. In addition, the desired polynucleotide insert and / or deletion region of the locus may, for example, be of any desired length, including, for example, 10-100 nucleotides in length, 100-500 nucleotides in length, 500-1 kbp. nucleotides in length, from 1 kb. up to 1.5 kbp nucleotides in length, from 1.5 kb. up to 2 kb nucleotides in length, from 2 kb. up to 2.5 kbp nucleotides in length, from 2.5 kb. up to 3 kbp nucleotides in length, from 3 kbp. up to 5 kbp nucleotides in length, from 5 kbp. up to 8 so. nucleotides in length, from 8 kbp. up to 10 kbp nucleotides in length or more. In other cases, the size of the insert or replacement is from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. to about 350 kbp, from about 350 kbp. to about 400 kbp, from about 400 kbp. to about 800 kbp, from about 800 kbp. up to 1 m.p., from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 mpo, from about 2.5 mpo. up to about 2.8 ppm, from about 2.8 ppm. up to about 3 m.p. In other embodiments, the desired polynucleotide insert and / or deletion region of the locus is at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides, or at least 1 kb, 2 kb kbp, 3 kbp, 4 kbp, 5 kbp, 6 kbp, 7 kbp, 8 kbp kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb ., 15 kbp, 16 kbp. or more. In other embodiments of the invention, the desired polynucleotide insert and / or deletion region of the locus is at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb b.p., at least 50 kbp, at least 60 kbp, at least 70 kbp, at least 80 kbp, at least 90 tbp kbp, at least 100 kbp, at least 150 kbp, at least 200 kbp, at least 250 kbp. or at least 300 kb. or more.

[00156] A given polynucleotide insert can be from any organism, including, for example, a non-rat rodent, rat, mouse, hamster, non-human mammal, eukaryote, non-rat eukaryotic, human, farm animal, or companion animal.

[00157] As discussed in more detail herein, various methods are proposed for making targeted modifications to any locus of interest, including, for example, targeted modifications to the ApoE locus, the interleukin-2 gamma receptor locus (Il2rg), the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1. Additionally, genetically modified non-human animals, genetically modified non-human mammals, genetically modified non-rat eukaryotes, genetically modified non-pluripotent cells, or genetically modified pluripotent cells (e.g., pluripotent cell, non-human pluripotent cell, human pluripotent cell, human ES human adult stem cell, human progenitor cell with limited development or human iPS cell) that contain deletion, insertion, substitution and / or any combination thereof at the interleukin-2 gamma receptor locus, at the ApoE locus, at the Rag2 locus, at the Rag1 locus and / or at the Rag2 / Rag1 locus. Such genetic modifications (including those that result in the absence, decrease, increase, or modulation of the target locus activity) are also capable of germline transmission. In specific embodiments of the invention, genetic modifications result in the exclusion of the desired target locus. Such non-human animals find use, for example, in various experimental systems, as discussed elsewhere in this document.

[00158] For example, turning off ApoE (apolipoprotein E) allows an animal model to study endothelial function, including, but not limited to, plaque formation, transcriptional changes (full transcriptome shotgun sequencing (RNA sequencing)), and ex vivo function. ApoE is an important transport molecule and can transport lipids such as cholesterol through the bloodstream. ApoE can also function in the nervous system, for example, to clear β-amyloid from the brain. Modifications of ApoE have been implicated in a variety of pathological conditions including, for example, atherosclerosis, hyperlipidemia, and Alzheimer's disease. Animals with disabled ApoE show impaired clearance of lipoproteins from the blood and are susceptible to the development of atherosclerosis. Thus, ApoE-disabled animals provide an animal model for studying pathological conditions and / or processes such as, for example, endothelial function, plaque formation, transcriptional changes (RNA sequencing), hyperlipidemia, atherosclerosis, and Alzheimer's disease. Assay methods for measuring APOE activity are known in the art. For example, a decrease in ApoE activity can be measured by assaying a decrease in ApoE levels in a blood sample obtained from a subject using an immunoassay such as ELISA or immunoblotting. In addition, the large size of the rats facilitates such analyzes and improves the quality of the data.

[00159] RAG1 (recombination activating gene 1) and RAG2 (recombination activating gene 2) are enzymes that are part of a multi-subunit complex with VDJ recombination activity and play an important role in the rearrangement and recombination of immunoglobulin and T- receptor genes. cells in lymphocytes. RAG1 and RAG2 induce cleavage of two stranded DNA to facilitate recombination and joining of T cell receptor and B cell receptor (ie, immunoglobulin) gene segments. Turning off RAG1 and / or RAG2 results in a loss of B cells and T cells in the animal's body, resulting in severe immunodeficiency. RAG1 and / or RAG2 disabled animals find use, for example, in the study of xenografts (ie, xenografts of human cells in the rat), cancer, vaccine development, autoimmune diseases, infectious diseases, and graft versus host disease (GVHD). Various assay methods for measuring RAG1 and / or RAG2 activity are known in the art and include, for example, measuring recombination efficiency or assaying for the presence or absence of B cells and / or T cells in a subject.

[00160] The IL-2 receptor (IL-2R) is expressed on the surface of certain immune cells and binds to the cytokine interleukin-2 (IL-2). TL-2R is an integral membrane protein containing at least three separate subunit chains, including an alpha chain (IL-2Ra, CD25), a beta chain (IL-2Rb, CD122), and a gamma chain (IL2-Rg, CD132 ). The TL-2 gamma receptor chain (also called IL2r-γ or IL2Rg) is a common gamma chain common to many cytokine receptors including, for example, IL-2, IL-4, IL-7, IL-9, IL -15 and IL-21. IL-2Rg contains an ectodomain on the outer surface of the cell, which is responsible for ligand binding, a transmembrane domain, and an intracellular domain that can interact with various molecules and induce intracellular signal transduction pathways. The Il2rg gene is found in mammals on the X chromosome, and some mutations in the gamma chain gene in humans can lead to X-linked severe combined human immunodeficiency (CTCID), which is characterized by severe disruption of T-cell function. In addition, the gamma chain ectodomain can be detached from the transmembrane receptor and released as a soluble gamma chain receptor. The soluble gamma chain receptor is found in the blood of a subject and may function to regulate cytokine signaling.

[00161] In some embodiments, the non-human IL-2Rg chain is replaced with the human IL2-Rg chain such that the genetically modified animal expresses the fully human IL-2Rg chain. In other cases, it is advisable to replace only the ectodomain of the non-human IL-2Rg chain with the ectodomain of the human IL-2Rg chain. In such cases, the resulting humanized IL-2Rg chain, expressed in a non-human organism, contains a human ectodomain, and the remainder of the molecule belongs to the native organism.

[00162] Full-length humanization of the IL-2Rg is desirable because non-human mammals containing such a modified locus will produce human IL-2Rg. This will enable the detection of human IL-2Rg in non-human mammals using antibodies specific for human IL-2Rg. Ectogumanization (i.e., replacement of the ectodomain of IL-2Rg with a non-human mammalian ectodomain of human IL-2Rg) will result in an IL-2Rg polypeptide that will bind ligands of human IL2-Rg, but since the cytoplasmic domain belongs to a non-human mammal, ectogumanized the IL-2Rg form will also interact with the signaling apparatus of a non-human mammal.

2. Modification of the target locus

A. Targeting vectors and nucleotide inserts

i. Nucleotide insert

[00163] In the context of this document, "nucleotide insert" includes a segment of DNA that needs to be integrated into the target locus. In one embodiment, the nucleotide insert comprises one or more polynucleotides of interest. In other embodiments of the invention, the nucleotide insert may contain one or more expression cassettes. This expression cassette may contain a polynucleotide of interest, a polynucleotide encoding a selectable marker and / or a reporter gene, along with various regulatory components that affect expression. Non-limiting examples of polynucleotides of interest, selectable markers and reporter genes that can be included in a nucleotide insert are discussed in more detail elsewhere in this document.

[00164] In certain embodiments, the nucleotide insert may comprise rat nucleic acid, which may comprise a genomic DNA segment, cDNA, regulatory region, or any portion or combination thereof. In other embodiments, the nucleotide insert may comprise nucleic acid from a eukaryote, a non-rat eukaryote, a mammal, a human, a non-human mammal, a rodent, a non-rat rodent, a human, a rat, a mouse, a hamster, a rabbit, a pig, a bull, a deer, a sheep, goat, chicken, cat, dog, ferret, primate (eg, marmoset, rhesus monkey), domestic mammal or agricultural mammal, or any other organism of interest. As described in more detail herein, a nucleotide insert used in various methods and compositions can result in "humanization" of the target locus of interest.

[00165] In one embodiment, the nucleotide insert comprises an inserted allele of at least one exon of an endogenous gene. In one embodiment of the invention, the nucleotide insert comprises an included allele of a whole endogenous gene (ie, “included gene replacement”).

[00166] In one embodiment, the nucleotide insert comprises a regulatory element, including, for example, a promoter, enhancer, or transcriptional repressor binding element.

[00167] In additional embodiments of the invention, the nucleotide insert comprises a conditioned allele. In one embodiment, the conditioning allele is a multifunctional allele as described in US 2011/0104799, which is incorporated by reference in its entirety. In specific embodiments of the invention, the conditional allele comprises: (a) an initiation sequence in a sense orientation with respect to transcription of a target gene and a drug susceptibility selection cassette in a sense or antisense orientation; (b) a nucleotide sequence of interest (NSI) and a conditioning component represented by an inversion module (COIN, which uses an exon separating intron and an inverted gene trap-like module; see, for example, US 2011/0104799, which is incorporated by reference in its entirety) ; and (c) recombining units that recombine after exposure to the first recombinase to form a conditioned allele, (i) lacking an initiation sequence and DSC, and (ii) containing NSI in sense orientation and COIN in antisense orientation.

[00168] The range of the nucleotide insertion is from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp

[00169] In one embodiment, the nucleotide insert comprises the deletion of a genomic DNA sequence, eg, eukaryotic cell, non-rat eukaryotic cell, mammalian cell, human cell, or non-human mammalian cell, in the range of about 1 kb. to about 200 kb, from about 2 kb. up to about 20 kbp or from about 0.5 kbp. up to about 3 m.p. In one embodiment, the size of the genomic DNA sequence deletion exceeds the total length of the 5 'homology arm and the 3' homology arm. In one embodiment, the size of the genomic DNA sequence deletion ranges from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 20 kbp. up to about 30 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kb, from about 50 kb. to about 60 kbp, from about 60 kbp. to about 70 kbp, from about 70 kbp. to about 80 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 110 kbp. to about 120 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. to about 190 kbp, from about 190 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. to about 350 kbp, from about 350 kbp. to about 400 kbp, from about 400 kbp. to about 800 kbp, from about 800 kbp. up to 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. to about 2 mpo, from about 2 mpo, to about 2.5 mpo, from about 2.5 mpo. up to about 2.8 ppm, from about 2.8 ppm. up to about 3 ppm, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p.

[00170] In one embodiment, the nucleotide insert comprises an insertion or substitution of the nucleotide sequence of a non-rat eukaryote, mammalian, human, or non-human mammalian eukaryote with a homologous or orthologous human nucleotide sequence. In one embodiment of the invention

a nucleotide insert comprises an insert or substitution of a DNA sequence with a homologous or orthologous human nucleotide sequence at an endogenous locus that contains the corresponding DNA sequence.

[00171] In one embodiment, the genetic modification is the addition of a nucleotide sequence. In one embodiment, the added nucleotide sequence is 5 kb in length. up to 200 kbp

[00172] In one embodiment, the nucleotide insert comprises a genetic modification in the coding sequence. In one embodiment, the genetic modification comprises a deletion mutation in a coding sequence. In one embodiment, the genetic modification comprises the fusion of two endogenous coding sequences.

In one embodiment, the nucleotide insert comprises an insertion or substitution of the nucleotide sequence of a non-rat eukaryote, mammalian, human, or non-human mammal with a homologous or orthologous human nucleotide sequence. In one embodiment, the nucleotide insert comprises the insertion or substitution of a DNA sequence with a homologous or orthologous human nucleotide sequence at an endogenous rat locus that contains the corresponding rat DNA sequence.

[00173] In one embodiment, the genetic modification comprises deletion of a non-protein coding sequence, but does not include deletion of a protein coding sequence. In one embodiment, the deletion of a non-protein coding sequence comprises deletion of a regulatory element. In one embodiment, the genetic modification comprises deleting a promoter. In one embodiment, the genetic modification comprises the addition of a promoter or regulatory element. In one embodiment, the genetic modification comprises substitution of a promoter or regulatory element.

[00174] In one embodiment, the nucleotide sequence of the targeting vector may comprise a polynucleotide that, upon integration into the genome, will provide genetic modification to a region of the ApoE locus of a mammalian, human, or non-human mammal, wherein genetic modification at the ApoE locus results in a decrease in ApoE activity, increasing the activity of ApoE or modulation of the activity of ApoE. In one embodiment of the invention, ApoE is turned off (creating a "null allele").

[00175] In one embodiment of the invention, the nucleotide sequence of the targeting vector may comprise a polynucleotide that, upon integration into the genome, will provide a genetic modification to the region of the interleukin-2 receptor locus of a mammalian, human, or non-human mammal, while the genetic modification at the interleukin-2 receptor locus leads to a decrease in the activity of the interleukin-2 receptor. In one embodiment, the interleukin-2 receptor is turned off (creating a "null allele").

[00176] In additional embodiments of the invention, the nucleotide insertion results in replacement of a portion of the ApoE locus, the interleukin-2 gamma receptor locus and / or the Rag2 locus, and / or the Rag1 locus and / or the Rag2 / Rag1 locus of a mammal, human, or non-human mammal the corresponding homologous or orthologous portion of the ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus and / or the Rag2 / Rag1 locus of another organism.

[00177] In other embodiments, the nucleotide insert comprises a polynucleotide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity along its entire length, 97%, 98%, 99% with part of the ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus and / or the Rag2 / Rag1 locus that it replaces.

[00178] The specified polynucleotide insert and the corresponding replaceable region of the mammalian, human or non-human mammalian locus can be a coding region, an intron, an exon, a non-translated region, a regulatory region, a promoter or enhancer, or any combination thereof. In addition, the desired polynucleotide insert and / or deletion region of the mammalian, human, or non-human mammalian locus can be of any desired length, including, for example, 10-100 nucleotides in length, 100-500 nucleotides in length, 500-1 kb. .O. nucleotides in length, from 1 kb. up to 1.5 kbp nucleotides in length, from 1.5 kb. up to 2 kb nucleotides in length, from 2 kb. up to 2.5 kbp nucleotides in length, from 2.5 kb. up to 3 kbp nucleotides in length, from 3 kbp. up to 5 kbp nucleotides in length, from 5 kbp. up to 8 so. nucleotides in length, from 8 kbp. up to 10 kbp nucleotides in length or more. In other cases, the size of the insert or replacement is from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. to about 350 kbp, from about 350 kbp. to about 400 kbp, from about 400 kbp. to about 800 kbp, from about 800 kbp. up to 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. to about 2 mpo, from about 2 mpo, to about 2.5 mpo, from about 2.5 mpo. up to about 2.8 ppm, from about 2.8 ppm. up to about 3 m.p. In other embodiments, a given polynucleotide insert and / or deletion region of a mammalian, human, or non-human mammalian locus is at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides, or at least 1 ton kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb ., 14 kbp, 15 kbp, 16 kbp. or more.

[00179] In one embodiment, a promoter is a constitutively active promoter.

[00180] In one embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter is a chemically regulated promoter. In one embodiment, the chemically regulated promoter is an alcohol-regulated promoter. In one embodiment, the alcohol-regulated promoter is an alcohol dehydrogenase (alcA) gene promoter. In one embodiment, the chemically regulated promoter is a tetracycline-regulated promoter. In one embodiment, the tetracycline-regulated promoter is a tetracycline-responsive promoter. In one embodiment, the tetracycline-regulated promoter is a tetracycline operator (tetO) sequence. In one embodiment, the tetracycline-regulated promoter is a tet-On promoter. In one embodiment, the tetracycline-regulated promoter is a tet-Off promoter. In one embodiment of the invention, the chemically regulated promoter is a steroid-regulated promoter. In one embodiment, the steroid-regulated promoter is a rat glucocorticoid receptor promoter. In one embodiment, the steroid-regulated promoter is an estrogen receptor promoter. In one embodiment, the steroid-regulated promoter is an ecdysone receptor promoter. In one embodiment, the chemically regulated promoter is a metal regulated promoter. In one embodiment, the metal-regulated promoter is a metalloprotein promoter. In one embodiment, the inducible promoter is a physically regulated promoter. In one embodiment, the physically regulated promoter is a temperature regulated promoter. In one embodiment, the temperature controlled promoter is a heat shock promoter. In one embodiment, the physically regulated promoter is a photoregulated promoter. In one embodiment, the photoregulated promoter is a photoinducible promoter. In one embodiment, the photoregulated promoter is a photorepressible promoter.

[00181] In one embodiment, the promoter is a tissue-specific promoter. In one embodiment, the promoter is a neuron-specific promoter. In one embodiment, the promoter is a neuroglia-specific promoter. In one embodiment, the promoter is a muscle cell specific promoter. In one embodiment, the promoter is a heart cell specific promoter. In one embodiment, the promoter is a renal cell specific promoter. In one embodiment, the promoter is a bone cell specific promoter. In one embodiment, the promoter is an endothelial cell specific promoter. In one embodiment, the promoter is an immune cell specific promoter. In one embodiment, the immune cell promoter is a B cell promoter. In one embodiment, the immune cell promoter is a T cell promoter.

[00182] In one embodiment, the promoter is a developmentally controlled promoter. In one embodiment, the developmentally regulated promoter is only active during the embryonic developmental stage. In one embodiment, the developmentally regulated promoter is active only in an adult cell.

[00183] In specific embodiments of the invention, the promoter can be selected based on cell type. Thus, various promoters find use in eukaryotic cell, non-rat eukaryotic cell, mammalian cell, non-human mammalian cell, pluripotent cell, non-pluripotent cell, non-human pluripotent cell, human pluripotent cell, human ES cell, human adult stem cell. a restricted developmental progenitor, human iPS cell, human cell, rodent cell, non-rat rodent cell, rat cell, mouse cell, hamster cell, fibroblast, or CHO cell.

[00184] In some embodiments, the nucleotide insert comprises a nucleic acid flanked by target site-specific recombination sequences. It should be understood that although an entire nucleotide insert may be flanked by such site-specific recombination target sequences, any region or individual polynucleotide of interest within a nucleotide insert may also be flanked by such regions. Site-specific recombinase can be introduced into a cell by any means, including introducing a recombinase polypeptide into a cell, or introducing a polynucleotide encoding a site-specific recombinase into a host cell. A polynucleotide encoding a site-specific recombinase can be located within a nucleotide insert or in a separate polynucleotide. The site-specific recombinase can be operably linked to a promoter active in a cell, including, for example, an inducible promoter, a cell-endogenous promoter, a cell-heterologous promoter, a cell-specific promoter, a tissue-specific promoter, or a developmental-specific promoter. Target site-specific recombination sequences that can flank a nucleotide insert or any polynucleotide of interest in a nucleotide insert may include, but are not limited to, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp , att, FRT, rox, and a combination of these.

[00185] In some embodiments, site-specific recombination sites flank a polynucleotide encoding a selectable marker and / or reporter gene that is in the nucleotide insert. In such cases, after integration of the nucleotide insert into the target locus, the sequence between the sites of site-specific recombination can be removed.

[00186] In one embodiment, the nucleotide insert comprises a polynucleotide encoding a selectable marker. The selectable marker can be in a selection cassette. Such selectable markers include, but are not limited to, neomycin phosphotransferase (neo ^r ), hygromycin B phosphotransferase (hyg ^r ), puromycin N acetyltransferase (puro ^r ), blasticidin S deaminase (bsr ^r ), xanthine / guanine phosphoribosyltransferase (gpt) or herpes simplex virus thymidine kinase (HSV-k), or a combination thereof. In one embodiment, a polynucleotide encoding a selectable marker is operably linked to a promoter active in a cell, rat cell, pluripotent rat cell, rat ES cell, eukaryotic cell, non-rat eukaryotic cell, pluripotent cell, non-pluripotent human cell, non-human pluripotent cell cell, human ES cell, human adult stem cell, human progenitor cell with limited development, human iPS cell, mammalian cell, non-human mammalian cell, human cell, rodent cell, non-rat rodent cell, mouse cell, hamster cell, fibroblast or CHO cell. In the case of serial placement of polynucleotides of interest at the target locus, the selectable marker may contain a nuclease agent recognition site as described above. In one embodiment, a polynucleotide encoding a selectable marker is flanked by target site-specific recombination sequences.

[00187] The nucleotide insert may further comprise a reporter gene operably linked to a promoter, wherein the reporter gene encodes a reporter protein selected from the group consisting of or including LacZ, mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (uLPP), Emerald, enhanced green fluorescent protein (uZPP), CyPet, blue fluorescent protein (GBP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, and / or a combination thereof ... Such reporter genes can be operably linked to a promoter active in the cell. Such promoters can be inducible promoters, promoters endogenous to a reporter gene or cell, promoters heterologous to a reporter gene or cell, cell-specific promoters, tissue-specific promoters, or developmental stage-specific promoters.

[00188] In one embodiment, the nucleotide insert may comprise a mammalian nucleic acid that contains a genomic locus that encodes a protein expressed in the nervous system, skeletal system, digestive system, circulatory system, muscular system, respiratory system, cardiovascular system, lymphatic system , endocrine system, urinary system, reproductive system, or a combination thereof. In one embodiment, the mammalian nucleic acid comprises a genomic locus that encodes a protein expressed in bone marrow or a bone marrow cell. In one embodiment, the nucleic acid comprises a genomic locus that encodes a protein expressed in a spleen cell.

[00189] In one embodiment, the mammalian nucleic acid comprises a genomic locus that encodes a protein expressed in the nervous system, skeletal system, digestive system, circulatory system, muscular system, respiratory system, cardiovascular system, lymphatic system, endocrine system, urinary system , the reproductive system, or a combination thereof. In one embodiment, the mammalian nucleic acid comprises a genomic locus that encodes a protein expressed in bone marrow or a bone marrow cell. In one embodiment, the nucleic acid comprises a genomic locus that encodes a protein expressed in a spleen cell. In one embodiment, the genomic locus comprises a murine genomic DNA sequence, a rat genomic DNA sequence, a eukaryotic genomic DNA sequence, a non-rat eukaryote genomic DNA sequence, a mammalian genomic DNA sequence, a human genomic DNA sequence, or a non-human mammalian genomic DNA sequence, or the combination. In one embodiment, the genomic locus contains, in any order, rat and human genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, murine and human genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, murine and rat genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, rat, murine, and human genomic DNA sequences.

[00190] In one embodiment, the genomic locus comprises a murine genomic DNA sequence, a rat genomic DNA sequence, a hamster genomic DNA sequence, a human genomic DNA sequence, a eukaryotic genomic DNA sequence, a non-rat eukaryotic genomic DNA sequence, a mammalian genomic DNA sequence, or a sequence genomic DNA of a non-human mammal; or a combination thereof. In one embodiment, the genomic locus contains, in any order, rat and human genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, murine and human genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, murine and rat genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, rat, murine, and human genomic DNA sequences.

[00191] In one embodiment, the genetic modification comprises at least one human disease allele from a human gene. In one embodiment, the human disease is a neurological disease. In one embodiment, the human disease is a cardiovascular disease. In one embodiment, the human disease is kidney disease. In one embodiment, the human disease is a muscle disease. In one embodiment, the human disease is a blood disorder. In one embodiment, the human disease is cancer. In one embodiment, the human disease is a disease of the immune system.

[00192] In one embodiment, the human disease allele is the dominant allele. In one embodiment, the human disease allele is a recessive allele. In one embodiment, the human disease allele comprises a single nucleotide polymorphism (SNP) allele.

[00193] In one embodiment, genetic modification results in a mutant form of the protein with altered binding characteristics, altered localization, altered expression and / or altered expression profile.

[00194] In one embodiment, the nucleotide insert comprises a selection cassette. In one embodiment of the invention, the selection cassette contains a nucleotide sequence encoding a selectable marker, the nucleotide sequence being operably linked to a promoter active in ES cells. In one embodiment, the selectable marker is selected from or comprises a hygromycin resistance gene or a neomycin resistance gene.

[00195] In one embodiment, the nucleic acid comprises a genomic locus that encodes a protein expressed in a B cell. In one embodiment, the nucleic acid comprises a genomic locus that encodes a protein expressed in an immature B cell. In one embodiment, the nucleic acid comprises a genomic locus that encodes a protein expressed in a mature B cell.

[00196] In one embodiment, the nucleotide insert comprises a regulatory element. In one embodiment, the regulatory element is a promoter. In one embodiment, the regulatory element is an enhancer. In one embodiment, the regulatory element is a transcriptional repressor binding element.

[00197] In one embodiment, the genetic modification comprises deletion of a non-protein coding sequence, but does not include deletion of a protein coding sequence. In one embodiment, the deletion of a non-protein coding sequence comprises deletion of a regulatory element. In one embodiment, the deletion of a non-protein coding sequence comprises deletion of a regulatory element. In one embodiment, the genetic modification comprises the addition of a promoter or regulatory element. In one embodiment, the genetic modification comprises substitution of a promoter or regulatory element.

ii. Expression cassettes

[00198] Provided herein are polynucleotides or nucleic acid molecules containing various components used in a targeted genomic integration system (i.e., any or any combination of nuclease agents, recognition sites, nucleotide insertions, polynucleotides of interest, targeting vectors, selective markers and other components).

[00199] The terms "polynucleotide", "polynucleotide sequence", "nucleotide sequence" and "nucleic acid fragment" are used interchangeably herein. These terms include nucleotide sequences and the like. The polynucleotide can be a polymer of RNA or DNA that is single or double stranded, which optionally contains synthetic, unnatural or altered nucleotide bases. A polynucleotide in the form of a DNA polymer can be composed of one or more segments of cDNA, genomic DNA, synthetic DNA, or combinations thereof. Polynucleotides can include deoxyribonucleotides and ribonucleotides, including both naturally occurring molecules and synthetic analogs, and any combinations thereof. Polynucleotides as provided herein also include sequence forms, including, but not limited to, single-stranded forms, two-stranded forms, hairpins, loop-on-stem structures, and the like.

[00200] Additionally, there are proposed recombinant polynucleotides containing various components of a targeted genomic integration system. The terms "recombinant polynucleotide" and "recombinant DNA construct" are used interchangeably herein. A recombinant construct contains an artificial or heterologous combination of nucleotide sequences, for example, regulatory and coding sequences, that cannot be found together in a natural state. In other embodiments of the invention, the recombinant construct may contain regulatory sequences and coding sequences obtained from different sources, or regulatory sequences and coding sequences obtained from the same source, but ordered in a way that does not occur in nature. This construct can be used by itself or used in combination with a vector. Where a vector is used, the choice of vector will depend on the method used to transform the host cells, as is well known to those skilled in the art. For example, a plasmid vector can be used. Also provided are the genetic elements required for transformation, selection and propagation of host cells containing any of the isolated nucleic acid fragments provided herein. Screening can be performed, but is not limited to Southern blotting DNA, Northern blotting mRNA expression, immunoblotting protein expression, or phenotypic analysis.

[00201] In certain embodiments of the invention, one or more components of the directed genomic integration system described herein may be in an expression cassette for expression in a prokaryotic cell, eukaryotic cell, non-rat eukaryotic cell, bacterial, yeast, or mammalian cell or other organism of interest or cell type. The cassette may contain 5 'and 3' regulatory sequences operably linked to the polynucleotide provided herein. “Operatively linked” represents the relationship in which the functionally related components function as intended. For example, a functional link between a polynucleotide of interest and a regulatory sequence (ie, a promoter) is a functional link that allows expression of the polynucleotide of interest. Functionally related items can be contiguous or non-contiguous. When used in relation to joining two protein coding regions, operably linked means that the coding regions are in the same reading frame. Alternatively, the nucleotide sequence encoding the protein can be operably linked to regulatory sequences (eg, promoter, enhancer, silencer sequence, etc.) so as to maintain proper transcriptional regulation. In one instance, an immunoglobulin variable region nucleotide sequence (or V (D) J segments) may be operably linked to an immunoglobulin constant region nucleotide sequence so as to allow proper recombination between sequences to form an immunoglobulin heavy or light chain sequence.

[00202] The cassette may further comprise at least one additional polynucleotide of interest for co-introduction into the body. Alternatively, the additional polynucleotide of interest may be present on multiple expression cassettes. Such an expression cassette contains multiple restriction sites and / or recombination sites for the insertion of a recombinant polynucleotide to be transcriptionally regulated by regulatory regions. The expression cassette may further contain selectable marker genes.

[00203] The expression cassette may contain, in the 5'-3 'direction of transcription, a transcription and translation initiation site (ie, a promoter), a recombinant polynucleotide as provided herein, and a transcription and translation termination site (ie, a termination site), functional in a mammalian cell of interest or a host cell. Regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and / or a polynucleotide as provided herein may be native / analogous to the host cell or to each other. Alternatively, the regulatory regions and / or the polynucleotide provided herein may be heterologous with respect to the host cell or one another. For example, a promoter operably linked to a heterologous polynucleotide belongs to a species other than the species from which the polynucleotide was derived, or, if they belong to the same species / similar species, one or both elements are significantly modified from their original form and / or genomic locus, or the promoter is not a native promoter with respect to the polynucleotide operably linked thereto. Alternatively, the regulatory regions and / or the polynucleotide provided herein can be fully synthetic.

[00204] The termination region can be native to the transcription initiation region, can be native to an operably linked recombinant polynucleotide, can be native to a host cell, or can be obtained from another source (i.e., foreign or heterologous ) than a promoter, recombinant polynucleotide, host cell, or any combination of these variants.

[00205] Upon receipt of an expression cassette, various DNA fragments can be manipulated to obtain DNA sequences in the correct orientation. For this purpose, adapters or linkers can be used to join DNA fragments or perform other manipulations to provide convenient restriction sites, removal of excess DNA, removal of restriction sites, and the like. For this purpose, in vitro mutagenesis, repair using primers, restriction, hybridization, repeated substitutions, for example, transitions and transversions, can be used.

[00206] A wide variety of promoters can be used in the expression cassettes provided herein. Promoters can be selected based on the desired result. It is known that different applications can be made more efficient by using different promoters in expression cassettes to modulate the timing, localization and / or expression level of the polynucleotide of interest. Such expression constructs can also contain, if necessary, promoter regulatory regions (for example, providing inducible, constitutive, regulated by the environment or level of development, or cell or tissue specific / selective expression), a site for transcription initiation, a ribosome binding site, an RNA processing signal , a transcription termination site and / or a polyadenylation signal.

[00207] An expression cassette containing polynucleotides as provided herein may also contain a selectable marker gene for selecting transformed cells. Selectable marker genes are used to select transformed cells or tissues.

[00208] Where appropriate, the sequences used in the methods and compositions (ie, polynucleotide of interest, nuclease agent, etc.) can be optimized to enhance expression in a cell. This means that genes can be synthesized using codons preferred in a given cell of interest, including, for example, mammalian preferred codons, human preferred codons, rodent preferred codons, non-rat rodent preferred codons, codons mice preferred, rat preferred codons, hamster preferred codons, etc. to improve expression.

[00209] Selectable markers can be used in various methods and compositions provided herein. Various selectable markers can be used in the methods and compositions disclosed herein. Such selectable markers can, for example, confer resistance to antibiotics such as G418, hygromycin, blasticidin, neomycin, or puromycin. Such selectable markers include neomycin phosphotransferase (neo ^r ), hygromycin B phosphotransferase (hyg ^r ), puromycin N acetyltransferase (puro ^r ), and blasticidin S deaminase (bsr ^r ). In other embodiments, the selectable marker is operably linked to an inducible promoter, and the expression of the selectable marker is toxic to the cell. Non-limiting examples of such selectable markers include xanthine / guanine phosphoribosyl transferase (gpt), hypoxanthine guanine phosphoribosyl transferase (HGPRT), or herpes simplex virus thymidine kinase (HSV-TK). Polynucleotides encoding selectable markers are operably linked to a promoter active in the cell.

iii. Direction vectors

[00210] Targeting vectors are used to introduce a nucleotide insert into a target nucleic acid locus of a rat, eukaryote, non-rat, eukaryote, non-human mammal, human, rodent, non-rat, mouse, or hamster. The targeting vector contains a nucleotide insert and further contains 5 'and 3' homology arms that flank the nucleotide insert. The homology arms that flank the nucleotide insert correspond to regions in the target nucleic acid locus of the rat, eukaryote, non-rat, eukaryote, non-human mammal, human, rodent, non-rat, mouse, or hamster. For convenience, the corresponding cognate genomic regions within the target genomic locus are referred to herein as "target regions". For example, the targeting vector may contain a first nucleotide insert flanked by first and second homology arms complementary to the first and second target regions. Thus, the targeting vector facilitates the integration of the nucleotide insert into the target nucleic acid locus of the rat, eukaryote, non-rat eukaryote, non-human mammal, human, non-rat, mouse, or hamster through homologous recombination that occurs between the shoulders homology and complementary target regions in the cell genome.

[00211] In one embodiment, the target nucleic acid locus of a rat, eukaryote, non-rat eukaryote, non-human mammal, human, rodent, non-rat, mouse, or hamster nucleic acid locus comprises a first nucleotide sequence that is complementary to the 5 'arm homology, and a second nucleotide sequence that is complementary to the 3 'arm of homology. In one embodiment, the first and second nucleotide sequences are separated by at least 5 kb. In another embodiment, the first and second nucleotide sequences are separated by at least 5 kb, but less than 200 kb. In one embodiment, the first and second nucleotide sequences are separated by at least 10 kb. In one embodiment, the first and second nucleotide sequences are separated by at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb. bp, 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 at least 190 kbp or at least 200 kbp. In other embodiments, the first and second nucleotide sequences are separated by at least 5 kb, but less than 10 kb, at least 5 kb, but less than 3 m. bp, at least 10 kb, but less than 20 kb, at least 20 kb, but less than 40 kb, by at least 40 kb but less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb bp, but less than 100 kbp, at least 100 kbp, but less than 150 kbp, or at least 150 kbp, but less than 200 kbp, at least about 200 kbp, but less than about 300 kbp, at least about 300 kbp, but less than about 400 kbp, at least about 400 kbp, but less than about 500 kbp, at least about 500 kbp, but less than about 1 ppm, at least about 1.5 ppm, but less than about 2 ppm, at least about 1 ppm, but less than about 1 , 5 p.p., at least about 2 p.p., but less than 2.5 ppm, at least about 2.5 ppm but less than 3 ppm, or at least about 2 ppm but less than about 3 m.p.

[00212] The targeting vector homology arm can be of any length that is sufficient to stimulate homologous recombination with the corresponding target region, including, for example, a length of at least 5-10 kb, 5-15 kb kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb .o., 60-70 kbp, 70-80 kbp, 80-90 kbp, 90-100 kbp, 100-110 kbp kb, 110-120 kb, 120-130 kb, 130-140 kb, 140-150 kb, 150-160 kb .about., 160-170 kbp, 170-180 kbp, 180-190 kbp, 190-200 kbp. or more. As described in more detail below, larger guide vectors can use longer guide arms. In a particular embodiment, the 5 'homology arm and the 3' homology arm add up to at least 10 kbp. or the 5 'arm of homology and the 3' arm of homology add up to at least about 16 kbp. up to about 100 kbp or from about 30 kbp. up to about 100 kbp In other embodiments, the total size of the 5 'and 3' arms of LTVEC homology is from about 10 kbp. to about 150 kbp, from about 10 kbp. to about 100 kbp, from about 10 kbp. to about 75 kbp, from about 20 kbp. to about 150 kbp, from about 20 kbp. to about 100 kbp, from about 20 kbp. to about 75 kbp, from about 30 kbp. to about 150 kbp, from about 30 kbp. to about 100 kbp, from about 30 kbp. to about 75 kbp, from about 40 kbp. to about 150 kbp, from about 40 kbp. to about 100 kbp, from about 40 kbp. to about 75 kbp, from about 50 kbp. to about 150 kbp, from about 50 kbp. up to about 100 kbp or from about 50 kbp. to about 75 kbp, from about 10 kbp. to about 30 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to about 150 kbp In one embodiment, the size of the deletion is the same or similar to the overall size of the 5 'and 3' arms of LTVEC homology.

[00213] In one embodiment, the genomic locus of interest comprises (i) a 5 'target sequence that is homologous to the 5' arm of homology; and (ii) a 3 'target sequence that is homologous to the 3' homology arm. In one embodiment, the 5 'target sequence and the 3' target sequence are separated by at least 5 kb, but less than 3 ppm. In other embodiments, the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 10 kb, at least 5 kb, but less than 3 ppm, at least 10 kb, but less than 20 kb, at least 20 kb, but less than 40 kb. v., at least 40 kb, but less than 60 kb, at least 60 kb, but less than 80 kb, at least about 80 kb but less than 100 kb, at least 100 kb but less than 150 kb or at least 150 kb but less than 200 kb, at least about 200 kb but less than about 300 kb, at least about 300 kb but less than about 400 kb, at least about 400 kb but less than about 500 kb, at least about 500 kbp, but less than about 1 ppm, at least about 1.5 ppm, but less than about 2 ppm, at least about 1 ppm but less than about 1.5 ppm, at least about 2 ppm, but less than 2.5 ppm, at least about 2.5 ppm but less than about 3 ppm, or at least about 2 ppm but less than about 3 ppm.

[00214] In the case of nuclease agents, the cognate genomic regions corresponding to the 5 'and 3' homology arms of the targeting vector are "located close enough" to the nuclease target regions to stimulate homologous recombination between cognate genomic regions and homology arms after single-stranded or double-stranded break in the recognition area. For example, nuclease target regions can be located anywhere between the cognate genomic regions corresponding to the 5 'and 3' arms of homology. In specific embodiments of the invention, the recognition site is directly adjacent to at least one or both of the cognate genomic regions.

[00215] As used herein, the homology arm and the target region (ie, the cognate genomic region) are "complementary" or "complementary" to each other if the two regions have a sufficient degree of sequence identity with respect to each other that serve as substrates for homologous recombination reactions. By "homologous" is meant DNA sequences that are identical or have sequence identity with a corresponding or "complementary" sequence. The sequence identity between a given target region and the corresponding homology arm in the targeting vector can have any degree of sequence identity that allows homologous recombination. For example, the degree of sequence identity between the homology arm of the targeting vector (or its fragment) and the target region (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%, so that the sequences are subject to homologous recombination. In addition, the complementary region of homology between the arm of homology and the complementary target region can be of any length that is sufficient to stimulate homologous recombination in the cleavable recognition region. The predetermined homology arm and / or complementary target region may contain complementary homology regions, the length of which is at least 5-10 kb, 5-15 kb, 10-20 kb. , 20-30 kbp, 30-40 kbp, 40-50 kbp, 50-60 kbp, 60-70 kbp. , 70-80 kbp, 80-90 kbp, 90-100 kbp, 100-110 kbp, 110-120 kbp. , 120-130 kbp, 130-140 kbp, 140-150 kbp, 150-160 kbp, 160-170 kbp. , 170-180 kbp, 180-190 kbp, 190-200 kbp, 200 kbp. up to 300 kbp or more (as described for the LTVEC vectors described elsewhere in this document), so that the homology arm has sufficient homology to undergo homologous recombination with the corresponding target regions within the cell genome. For convenience, the homology arms are referred to herein as 5 'and 3' homology arms. This terminology is associated with the relative position of the arms of homology with respect to the nucleotide insert within the targeting vector.

[00216] Therefore, the homology arms of the targeting vector are designed to be complementary to the target region with the targeted locus. Thus, the homology arms can be complementary to a locus that is native to the cell, or, alternatively, they can be complementary to a region of a heterologous or exogenous DNA segment that has been integrated into the cell's genome, including, but not limited to, transgenes , expression cassettes, or heterologous or exogenous regions of genomic DNA. Alternatively, the homology arms of the targeting vector may be complementary to a region of a human artificial chromosome or any other engineered genomic region contained in an appropriate host cell. In addition, the homology arms of the targeting vector can be complementary to or derived from the IIR library region, the cosmid library, or the P1 phage library. Thus, in specific embodiments of the invention, the homology arms of the targeting vector are complementary to the genomic locus of the rat, eukaryote, non-rat eukaryote, non-human mammal, human, non-rat rodent, mouse, or hamster that is native, heterologous, or exogenous for a given cell. In additional embodiments, the targeting vector homology arms are complementary to the genomic locus of the rat, eukaryote, non-rat eukaryote, non-human mammal, human, non-rat rodent, mouse, or hamster that cannot be conventionally targeted, or which may be targeted only incorrectly or with very low efficacy in the absence of a single-stranded or double-stranded break induced by a nuclease agent. In one embodiment, the homology arms are derived from synthetic DNA.

[00217] In other embodiments, the 5 'and 3' homology arms are complementary to the same genome as the target genome. In one embodiment, the homology arms are derived from a related genome, for example, the targeted genome is a rat genome from a first strain, and the homology arms are derived from a rat genome from a second strain, wherein the first strain and the second strain are different. In other embodiments, the homology arms are derived from the genome of a single animal or are derived from the genome of a single strain, for example, the targeted genome is a rat genome from a first strain, and the homology arms are derived from a rat genome of the same rat or the same strain.

[00218] A targeting vector (such as a large targeting vector) can also contain a selection cassette or reporter gene, as discussed elsewhere in this document. The selection cassette may contain a nucleotide sequence encoding a selection marker, the nucleotide sequence being operably linked to a promoter. The promoter can be active in the prokaryotic cell of interest and / or be active in the eukaryotic cell of interest. Such promoters can be inducible promoters, promoters endogenous to a reporter gene or to a cell, promoters heterologous to a reporter gene or a cell, cell-specific promoters, tissue-specific promoters, or developmental stage-specific promoters. In one embodiment, the selectable marker is selected from or includes neomycin phosphotransferase (neo ^r ), hygromycin B phosphotransferase (hyg ^r ), puromycin N acetyltransferase (puro ^r ), blasticidin S deaminase (bsr ^r ), xanthine / guanine phosphoribosyltransferase (gpt) and herpes simplex virus thymidine kinase (HSV-k) and / or a combination thereof. The targeting vector selectable marker can be flanked by 5 'and 3' arms of homology, or be located 5 'or 3' in relation to the arms of homology.

[00219] In one embodiment, a targeting vector (such as a large targeting vector) comprises a reporter gene operably linked to a promoter, the reporter gene encoding a reporter protein selected from the group consisting of or including LacZ, mPlum, mCherry, tdTomato, m Strawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (uLPP), Emerald, enhanced green fluorescent protein (uZPP), CyPet, blue fluorescent protein (HPP), Cerulean, T- Sapphire, luciferase, alkaline phosphatase, and / or a combination thereof. Such reporter genes can be operably linked to a promoter active in the cell. Such promoters can be inducible promoters, promoters endogenous to a reporter gene or cell, promoters heterologous to a reporter gene or cell, cell-specific promoters, tissue-specific promoters, or developmental stage-specific promoters.

[00220] In one embodiment of the invention, the combined use of a targeting vector (including, for example, a large targeting vector) with nuclease agents results in an increase in targeting efficiency compared to using a single targeting vector. In one embodiment, when a targeting vector is used in combination with a nuclease agent, the targeting efficiency of the targeting vector is increased at least two-fold, at least three-fold, or at least 4-fold compared to the use of a single targeting vector.

[00221] When using a targeting vector, the vector design may be such as to allow insertion of a predetermined sequence of about 5 kb. up to about 200 kbp as described herein. In one embodiment, the insert is from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. up to about 30 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kb, from about 50 kb. to about 60 kbp, from about 60 kbp. to about 70 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 110 kbp. to about 120 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. up to about 190 kbp or from about 190 kbp. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp

[00222] When using a targeting vector, the vector design may be such as to allow substitution for a predetermined sequence of about 5 kbp. up to about 200 kbp or from about 5 kbp. up to about 3.0 mpo, as described in this document. In one embodiment, the substitution is from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. up to about 30 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kb, from about 50 kb. to about 60 kbp, from about 60 kbp. to about 70 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 110 kbp. to about 120 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. to about 190 kbp, from about 190 kbp. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p.

[00223] In one embodiment, the targeting vector comprises a site-specific recombinase gene. In one embodiment, the site-specific recombinase gene encodes Cre recombinase. In one embodiment, the Cre recombinase gene is Crei, in which two exons encoding Cre recombinase are separated by an intron to prevent its expression in a prokaryotic cell.

[00224] In one embodiment, the Cre recombinase gene further comprises a nuclear localization signal to facilitate localization of Cre (or any recombinase or nuclease agent) to the nucleus (eg, the gene is an NL-Cre gene). In a specific embodiment, the Cre recombinase gene further comprises a nuclear localization signal and an intron (eg, NL-Crei).

[00225] In various embodiments, a promoter suitable for expression of a nuclease agent (including the Cre or Crei recombinase discussed above) is selected from or includes Prm1, Blimp1, Gata6, Gata4, Igƒ2, Lhx2, Lhx5 and / or Pax3. In a specific embodiment, the promoter is a Gata6 or Gata4 promoter. Various promoters can be obtained from any organism, including, for example, a rodent such as a mouse or a rat, a non-rat rodent, a eukaryote, a non-rat eukaryote, a non-human mammal, a mammal, a human, or a hamster. In another specific embodiment, the promoter is a Prm1 promoter. In another specific embodiment, the promoter is a rat Prm1 promoter. In another specific embodiment, the promoter is a murine Prm1 promoter. In another specific embodiment of the invention, the promoter is the Blimp1 promoter or a fragment thereof, for example, containing 1 kb. or 2 like. fragment of the Blimp1 promoter. See, for example, US patent 8697851 and US published patent application 2013-0312129, which are fully incorporated into this document by reference.

iv. Large guide vectors

[00226] As used herein, the term "large targeting vector" or "LTVEC" includes large targeting vectors that contain arms of homology that correspond to or are derived from nucleotide sequences larger than those commonly used in other approaches to perform homologous targeting in cells , and / or containing nucleotide insertions containing nucleotide sequences larger than those commonly used in other approaches to carry out homologous recombination targeting in cells. For example, LTVEC allows modification of large loci that is not possible with traditional plasmid-based targeting vectors due to size limitations. In specific embodiments, the homology arms and / or nucleotide insert of the LTVEC contains the genomic sequence of a eukaryotic cell or non-rat eukaryotic cell. The size of the LTVEC is too large to be screened for targeting by conventional assays such as Southern blotting and long-fragment PCR (eg, 1 kb - 5 kb). Examples of LTVECs include, but are not limited to, vectors derived from bacterial artificial chromosome (IIR), human artificial chromosome, and yeast artificial chromosome (DIR). Non-limiting examples of LTVECs and methods for their preparation are described, for example, in US patents No. 6586251, 6596541, 7105348 and WO 2002/036789 (PCT / US 01/45375), and US 2013/0137101, all of which are incorporated herein by reference.

[00227] LTVEC can be of any length, including, but not limited to, from about 20 kbp. to about 400 kbp, from about 20 kbp. up to about 30 kbp, from about 30 kbp. up to 40 kbp, from about 40 kbp. to about 50 kb, from about 50 kb. to about 75 kbp, from about 75 kbp. to about 100 kbp, from about 100 kbp. to 125 kbp, from about 125 kbp. to about 150 kbp, from about 150 kbp. to about 175 kbp, from about 175 kbp. to about 200 kbp, from about 200 kbp. to about 225 kbp, from about 225 kbp. to about 250 kbp, from about 250 kbp. up to about 275 kbp or from about 275 kbp. to about 300 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 350 kbp, from about 350 kbp. to about 400 kbp, from about 350 kbp. up to about 550 kbp In one embodiment, the LTVEC is about 100 kbp.

[00228] In some embodiments, the LTVEC is 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 at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb or at least 200 kbp.

[00229] In some embodiments, the LTVEC comprises a nucleotide sequence of 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 kbp, at least 150 kbp. or at least 200 kbp.

[00230] In one embodiment, the LTVEC contains a nucleotide insert in the range of about 5 kbp. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 30 kbp, from about 0.5 kbp to about 30 kbp, from about 0.5 kbp to about 40 kbp, from about 30 kbp. to about 150 kbp, from about 0.5 kbp. to about 150 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kbp, from about 60 kbp. to about 70 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. up to about 190 kbp or from about 190 kbp. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp

[00231] In one embodiment, the LTVEC comprises a nucleotide sequence of at least 100 kb, at least 150 kb. or at least 200 kbp.

[00232] When using LTVEC, the vector design may be such as to allow substitution for a predetermined sequence of about 5 kb. up to about 200 kbp or from about 5 kbp. up to about 3 mpo, as described in this document. In one embodiment, the substitution is from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. up to about 30 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kb, from about 50 kb. to about 60 kbp, from about 60 kbp. to about 70 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 110 kbp. to about 120 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. to about 190 kbp, from about 190 kbp. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p.

[00233] In one embodiment, the LTVEC homology arms are derived from an IIR library, a cosmid library, or a P1 phage library. In other embodiments of the invention, the homology arms are derived from the target genomic locus of the cell, and in some cases, targeting the target genomic locus to which LTVEC is targeted cannot be accomplished in a conventional manner. In other embodiments, the homology arms are derived from synthetic DNA.

[00234] In one embodiment, the 5 'homology arm and the 3' homology arm in LTVEC add up to at least 10 kbp. In other embodiments, the 5 'and 3' arms of LTVEC homology total from about 10 kbp. to about 30 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. up to about 100 kbp, from 100 kbp. to about 120 kbp, from about 120 kbp. to about 140 kbp, from about 140 kbp. to about 160 kbp, from about 160 kbp. to about 180 kbp, from about 180 kbp. up to about 200 kbp In one embodiment, the 5 'and 3' arms of LTVEC homology total from about 30 kbp. up to about 100 kbp In other embodiments, the total size of the 5 'and 3' arms of LTVEC homology is from about 10 kbp. to about 150 kbp, from about 10 kbp. to about 100 kbp, from about 10 kbp. to about 75 kbp, from about 20 kbp. to about 150 kbp, from about 20 kbp. to about 100 kbp, from about 20 kbp. to about 75 kbp, from about 30 kbp. to about 150 kbp, from about 30 kbp. to about 100 kbp, from about 30 kbp. to about 75 kbp, from about 40 kbp. to about 150 kbp, from about 40 kbp. to about 100 kbp, from about 40 kbp. to about 75 kbp, from about 50 kbp. to about 150 kbp, from about 50 kbp. up to about 100 kbp or from about 50 kbp. to about 75 kbp, from about 10 kbp. to about 30 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to about 150 kbp In one embodiment, the size of the deletion is the same or similar to the overall size of the 5 'and 3' arms of LTVEC homology.

[00235] In other embodiments of the invention, the range of the 5 'arm of homology is from about 5 kbp. up to about 100 kbp In one embodiment, the range of the 3 'arm of homology is from about 5 kbp. up to about 100 kbp In other embodiments, the 5 'and 3' homology arms total between about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. up to about 30 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kb, from about 50 kb. to about 60 kbp, from about 60 kbp. to about 70 kbp, from about 70 kbp. to about 80 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 110 kbp. to about 120 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. to about 190 kbp, from about 190 kbp. up to about 200 kbp or from about 30 kbp. to about 100 kbp, from about 10 kbp. to about 30 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to about 150 kbp

[00236] In one embodiment, the LTVEC contains a nucleotide insert that is homologous or orthologous to the rat nucleotide sequence flanked by the LTVEC homology arms. In one embodiment, the nucleotide insert sequence is from a non-rat species. In one embodiment, the insert sequence is derived from a eukaryote. In one embodiment, the nucleotide insert that is homologous or orthologous to the rat nucleotide sequence is a mammalian nucleic acid. In one embodiment, the nucleotide insert that is homologous to or orthologous to the rat nucleotide sequence is a non-human mammalian nucleic acid. In one embodiment, the mammalian nucleic acid is a mouse nucleic acid. In one embodiment, the mammalian nucleic acid is a human nucleic acid. In one embodiment, the mammalian nucleic acid is a hamster nucleic acid. In one embodiment, the nucleotide insert is genomic DNA. In one embodiment, the insert is 5 kbp. up to 200 kbp as described above.

[00237] In one embodiment, the LTVEC comprises a selection cassette or reporter gene. The various forms of selection cassettes or reporter genes that can be used are discussed elsewhere in this document.

As described elsewhere in this document, LTVEC can also be used in the methods provided herein in combination with a nuclease agent that promotes homologous recombination between the targeting vector and the target nucleic acid locus of a rat, eukaryote, non-rat, eukaryote, non-human mammal mammal, human, non-rat rodent, mouse or hamster in a pluripotent or non-pluripotent rat cell, eukaryote, non-rat eukaryote, non-human mammal, human, rodent, non-rat rodent, mouse or hamster.

[00238] In one embodiment, a large targeting vector (LTVEC) contains a site-specific recombinase gene. In one embodiment, the site-specific recombinase gene encodes Cre recombinase. In one embodiment, the Cre recombinase gene is Crei, in which two exons encoding Cre recombinase are separated by a nitrone to prevent its expression in a prokaryotic cell. In one embodiment, the Cre recombinase gene further comprises a nuclear localization signal to facilitate localization of Cre (or any recombinase or nuclease agent) to the nucleus (eg, the gene is an NL-Cre gene). In a specific embodiment, the Cre recombinase gene further comprises a nuclear localization signal and an intron (eg, NL-Crei).

[00239] In various embodiments, a promoter suitable for expression of a nuclease agent (including the Cre or Crei recombinase discussed above) is selected from or includes Prm1, Blimp1, Gata6, Gata4, Igƒ2, Lhx2, Lhx5 and / or Pax3. In a specific embodiment, the promoter is a Gata6 or Gata4 promoter. Various promoters can be obtained from any organism, including, for example, a rodent such as a mouse or a rat, a non-rat rodent, a eukaryote, a non-rat eukaryote, a non-human mammal, a mammal, a human, or a hamster. In another specific embodiment, the promoter is a Prm1 promoter. In another specific embodiment, the promoter is a rat Prm1 promoter. In another specific embodiment, the promoter is a murine Prm1 promoter. In another specific embodiment of the invention, the promoter is the Blimp1 promoter or a fragment thereof, for example, containing 1 kb. or 2 like. fragment of the Blimp1 promoter. See, for example, US patent 8697851 and US published patent application 2013-0312129, which are fully incorporated into this document by reference.

[00240] In one embodiment, the LTVEC comprises a nucleotide insert that can remove, add, substitute, or a combination of the region of the ApoE locus, Il2rg locus, Rag2 locus, Rag1 locus and / or rat, non-rat eukaryotic Rag2 / Rag1 locus eukaryote, non-human mammal, human, non-rat rodent, mouse, or hamster, as discussed in more detail elsewhere in this document. In specific embodiments, genetic modification at the ApoE locus results in a decrease, increase, or modulation of ApoE activity, IL-2Rg activity, Rag2 activity, Rag1 activity, and / or Rag2 and Rag1 activity. In one embodiment, APOE is turned off, Il2rg turned off, Rag2 turned off, Rag1 turned off, Rag2 / Rag1 turned off. As discussed below, nuclease agents can be used with any of the LTVEC targeting systems to target any genomic locus of interest.

[00241] In another embodiment, the genome is exposed to Cas protein and CRISPR RNA in the presence of a large targeting vector (LTVEC) containing a nucleotide sequence of at least 10 kb. In such cases, after exposure to Cas protein, CRISPR RNA and LTVEC, the genome is modified so that it contains at least 10 kbp. nucleotide sequence. In specific embodiments, the LTVEC comprises a nucleotide sequence of at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb. bp, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kbp, at least 150 kbp or at least 200 kbp.

v. Nuclease agents and recognition sites for nuclease agents

[00242] As detailed above, nuclease agents can be used in the methods and compositions disclosed herein to facilitate modification of a target locus in both a prokaryotic cell and a pluripotent or non-pluripotent rat, eukaryote, non-rat, eukaryote, non-rat, mammalian a human mammal, a human, a rodent other than a rat, a rodent, a mouse, or a hamster. Such a nuclease agent can stimulate homologous recombination between the targeting vector and the target locus. In one embodiment, the nuclease agent comprises an endonuclease agent.

[00243] As used herein, the term "nuclease agent recognition site" includes a DNA sequence in which the nuclease agent induces a single or double strand break. The recognition site of the nuclease agent can be endogenous (or native) to the cell, or the recognition site can be exogenous to the cell. In specific embodiments of the invention, the recognition site is exogenous to the cell and, therefore, is not naturally present in the genome of the cell. In additional embodiments of the invention, the recognition site is exogenous to the cell and to the polynucleotides of interest to be placed at the target genomic locus. In additional embodiments of the invention, the exogenous or endogenous recognition site is present in only one copy in the genome of the host cell. In specific embodiments of the invention, an endogenous or native region is identified that is found in only one copy in the genome. Subsequently, such a site can be used to design nuclease agents that will create a single-stranded or double-stranded break in the endogenous recognition site.

[00244] The length of the recognition portion may vary and includes, for example, recognition portions that are at least 4, 6, 8, 10, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24 in length, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides. In one embodiment, each nuclease agent monomer recognizes a recognition site of at least 9 nucleotides. In other embodiments, the length of the recognition region is from about 9 to about 12 nucleotides, from about 12 to about 15 nucleotides, from about 15 to about 18 nucleotides, or from about 18 to about 21 nucleotides, and any combination of such subranges (e.g., 9- 18 nucleotides). The recognition site can be palindromic, which means that the sequence of one strand is read identically with the complementary strand in the opposite direction. It is known that a nuclease agent can bind to and cleave a recognition site, or, alternatively, a nuclease agent can bind to a sequence that is different from the recognition site. In addition, the term recognition site includes both a nuclease binding site and a single-strand break / cleavage site, whether the single-strand break / cleavage site is within or outside the nuclease agent binding site. Alternatively, cleavage with a nuclease agent can occur at nucleotide positions directly opposite to each other to produce a blunt cut or, in other cases, the cuts can be made offset to form single-stranded protrusions, also called "sticky ends", which can be both 5 'sticky ends and 3' sticky ends.

[00245] Any nuclease agent that induces a single-stranded or double-stranded break in the desired recognition site can be used in the methods and compositions disclosed herein. A natural or native nuclease agent can be used as long as the nuclease agent induces a single-stranded or double-stranded break at the desired recognition site. Alternatively, a modified or engineered nuclease agent may be used. A "engineered nuclease agent" includes a nuclease that has been engineered (modified or produced) from its native form to specifically recognize and induce a single-stranded or double-stranded break at a desired recognition site. Thus, a designed nuclease agent can be obtained from a native nuclease agent of natural origin, or it can be artificially created or synthesized. The modification of the nuclease agent can be at least one amino acid in the cleavage agent protein or one nucleotide in the cleavage agent nucleic acid. In some embodiments, the engineered nuclease agent induces a single-stranded or double-stranded break in the recognition site, where the recognition site is not a sequence that a native (undesigned or unmodified) nuclease agent would recognize. Creating a single-stranded or double-stranded break in the recognition site or other DNA may be referred to herein as "cutting" or "cleaving" the recognition site or other DNA.

[00246] Active variants and fragments of typical recognition regions are also provided. Such active variants may have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a given recognition site, while active variants retain biological activity and, therefore, can be recognized and cleaved by a nuclease agent in a sequence-specific manner. Assays are known in the art for determining the presence of a double-stranded break in the recognition site due to the action of a nuclease agent, and generally determine the ability of the nuclease to cut the recognition site.

[00247] The nuclease agent recognition site can be located anywhere or near the target locus. The recognition site can be located within the coding region of a gene or within regulatory regions that affect gene expression. Thus, the recognition site of the nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region.

[00248] In one embodiment, the nuclease agent is a transcription activator-like effector nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to create double-stranded breaks in specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases are generated by fusing a native or engineered transcription activator-like (TAL) effector or functional portion thereof with a catalytic endonuclease domain such as, for example, FokI. The unique, modular DNA-binding domain of the TAL effector allows the design of proteins with the specificity of recognizing potentially any given DNA. Thus, the DNA-binding domains of TAL effector nucleases can be engineered to recognize specific target DNA regions and, therefore, be used to create double-stranded breaks in the desired target sequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1: 428-432; Christian et al. Genetics (2010) 186: 757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi: 10.1093 / nar / gkq704; and Miller et al. (2011) Nature Biotechnology 29: 143-148; which are all incorporated herein by reference.

[00249] Examples of suitable TAL nucleases and methods for preparing suitable TAL nucleases are disclosed, for example, in US patent applications No. 2011/0239315 A1, 2011/0269234 A1, 2011/0145940 A1, 2003/0232410 A1, 2005/0208489 A1, 2005 / 0026157 A1, 2005/0064474 A1, 2006/0188987 A1 and 2006/0063231 A1 (each of which is incorporated herein by reference). In various embodiments, TAL effector nucleases are constructed that cut at or near the target nucleotide sequence, for example, at the genomic locus of interest, with the target nucleotide sequence at or near the sequence to be modified by the targeting vector. TAL nucleases suitable for use in the various methods and compositions provided herein include those designed specifically to bind at or near target nucleotide sequences to be modified with targeting vectors as described herein.

[00250] In one embodiment, each TALEN monomer contains 12-25 TAL repeats, with each TAL repeat linking a 1 bp subsite. In one embodiment, the nuclease agent is a chimeric protein comprising a TAL repeat-based DNA binding domain operably linked to an independent nuclease. In one embodiment, the independent nuclease is a FokI endonuclease. In one embodiment, the nuclease agent comprises a first TAL repeat based DNA binding domain and a second TAL repeat based DNA binding domain, wherein each of the first and second TAL repeat based DNA binding domains is operably linked to a FokI nuclease, when the first and second DNA-binding domains based on TAL repeats recognize two adjacent target DNA sequences on each strand of the target DNA sequence, separated by a cleavage site of about 6 bp. to about 40 bp, and the FokI nucleases dimerize and create a double-stranded break in the target sequence.

[00251] In one embodiment, the nuclease agent comprises a first TAL repeat based DNA binding domain and a second TAL repeat based DNA binding domain, wherein each of the first and second TAL repeat based DNA binding domains is operably linked to a nuclease FokI, while the first and second DNA-binding domains based on TAL repeats recognize two adjacent target DNA sequences on each strand of the target DNA sequence, separated by a cleavage site of from 5 bp. to about 6 bp, and the FokI nucleases dimerize and create a double-stranded break.

[00252] The nuclease agent used in the various methods and compositions described herein may further comprise zinc finger nuclease (ZFN). In one embodiment, each ZFN monomer contains 3 or more zinc finger based DNA binding domains, with each zinc finger based DNA binding domain being associated with a 3 bp subsite. In other embodiments, the ZFN is a chimeric protein comprising a zinc finger DNA binding domain operably linked to an independent nuclease. In one embodiment, the independent nuclease is a FokI endonuclease. In one embodiment of the invention, the nuclease agent comprises a first ZFN and a second ZFN, each of the first ZFN and the second ZFN being operably linked to a FokI nuclease, wherein the first and second ZFN recognize two adjacent target DNA sequences on each strand of the target DNA sequence separated by a region cleavage from about 6 bp. up to about 40 bp or a cleavage site from about 5 bp. to about 6 bp, and the FokI nucleases dimerize and create a double-stranded break. See, for example, US 20060246567; US 20080182332; US 20020081614; US 20030021776; WO / 2002/057308 A2; US 20130123484; US 20100291048; and WO / 2011/017293 A2, each of which is incorporated herein by reference.

[00253] In one embodiment of the methods provided herein, the nuclease agent comprises (a) a chimeric protein comprising a zinc finger DNA binding domain fused to a FokI endonuclease; or (b) a chimeric protein comprising a transcription activator-like effector nuclease (TALEN) fused to a FokI endonuclease.

[00254] In another embodiment, the nuclease agent is a meganuclease. Meganucleases have been divided into four families based on conserved sequence motifs, and these families are the LAGLIDADG (SEQ ID no: 16), GIY-YIG, H-N-H and His-Cys boxing families. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. HEases are known for their long recognition regions and for tolerating some degree of sequence polymorphism in their DNA substrates. The domains, structure and function of meganucleases are known, see, for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38: 199-248; Lucas et al., (2001) Nucleic Acids Res 29: 960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55: 1304-26; Stoddard, (2006) Q Rev Biophys 38: 49-95; and Moure et al., (2002) Nat Struct Biol 9: 764. In some examples, a naturally occurring variant and / or an engineered meganuclease derivative is used. Methods for altering kinetics, interaction of cofactors, expression, optimal conditions and / or specificity of recognition sites, and screening for activity are known, see, for example, Epinat et al., (2003) Nucleic Acids Res 31: 2952-62; Chevalier et al., (2002) Mol Cell 10: 895-905; Gimble et al., (2003) Mol Biol 334: 993-1008; Seligman et al., (2002) Nucleic Acids Res 30: 3870-9; Sussman et al., (2004) J Mol Biol 342: 31-41; Rosen et al., (2006) Nucleic Acids Res 34: 4791-800; Chames et al., (2005) Nucleic Acids Res 33: e178; Smith et al., (2006) Nucleic Acids Res 34: e149; Gruen et al., (2002) Nucleic Acids Res 30: e29; Chen and Zhao, (2005) Nucleic Acids Res 33: e154; WO 2005105989; WO 2003078619; WO 2006097854; WO 2006097853; WO 2006097784; and WO 2004031346.

[00255] Any meganucleases can be used in this invention, including but not limited to I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI , I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F -TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII , I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I -NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP , I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I -TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliI I or any active variants or fragments thereof.

[00256] In one embodiment, the meganuclease recognizes 12 to 40 bp double stranded DNA sequences. In one embodiment, the meganuclease recognizes one perfectly matching target sequence in the genome. In one embodiment, the meganuclease is a homing nuclease. In one embodiment, the homing nuclease belongs to the LAGLIDADG family (SEQ ID NO: 16) of homing nucleases. In one embodiment, the LAGLIDADG (SEQ ID NO: 16) family of homing nucleases is selected from I-SceI, I-CreI, and I-DmoI.

[00257] The nuclease agents may further include restriction endonucleases, which include type I, type II, type III, and type IV endonucleases. Restriction endonucleases of type I and type III recognize specific recognition sites, but, as a rule, are cleaved at an arbitrary position relative to the binding site, which can be hundreds of base pairs from the cleavage site (recognition site). In type II systems, restriction activity is independent of any methylase activity, and cleavage typically occurs at specific sites within or near the binding site. Most type II enzymes cut palindromic sequences, with type IIa enzymes recognize non-palindromic recognition sites and cleave outside the recognition site, type IIb enzymes cut sequences twice on both sides outside the recognition site, and type IIs enzymes recognize an asymmetric recognition site and cleave with one side and at a certain distance of about 1-20 nucleotides from the recognition site. Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, for example, in the REBASE database (website at rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31: 418-20), Roberts et al., (2003) Nucleic Acids Res 31: 1805-12, and Belfort et al., (2002) in Mobile DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press, Washington, DC).

[00258] The nuclease agent used in various methods and compositions can also include a CRISPR / Cas system. In such systems, for example, Cas9 nuclease can be used, which in some cases is codon-optimized for the required type of cells in which it is to be expressed. Such systems can also use guide RNA (nRNA), which contains two separate molecules. A typical two-molecule nRNA contains a crRNA-like ("CRISPR RNA" or "targeting RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA" or "activating RNA" , "TracrRNA, or" scaffold ") molecule. crRNA contains both a DNA targeting (single-stranded) nRNA segment and a group of nucleotides that forms one half of the double-stranded RNA helix (dsRNA) of the nRNA protein-binding segment. The corresponding tracrRNA (activating RNA) contains a group of nucleotides that forms the other half of the dsRNA helix of the nRNA protein-binding segment. Thus, the crRNA group of nucleotides is complementary and hybridizes with the tracrRNA group of nucleotides to form a dsRNA helix of the nRNA protein-binding domain. Therefore, we can say that crRNA corresponds to tracrRNA. crRNA additionally provides a single-stranded DNA targeting segment. Accordingly, the nRNA contains the sequence that hybridizes to the target sequence and tracrRNA. Thus, crRNA and tracrRNA (as a corresponding pair) hybridize to form nRNA. When used for modification in a cell, the exact sequence and / or length of a given crRNA or tracrRNA molecule can be selected so that they are specific with respect to the species to which the RNA molecules will be applied.

[00259] Genes of natural origin, encoding three elements (Cas9, tracrRNA and crRNA), as a rule, are part of the operon (s). Naturally occurring CRISPR RNAs differ depending on the Cas9 system and organism, but often contain a targeting segment of 21 to 72 nucleotides in length, flanked by two forward repeats (FRs) of 21 to 46 nucleotides in length (see, for example, WO 2014/131833). In the case of S. pyogenes, the length of the PN is 36 nucleotides, and the length of the targeting segment is 30 nucleotides. The 3 'located PP is complementary and hybridizes with the corresponding tracrRNA, which in turn binds to the Cas9 protein.

[00260] Alternatively, the system further employs a crRNA-tracrRNA fusion (ie, one transcript) that functions with a codon-optimized Cas9. This RNA is often referred to as guide RNA or nRNA. Within nRNA, part of crRNA is defined as the “target sequence” for a given recognition site, and tracrRNA is often called a “scaffold”. Briefly, a short DNA fragment containing the target sequence is introduced into a guide RNA expression plasmid. The guide RNA expression plasmid contains the target sequence (in some embodiments, about 20 nucleotides), a form of the tracrRNA sequence (scaffold), as well as a suitable active promoter in the cell and the necessary elements for proper processing in eukaryotic cells. Many of the systems are based on individually matched complementary oligos that hybridize to form double-stranded DNA and then cloned into an nRNA expression plasmid. Then, an nRNA expression cassette and a Cas9 expression cassette are introduced into the cell. See, for example, Mali P et al. (2013) Science 2013 Feb 15; 339 (6121): 823-6; Jinek M et al. Science 2012 Aug 17; 337 (6096): 816-21; Hwang WY et al. Nat Biotechnol 2013 Mar; 31 (3): 227-9; Jiang W et al. Nat Biotechnol 2013 Mar; 31 (3): 233-9; and Cong L et al. Science 2013 Feb 15; 339 (6121): 819-23, each of which is incorporated herein by reference. See also, for example, WO / 2013/176772 A1, WO / 2014/065596 A1, WO / 2014/089290 A1, WO / 2014/093622 A2, WO / 2014/099750 A2 and WO / 2013142578 A1, each of which is included in this document by reference.

[00261] In some embodiments, the Cas9 nuclease may be in the form of a protein. In some embodiments, the Cas9 protein may be in the form of a complex with an nRNA. In other embodiments, the Cas9 nuclease may be in the form of a nucleic acid encoding a protein. The nucleic acid encoding the Cas9 nuclease can be RNA (eg messenger RNA (mRNA)) or DNA.

[00262] In some embodiments, the nRNA may be in the form of RNA. In other embodiments, the nRNA may be in the form of DNA encoding the nRNA. In some embodiments, the nRNA may be in the form of separate crRNA and tracrRNA molecules or separate DNA molecules encoding crRNA and tracrRNA, respectively.

[00263] In one embodiment, a method for modifying a genomic locus of interest in a cell further comprises introducing into the cell: (a) a first expression construct comprising a first promoter operably linked to a first nucleotide sequence encoding a regularly spaced group associated with short palindromic repeats (CRISPR), protein (Cas); (b) a second expression construct comprising a second promoter operably linked to a target genomic sequence linked to a guide RNA (nRNA), the target genomic sequence being flanked by a motif adjacent to the protospacer. Optionally, the target genomic sequence is flanked at the 3 'end by a protospacer contiguous motif (PAM) sequence. In one embodiment, the cell includes a eukaryotic cell, a non-rat eukaryotic cell, a mammalian cell, a human cell, a non-human mammalian cell, a pluripotent cell, a non-pluripotent cell, a non-human pluripotent cell, a human pluripotent human cell, a human ES cell, a human a restricted developmental progenitor cell, a human iPS cell, a human cell, a rodent cell, a non-rat rodent cell, a rat cell, a mouse cell, a hamster cell, a fibroblast, or a CHO cell.

(GN_1-20GG; SEQ ID №1). В одном варианте реализации изобретения целевая геномная последовательность содержит SEQ ID №23, где длина N составляет от 1 до 20 нуклеотидов. В другом варианте реализации изобретения длина целевой геномной последовательности составляет от 14 до 20 нуклеотидов от SEQ ID №1.[00264] In one embodiment, the target genomic sequence comprises a nucleotide sequence

(GN _1-20 GG; SEQ ID No. 1). In one embodiment, the target genomic sequence comprises SEQ ID NO: 23, where N is 1 to 20 nucleotides in length. In another embodiment, the target genomic sequence is 14 to 20 nucleotides in length from SEQ ID No. 1.

[00265] In one embodiment, the nRNA comprises a third nucleotide sequence encoding RNA of short palindromic repeats, regularly spaced groups (CRISPR) (crRNA), and trans-activating RNA CRISPR (tracrRNA). In specific embodiments, the Cas protein is Cas9.

(SEQ ID №2); или (b) химерную РНК с нуклеотидной последовательностью

(SEQ ID №3).[00266] In some embodiments, the nRNA comprises (a) a chimeric RNA with a nucleotide sequence

(SEQ ID No. 2); or (b) chimeric RNA with a nucleotide sequence

(SEQ ID No. 3).

(SEQ ID №4);

(SEQ ID №5); или

(SEQ ID №6).[00267] In another embodiment, the crRNA comprises

(SEQ ID No. 4);

(SEQ ID No. 5); or

(SEQ ID No. 6).

(SEQ ID №7) или

(SEQ ID №8).[00268] In other embodiments, tracrRNA comprises

(SEQ ID No. 7) or

(SEQ ID No. 8).

[00269] In one embodiment, the Cas protein is a type I Cas protein. In one embodiment, the Cas protein is a type II Cas protein. In one embodiment, the Cas type II protein is Cas9. In one embodiment, the first nucleotide sequence encodes a codon-optimized Cas protein.

[00270] In certain embodiments, the Cas protein is a "nickase" that can create single-stranded breaks (ie, "nicks") in the target region without cutting both strands of double-stranded DNA (dsDNA). Cas9, for example, contains two nuclease domains, the RuvC-like nuclease domain and the HNH-like nuclease domain, which are responsible for cleaving opposite DNA strands. A mutation in any of these domains can lead to the creation of nickase. Examples of mutations leading to the creation of nickases can be found, for example, in WO / 2013/176772 A1 and WO / 2013/142578 A1, each of which is incorporated herein by reference.

[00271] In certain embodiments, two separate Cas proteins (eg, nickases) specific for a target region on each strand of dsDNA can create overhanging sequences complementary to overhanging sequences of another nucleic acid or a separate region of the same nucleic acid. The protruding ends resulting from the contact of the nucleic acid with two nickases specific for the target sites on both strands of dsDNA can be either 5 'or 3' protruding ends. For example, the first nickase can create a single-stranded break in the first strand of dsDNA, and the second nickase can create a single-stranded break in the second strand of dsDNA, resulting in protruding ends. The target regions of each nickase that creates a single strand break can be selected so that the resulting overhang sequences are complementary to the overhang sequences of another nucleic acid molecule. Hybridization of complementary overhangs of two different nucleic acid molecules can be performed by methods disclosed herein. In some embodiments, the nickase target site on the first strand is different from the nickase target site on the second strand.

[00272] In one embodiment, the first nucleic acid contains a mutation that leads to the destruction of at least one amino acid residue in the active sites of nucleases in the Cas protein, while the mutant Cas protein creates a break in only one strand of the target DNA region, and the mutation reduces the probability of non-homologous recombination in the target DNA region.

[00273] In one embodiment, the first nucleic acid that encodes the Cas protein further comprises a nuclear localization signal (NLS). In one embodiment, the nuclear localization signal is the SV40 nuclear localization signal.

[00274] In one embodiment, the second promoter that directs the expression of the target genomic sequence and guide RNA (nRNA) is an RNA polymerase III promoter. In one embodiment, the RNA polymerase III promoter is a human U6 promoter. In one embodiment, the RNA polymerase III promoter is a rat U6 polymerase III promoter. In one embodiment, the RNA polymerase III promoter is a mouse U6 polymerase III promoter.

[00275] In one embodiment, the nucleotide sequences encoding crRNA and tracrRNA are linked through a synthetic loop, and upon expression, crRNA and tracrRNA form a crRNA: tracrRNA helix.

[00276] The CRISPR / Cas system described above can be used in combination with large targeting vectors with any of the following cell types: eukaryotic cell, non-rat eukaryotic cell, mammalian cell, non-human mammalian cell, pluripotent cell, non-pluripotent cell, non-human pluripotent cell. human pluripotent cell, human ES cell, human adult stem cell, human restricted progenitor cell, human iPS cell, human cell, rodent cell, non-rat rodent cell, rat cell, mouse cell, hamster cell, fibroblast or CHO cell ...

[00277] In one embodiment, the first expression construct and the second expression construct are expressed from a single plasmid.

[00278] In one embodiment, the first and second expression constructs are co-introduced with the LTVEC. In one embodiment, the first and second expression constructs are applied separately from the LTVEC over time.

[00279] In one embodiment, the method comprises introducing multiple second constructs and multiple LTVECs for multiple editing of different target loci, as described herein.

[00280] Active variants and fragments of nuclease agents (ie, engineered nuclease agents) are also provided. Such active variants may have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the native nuclease agent, while the active variants retain the ability to cut the desired recognition site and, therefore, retain single- or double-stranded break-inducing activity. For example, any nuclease agents described herein can be modified with respect to the native endonuclease sequence and designed to recognize and induce a single-stranded or double-stranded break at a recognition site that was not recognized by the native nuclease agent. Thus, in some embodiments, the engineered nuclease has specificity to induce a single-stranded or double-stranded break at a recognition site that is different from the corresponding recognition site of the native nuclease agent. Methods for analyzing single-stranded or double-stranded break-inducing activity are known, and in general they determine the general activity and specificity of endonucleases on DNA substrates containing a recognition site.

[00281] The nuclease agent can be introduced into the cell by any means known in the art. A polypeptide encoding a nuclease agent can be introduced directly into a cell. Alternatively, a polynucleotide encoding a nuclease agent can be introduced into the cell. When a polynucleotide encoding a nuclease agent is introduced into a cell, the nuclease agent can be temporarily, under certain conditions, or constitutively expressed in the cell. Thus, a polynucleotide encoding a nuclease agent can be in an expression cassette and be operably linked to a conditioning promoter, an inducible promoter, a constitutive promoter, or a tissue-specific promoter. Such promoters of interest are discussed in more detail elsewhere in this document. Alternatively, the nuclease agent is introduced into the cell as mRNA encoding or containing the nuclease agent.

[00282] In one embodiment, crRNA and tracrRNA are expressed as separate RNA transcripts.

[00283] In certain embodiments, a polynucleotide encoding a nuclease agent is stably integrated into the genome of a cell and is operably linked to a promoter active in the cell. In other embodiments, the polynucleotide encoding the nuclease agent is in a single targeting vector containing the nucleotide insert, while in other cases, the polynucleotide encoding the nuclease agent is in a vector or plasmid other than the targeting vector containing the nucleotide insert.

[00284] If a nuclease agent is introduced into a cell by introducing a polynucleotide encoding a nuclease agent, such a polynucleotide encoding a nuclease agent can be modified to replace codons having a higher frequency of use in the cell of interest as compared to the naturally occurring polynucleotide sequence encoding the nuclease agent ... For example, a polynucleotide encoding a nuclease agent can be modified to replace codons having a high frequency of use in a given prokaryotic or eukaryotic cell of interest, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a non-rat eukaryotic cell, a mammalian cell, a rodent cell. , a non-rat rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.

[00285] In one embodiment, the endonuclease agent is co-administered with the LTVEC. In one embodiment, the endonuclease agent is administered separately from the LTVEC over time. In one embodiment, the endonuclease agent is added prior to the LTVEC. In one embodiment, the endonuclease agent is introduced into an ES cell of a rat, eukaryote, non-rat eukaryote, non-human mammal, human, rodent, non-rat, mouse, or hamster after LTVEC application.

[00286] In one embodiment, the endonuclease agent is an expression construct comprising a nucleotide sequence encoding an endonuclease, the nucleotide sequence being operably linked to a promoter. In one embodiment, the promoter is a constitutively active promoter. In one embodiment, the promoter is an inducible promoter. In one embodiment, the promoter is active in a pluripotent or non-pluripotent rat, eukaryotic, non-rat eukaryotic, non-human mammal, human, non-rat rodent, mouse, or hamster mammalian cell. In one embodiment, the endonuclease agent is an mRNA encoding an endonuclease.

B. Methods for Integrating a Polynucleotide of Interest into a Target Locus

[00287] Methods for modifying a target locus of interest are provided. In one embodiment, a target locus in a pluripotent or non-pluripotent cell of a rat, eukaryote, non-rat eukaryote, non-human mammal, human, non-rat rodent, mouse, or hamster is intended to be genetically modified. Such a method includes: (a) introducing into a pluripotent or non-pluripotent cell of a rat, a eukaryote, a non-rat eukaryote, a non-human mammal, a human, a non-rat rodent, mouse, or hamster, a targeting vector containing a nucleotide insert flanked by 5 'rat homology arm, eukaryote, non-rat eukaryote, non-human mammal, human, rodent, non-rat rodent, mouse or hamster and 3' homology arm of rat, eukaryote, non-rat eukaryote, non-human mammal a mammal, a human, a rodent other than a rat, a rodent, a mouse, or a hamster; and (b) detecting a genetically modified pluripotent or non-pluripotent rat cell, a eukaryote, a non-rat eukaryote, a non-human mammal, a human, a non-rat rodent, mouse or hamster containing a targeted genetic modification at a target locus, wherein targeted genetic modification can be transmitted through the germ line. In specific embodiments of the invention, the 5 'homology arm and the 3' homology arm add up to at least 10 kbp. and / or using a large directional vector.

[00288] In other embodiments, the total size of the 5 'and 3' arms of LTVEC homology is from about 10 kbp. to about 150 kbp, from about 10 kbp. to about 100 kbp, from about 10 kbp. to about 75 kbp, from about 20 kbp. to about 150 kbp, from about 20 kbp. to about 100 kbp, from about 20 kbp. to about 75 kbp, from about 30 kbp. to about 150 kbp, from about 30 kbp. to about 100 kbp, from about 30 kbp. to about 75 kbp, from about 40 kbp. to about 150 kbp, from about 40 kbp. to about 100 kbp, from about 40 kbp. to about 75 kbp, from about 50 kbp. to about 150 kbp, from about 50 kbp. up to about 100 kbp or from about 50 kbp. to about 75 kbp, from about 10 kbp. to about 30 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to about 150 kbp In one embodiment, the size of the deletion is the same or similar to the overall size of the 5 'and 3' arms of LTVEC homology.

[00289] The pluripotent cell, for example, a rat cell, may be an embryonic stem cell, for example, a rat embryonic stem cell. In a specific embodiment of the invention (a) a rat ES cell is obtained from a DA strain or an ACI strain; or (b) the rat ES cell is characterized by the expression of a pluripotency marker, including Oct-4, Sox-2, alkaline phosphatase, or combinations thereof. In other instances, the rat embryonic stem cell used includes the rat ES cell described in US Patent Application No. 14/185103, filed Feb. 20, 2014, which is hereby incorporated by reference in its entirety.

[00290] Any pluripotent or non-pluripotent cell can be used in the methods provided herein. For example, a pluripotent or non-pluripotent cell can be obtained from a non-rat eukaryote, a non-human mammal, a mammal, a rodent, a non-rat rodent, rat, mouse, human, or hamster.

[00291] As described elsewhere in this document, the nucleotide insert can be any nucleotide sequence. In non-limiting embodiments of the invention (a) the nucleotide insert comprises replacing an endogenous nucleotide sequence of a rat, eukaryote, non-rat eukaryote, mammal, human, rodent, non-rat, rodent, mouse, or hamster with a homologous or orthologous mammalian nucleotide sequence; (b) the nucleotide insert comprises deletion of an endogenous nucleotide sequence of a rat, eukaryote, non-rat eukaryote, mammal, human, rodent, non-rat, mouse, or hamster; (c) the nucleotide insert comprises deletion of an endogenous nucleotide sequence of a rat, eukaryote, non-rat eukaryote, mammal, non-human mammal, human, rodent, non-rat, mouse, or hamster, the removal range being from 5 kb. O. up to 200 kbp or from 5 kbp. up to 3 m.p. (as discussed in detail elsewhere in this document); (d) the nucleotide insert comprises the addition of an exogenous nucleotide sequence (including, for example, an exogenous nucleotide sequence ranging 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 bp, from about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to about 300 kbp, from about 300 kbp to about 350 kbp, or from about 350 kbp to about 400 kbp) ; (e) the nucleotide insert contains an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (f) a homologous or orthologous nucleotide sequence according to (a), wherein the nucleotide sequence is a human nucleotide sequence; (g) the nucleotide insert contains a homologous or orthologous nucleotide sequence according to (a), the nucleotide sequence being a chimeric nucleotide sequence comprising a human and rat nucleotide sequence; (h) the nucleotide insert contains the exogenous nucleotide sequence of (e), wherein the range of the nucleotide insert is from about 5 kb. up to about 200 kbp; (i) the nucleotide insert contains a conditioned allele flanked by the target sequences of the site-specific recombinase; (j) the nucleotide insert contains a reporter gene operably linked to a promoter; (k) the nucleotide insert contains one or more non-rearranged _{human immunoglobulin heavy chain V H} gene segments, one or more non-rearranged human immunoglobulin heavy chain D gene segments, and one or more non-rearranged _{human immunoglobulin heavy chain J H} gene segments that are operably linked to the nucleotide sequence the constant region of the rodent heavy chain; (1) the nucleotide insert contains the rearranged nucleotide sequence of the variable region of the heavy chain of a human immunoglobulin, operably linked to the nucleotide sequence of the constant region of the heavy chain of a rodent; (m) the nucleotide insert contains one or more non-rearranged _{human immunoglobulin V κ} or V _λ gene segments and one or more non-rearranged _{human immunoglobulin J κ} or J _λ gene segments that are operably linked to the nucleotide sequence of the constant λ or κ region of the mammalian immunoglobulin light chain; (n) the nucleotide insert contains a rearranged nucleotide sequence of the λ or κ light chain of a human immunoglobulin, operably linked to the nucleotide sequence of the constant region of λ or κ of the light chain of a mammalian immunoglobulin; (o) the mammalian heavy chain constant region nucleotide sequence according to (k) and / or (l) comprises a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof; or (p) the λ or κ light chain constant region nucleic acid of a mammalian immunoglobulin according to (m) and / or (n) comprises a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof.

[00292] In one embodiment, the nucleotide insert

contains one or more functional human V _H gene segments including V _H 1-2, V _H 1-3, V _H 1-8, V _H 1-18, V _H 1-24, V _H 1-45, V _H 1-46, V _H 1-58, V _H 1-69, V _H 2-5, V _H 2-26, V _H 2-70, V _H 3-7, V _H 3-9, V _H 3- 11, V _H 3-13, V _H 3-15, V _H 3-16, V _H 3-20, V _H 3-21, V _H 3-23, V _H 3-30, V _H 3-30- 3, V _H 3-30-5, V _H 3-33, V _H 3-35, V _H 3-38, V _H 3-43, V _H 3-48, V _H 3-49, V _H 3- 53, V _H 3-64, V _H 3-66, V _H 3-72, V _H 3-73, V _H 3-74, V _H 4-4, V _H 4-28, V _H 4-30- 1, V _H 4-30-2, V _H 4-30-4, V _H 4-31, V _H 4-34, V _H 4-39, V _H 4-59, V _H 4-61, V _H 5-51, V _H 6-1, V _H 7-4-1, V _H 7-81, or a combination thereof.

[00293] In one embodiment, the nucleotide insert comprises one or more functional human D gene segments including D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2 -15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5 , D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27, or a combination thereof.

[00294] In one embodiment, the nucleotide insert comprises one or more functional J _H gene segments, including J _H 1, J _H 2, J _H 3, J _H 4, J _H 5, J _H 6, or a combination thereof. In one embodiment, the nucleotide insert contains one or more human Vκ gene segments, including Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8, Vκ1 -9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21 , Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3 -34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40, or a combination thereof.

[00295] In one embodiment, the nucleotide insert comprises one or more human Vλ gene segments including Vλ3-1, Vλ4-3, Vλ2-8, Yλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2- 14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, or a combination thereof.

[00296] In one embodiment, the nucleotide insert comprises one or more human Jκ gene segments comprising Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, or a combination thereof.

[00297] In specific embodiments of the invention, after modification

target locus in a pluripotent or non-pluripotent cell of a rat, eukaryote, non-rat eukaryote, non-human mammal, human, non-rat rodent, mouse or hamster, the genetic modification is transmitted through the germ line.

[00298] In one embodiment, the inserted nucleotide sequence comprises a polynucleotide that, when integrated into the genome, will result in a genetic modification of the rat ApoE locus region, a non-rat eukaryote eukaryote, a non-human mammal, a human, a non-rat rodent rodent, mouse or hamster, while genetic modification at the ApoE locus leads to a decrease in the activity of ApoE, an increase in the activity of ApoE, or modulation of the activity of ApoE. In one embodiment, the APOE is turned off.

[00299] In one embodiment, the inserted nucleotide

the sequence contains a polynucleotide that, when integrated into the genome, will lead to a genetic modification of the region of the interleukin-2 gamma receptor locus of the rat, eukaryote, non-rat eukaryote, non-human mammal, human, rodent, non-rat rodent, mouse or hamster, Moreover, genetic modification at the locus of the interleukin-2 gamma receptor leads to a decrease in the activity of the interleukin-2 receptor, an increase in the activity of the interleukin-2 gamma receptor, or modulation of the activity of the interleukin-2 receptor. In one embodiment, the interleukin-2 receptor is turned off.

[00300] In another embodiment, the inserted nucleotide

the sequence contains a polynucleotide that, when integrated into the genome, will result in a genetic modification of a region of the rat Rag1 locus, a non-rat eukaryote eukaryote, a non-human mammal, a human, a non-rat rodent, mouse or hamster, a rat Rag2 locus, non-rat eukaryote eukaryote, non-human mammal, human, non-rat rodent rodent, mouse or hamster and / or rat Rag2 / Rag1 locus, non-rat eukaryote eukaryote, non-human mammal, human , a rodent other than a rat rodent, mouse or hamster, wherein a genetic modification at the rat Rag1, Rag2 and / or Rag2 / Rag1 locus, a eukaryote, a non-rat eukaryote, a non-human mammal, a human, a non-rat rodent rodent, mouse or hamster leads to a decrease in the protein activity of Rag1, Rag2 or Rag1 and Rag2, an increase in protein activity and Rag1, Rag2 or Rag1 and Rag2, or modulation of protein activity by Rag1, Rag2 or Rag1 and Rag2. In one embodiment, a Rag1, Rag2, or Rag2 / Rag1 shutdown is generated.

[00301] In additional embodiments of the invention, the nucleotide insert results in the replacement of a portion of the ApoE locus, the interleukin-2 gamma receptor locus and / or the Rag2 locus, and / or the Rag1 locus and / or the Rag2 / Rag1 locus of a rat, eukaryotic, non-rat eukaryotic , a non-human mammal, a human, a non-rat rodent, mouse or hamster with a corresponding orthologous portion of the ApoE locus, the interleukin-2 gamma receptor locus and / or the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1 locus of another organism ...

[00302] In other embodiments, the nucleotide insert comprises a polynucleotide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity along its entire length, 97%, 98%, 99% with part of the ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus and / or the Rag2 / Rag1 locus that it replaces.

[00303] A given polynucleotide insert and corresponding displacement region of a rat, eukaryote, non-rat eukaryote, mammal, non-human mammal, human, rodent, non-rat, mouse, or hamster can be a coding region, an intron, an exon, an untranslated region, regulatory region, promoter or enhancer, or any combination thereof. In addition, a given polynucleotide insert and / or replacement region of a rat, eukaryote, non-rat eukaryote, mammal, non-human mammal, human, rodent, non-rat, mouse, or hamster can be of any desired length, including, for example, 10 -100 nucleotides, 100-500 nucleotides, 500-1 kb nucleotides, from 1 kb. up to 1.5 kbp nucleotides, from 1.5 kb. up to 2 kb nucleotides, from 2 kb. up to 2.5 kbp nucleotides, from 2.5 kb. up to 3 kbp nucleotides, from 3 kb. up to 5 kbp nucleotides, from 5 kb. up to 8 so. nucleotides, from 8 kb. up to 10 kbp nucleotides or more. In other cases, the size of the insert or replacement is from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. to about 350 kbp, from about 350 kbp. to about 400 kbp, from about 400 kbp. to about 800 kbp, from about 800 kbp. up to 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. to about 2 mpo, from about 2 mpo, to about 2.5 mpo, from about 2.5 mpo. up to about 2.8 ppm, from about 2.8 ppm. up to about 3 m.p. In other embodiments, the desired polynucleotide insert and / or replacement region of a rat, eukaryote, non-rat eukaryote, non-human mammal, human, non-rat, mouse, or hamster 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 kbp, 6 kbp, 7 kbp, 8 kbp, 9 kbp, 10 kbp, 11 kbp, 11 kbp. bp, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or more.

i. Methods for Modifying the Target Nucleic Acid Locus by Bacterial Homologous Recombination (BHR)

[00304] Methods and compositions are provided for modifying a target nucleic acid locus of a non-rat eukaryote, mammalian, human, or non-human mammal by means of bacterial homologous recombination (BHR) in a prokaryotic cell. Such methods find use in the use of bacterial homologous recombination in a prokaryotic cell to genetically modify a target nucleic acid locus of a non-rat eukaryote, mammalian, human, or non-human mammal to generate a targeting vector. Such a targeting vector containing a genetically modified target locus can be introduced into a eukaryotic cell, for example, a eukaryotic cell, a non-rat eukaryotic cell, a mammalian cell, a human cell, a non-human mammalian cell, a pluripotent cell, a non-pluripotent cell, a non-human pluripotent cell , human ES cell, human adult stem cell, human restricted progenitor cell, human iPS cell, human cell, rodent cell, non-rat rodent cell, rat cell, mouse cell, hamster cell, fibroblast, or CHO cell. "Homologous recombination" involves the exchange of DNA fragments between two DNA molecules at crossover sites within regions of homology. Thus, "bacterial homologous recombination" or "BHR" includes homologous recombination that occurs in bacteria.

[00305] Methods are provided for modifying a target nucleic acid locus from a eukaryotic cell, non-rat eukaryotic cell, mammalian cell, human cell, non-human mammalian cell, pluripotent cell, non-pluripotent cell, non-human pluripotent cell, human pluripotent cell, human ES cell, human adult stem cell, human restricted progenitor cell, human iPS cell, human cell, rodent cell, non-rat cell, rat cell, mouse cell, hamster cell, fibroblast, or CHO cell via bacterial homologous recombination (BHR). The methods include introducing into a prokaryotic cell a targeting vector containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, wherein the prokaryotic cell contains the target nucleic acid locus and is capable of expressing recombinase that mediates BHR at the target locus. Such direction vectors can include any of the large direction vectors described herein.

[00306] In one embodiment, the method comprises introducing into a prokaryotic cell: (i) a first construct comprising a nucleic acid having a DNA sequence of interest; (ii) a second targeting construct containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, and (iii) a third construct encoding a recombinase that mediates bacterial homologous recombination. In one embodiment of the invention, the first, second and third constructs are introduced into the prokaryotic cell separately over time. In one embodiment, the prokaryotic cell contains a nucleic acid that encodes a recombinase, and the method does not require the introduction of a third construct. In one embodiment, the recombinase is expressed under the control of an inducible promoter.

[00307] In one embodiment of the invention, the first construct containing the nucleic acid is derived from a bacterial artificial chromosome (IIR) or yeast artificial chromosome (DIR). You can select a prokaryotic cell containing a nucleotide insert at the target genomic locus. This method can be serially repeated, as described herein, to effect the introduction of multiple nucleotide insertions at a target locus in a prokaryotic cell. After “insertion” of the target nucleic acid locus into the prokaryotic cell, the targeting vector containing the modified target locus can be isolated from the prokaryotic cell and introduced into the target genomic locus in a eukaryotic cell, non-rat eukaryotic cell, mammalian cell, human cell, non-human mammalian cell, pluripotent cell, non-pluripotent cell, non-human pluripotent cell, human pluripotent cell, human ES cell, human adult stem cell, human restricted progenitor cell, human iPS cell, human cell, rodent cell, non-rat rat cell, rodent cell mouse cell, hamster cell, fibroblast, or CHO cell.

[00308] Preferred rat cells for producing targeting vectors are described in US Patent Application No. 14/185703, filed Feb. 20, 2014, the contents of which are summarized herein. These rat cells are pluripotent rat cells, capable of maintaining their pluripotency after one or more directed genetic modifications in vitro and capable of transmitting targeted genetic modifications through the germ line.

[00309] Electroporated pluripotent cells, for example, are seeded at high density to select drug resistant cells containing a targeting vector. The selection process for drug susceptibility results in the removal of most of the seeded cells (~ 99%), leaving individual colonies, each of which is a clone derived from a single cell. Among the remaining cells, the majority of cells (~ 80-100%) contain a targeting vector (containing a drug sensitivity selection cassette) integrated into a random region of the genome. Therefore, colonies are picked individually and genotyped to identify ES cells containing the targeting vector in the correct region of the genome (eg, using the allele modification analysis described below).

[00310] For genotyping, you can use a high-throughput quantitative analysis, namely, allele modification analysis (MOA). This analysis allows large-scale screening of the modified allele (s) on the parental chromosome after genetic modification. MOA analysis can be performed using a variety of analytical methods, including, but not limited to, quantitative PCR, such as real-time PCR (qPCR). For example, real-time PCR involves the use of a first primer set that recognizes a target locus and a second primer set that recognizes a non-target control locus. In addition, the primer group contains a fluorescent probe that recognizes the amplified sequence. In one embodiment of the invention, quantitation is performed using Invader Probes®. In one embodiment of the invention, quantitation is performed using MMP assays®. In one embodiment, the quantitation is performed using a TaqMan® Molecular Beacon. In one embodiment, the quantitation is performed using the Eclipse ™ probe method. (See, for example, US 2005/0144655, which is fully incorporated into this document by reference).

[00311] The selected pluripotent cell (i.e., non-human pluripotent cell, non-human ES cell) containing the targeted genetic modification can then be introduced into a host embryo, for example, an embryo at the pre-morula or blastocyst stage, and implanted into a surrogate mother's uterus to obtain a founder animal (FO animal). Thereafter, the founder animal can be crossed with a wild-type animal to produce F1 offspring heterozygous for genetic modification. Mating of heterozygous F1 animals can produce offspring that are homozygous for genetic modification. Mating of heterozygous F1 animals can produce offspring that are homozygous for genetic modification. In some embodiments, various genetic modifications of target loci described herein can be made using a large targeting vector (LTVEC), as detailed elsewhere in this document. For example, LTVEC can be obtained from bacterial artificial chromosome (IIR) DNA using the VELOCIGENE® genetic engineering method (see, for example, U.S. Patent No. 6,586,251 and Valenzuela, D. M. et al. (2003), High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotechnology 21 (6): 652-659, which are hereby incorporated by reference in their entirety).

[00312] The use of bacterial homologous recombination (BHR) to obtain a large targeting vector (LTVEC) removes the limitations arising from the impossibility of placing large fragments of genomic DNA in plasmids and the associated low efficiency of introducing targeted modifications at the endogenous locus in pluripotent or non-pluripotent cells. By obtaining LTVEC, one or more targeted genetic modifications can be made. An exemplary LTVEC produced in a prokaryotic cell may contain a nucleotide insert that carries a genomic sequence with one or more genetic modifications, or an exogenous nucleic acid (e.g., a rat nucleic acid homolog or ortholog) that is flanked by arms of homology complementary to specific genomic regions.

[00313] Also provided are prokaryotic host cells comprising the various targeting vectors described herein. Such prokaryotic cells include, but are not limited to, bacteria such as E. coli. In one embodiment, the prokaryotic host cell comprises a targeting vector containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, wherein the range of the nucleotide insert is from about 5 kb. up to about 200 kbp

[00314] The prokaryotic host cell may further comprise a nucleic acid that encodes a recombinase polypeptide, or a nucleic acid that encodes a recombinase polypeptide is operably linked to an inducible promoter.

[00315] Additionally, various methods and compositions are provided that use the LTVEC described herein in combination with a prokaryotic cell to generate targeted genetic modifications. Such compositions and methods are discussed elsewhere in this document.

[00316] Methods for modifying a target nucleic acid locus by means of bacterial homologous recombination (BHR) are proposed, which include introducing into a prokaryotic cell a targeting vector containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, wherein the prokaryotic cell contains nucleic acids, corresponding to the 5 'and 3' arms of homology, and is capable of expressing recombinase that mediates BHR at the target locus. Such direction vectors can include any of the large direction vectors described herein. In such methods, LTVEC can be used as discussed in detail in this document, as well as additionally apply the CRISPR / Cas system, as discussed elsewhere in this document.

[00317] In one embodiment, the CRISPR / Cas system may be under the control of a promoter active in a prokaryotic cell, such as, for example, E. coli.

ii. Methods for modifying a target locus of interest in a pluripotent cell or a non-pluripotent cell.

[00318] Additionally, there is provided a method for modifying a target locus of interest in a pluripotent cell or non-pluripotent cell by targeted genetic modification, comprising (a) introducing into a pluripotent cell or non-pluripotent cell a targeting vector containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm wherein the 5 'homology arm and the 3' homology arm add up to at least 10 kb; and (b) identifying a genetically modified pluripotent or non-pluripotent cell containing targeted genetic modification at the genomic locus of interest. In one embodiment, the 5 'homology arm and the 3' homology arm add up to at least about 16 kbp. up to about 30 kbp In specific embodiments of the invention, targeted genetic modification can be transmitted through the germ line. Such direction vectors can include any of the large direction vectors described herein.

[00319] Methods for modifying a target locus of interest as provided herein can also use a variety of cells. In specific embodiments of the invention, the cell is a eukaryotic cell, a non-rat eukaryotic cell, a pluripotent cell, a non-pluripotent cell, a non-human pluripotent cell, a human pluripotent cell, a human ES cell, a human adult stem cell, a human progenitor cell with limited development, induced human iPS), mammalian cell, human cell, fibroblast, rodent cell, non-rat rodent cell, mouse cell, hamster cell, or CHO cell.

[00320] In one aspect, there is provided a method for modifying a target locus of interest in a pluripotent cell by targeted genetic modification, comprising: (a) obtaining a pluripotent cell that is capable of retaining its pluripotency after at least one directed genetic modification of its genome and is capable of transmitting targeted modification germ line generation F1; (b) introducing a large targeting vector (LTVEC) into a pluripotent cell, wherein the LTVEC contains a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, wherein the 5 'homology arm and 3' homology arm contain a genomic DNA fragment; and (c) identifying a genetically modified pluripotent cell containing targeted genetic modification.

[00321] Various methods can be used to detect cells containing a nucleotide insert integrated into a target locus of interest. Insertion of a nucleic acid at the target locus of interest results in "allele modification". The term "allele modification" and methods for detecting a modified allele are discussed in more detail elsewhere in this document.

[00322] In one aspect, there is provided a method of modifying a target locus of interest in a non-pluripotent cell or pluripotent cell through endonuclease-mediated gene targeting, comprising: (a) obtaining an isolated non-pluripotent cell or isolated pluripotent cell that is capable of transmitting a genetically modified germline genome F1; (b) introducing an endonuclease agent into a non-pluripotent cell or a pluripotent cell; whereby the endonuclease agent creates a single-stranded or double-stranded break in the target DNA sequence located at the genomic locus of interest, and the single-stranded or double-stranded break in the target DNA sequence in a non-pluripotent cell or pluripotent cell induces: (i) non-homologous end-joining-mediated (NHSC) DNA repair at the site of a single-stranded or double-strand break, while NHSC-mediated DNA repair leads to the creation of a mutant allele containing the insertion or deletion of a nucleotide sequence in the target DNA sequence; or (ii) mediated by homologous recombination DNA repair, which leads to the restoration of the nucleotide sequence of the wild type; and (c) identifying the modified genomic locus of interest.

[00323] In one aspect, there is provided a method of modifying a target locus of interest in an isolated embryonic stem cell (ES) with a nuclease agent, comprising: (a) providing an isolated ES cell that is capable of transmitting an F1 generation directed germline modification; (b) introducing into an ES cell: (i) a large targeting vector (LTVEC) containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, wherein the insert is a nucleotide sequence of at least 5 kb. .; and (ii) an endonuclease agent, wherein the endonuclease agent creates a single-stranded or double-stranded break in the target DNA sequence located at the genomic locus of interest, and the target sequence is not present in the nucleotide insert; and (c) identifying targeted genetic modification in the embryonic stem (ES) cell.

[00324] In one aspect, there is provided a method of modifying a target locus of interest in a non-pluripotent cell or pluripotent cell by RNA-guided genetic engineering, comprising: (a) providing a non-pluripotent cell or pluripotent cell that is capable of transmitting a genetically modified F1 germline genome; (b) introducing into a non-pluripotent cell or pluripotent cell: (i) a first expression construct containing a first promoter operably linked to a first nucleotide sequence encoding a protein (Cas) associated with short palindromic repeats, regularly spaced groups (CRISPR), (ii ) a second expression construct comprising a second promoter operably linked to a target genomic sequence linked to a guide RNA (nRNA), the target genomic sequence being flanked by a sequence of a protospacer adjacent motif (PAM). Optionally, the target genomic sequence is flanked at the 3 'end by a protospacer contiguous motif (PAM) sequence. In one embodiment, the Cas protein and CRISPR RNA and / or tracrRNA do not occur together in a natural state (for example, the Cas protein and CRISPR RNA do not occur together in a natural state). In one embodiment, the target genomic sequence comprises the nucleotide sequence GNNNNNNNNNNNNNNNNNNNNGG (GN _1-20 GG; SEQ ID No. 1). In one embodiment, the target genomic sequence comprises SEQ ID No. 1, where N is 14 to 20 nucleotides in length. In one embodiment, the nRNA comprises a third nucleotide sequence encoding RNA of short palindromic repeats in regular clusters (CRISPR) (crRNA) and a fourth nucleotide sequence encoding trans-activating CRISPR RNA (tracrRNA). In one embodiment, upon expression, the Cas protein forms a CRISPR-Cas complex containing crRNA and tracrRNA, and the CRISPR-Cas complex creates a single-stranded or double-stranded break in the target DNA sequence located at the genomic locus of interest, and a single-stranded or double-stranded break at the target DNA sequence in a non-pluripotent cell or pluripotent cell induces: (i) non-homologous end-joining-mediated (NHSC) repair of DNA at the site of a single-stranded or double-stranded break created by the CRISPR-Cas complex, while the NHSC leads to the creation of a mutant allele containing an insertion or deletion of a nucleotide sequence in the target DNA sequence; or (ii) mediated by homologous recombination DNA repair, which leads to the restoration of the nucleotide sequence of the wild type; and (c) identifying the modified genomic locus of interest.

[00325] In one aspect, there is provided a method for modifying a target locus of interest in a non-pluripotent cell or pluripotent cell by RNA-guided genetic engineering, comprising introducing into a non-pluripotent cell or pluripotent cell that is capable of transmitting a genetically modified genome through the germ line: (i) associated with short palindromic repeats, regularly spaced groups (CRISPR), protein (Cas) or nucleic acid encoding the Cas protein; and (ii) an nRNA or DNA encoding an nRNA, the nRNA comprising a nucleotide sequence that hybridizes to a target genomic sequence and a trans-activating CRISPR RNA (tracrRNA); while the target genomic sequence is flanked by the sequence of the motif adjacent to the protospacer (PAM).

[00326] In some embodiments, the Cas protein can be introduced into a non-pluripotent cell or a pluripotent cell as an isolated protein. In some embodiments, the Cas protein further comprises a cell-penetrating domain that facilitates uptake of the protein by the cell. In other embodiments, the Cas protein can be introduced into the cell as a messenger RNA (mRNA) molecule encoding the Cas protein. In other embodiments, the Cas protein can be introduced into the cell as a DNA molecule encoding the Cas protein. For example, a DNA molecule encoding a Cas protein may be in a construct and operably linked to a promoter capable of expression in a non-pluripotent cell or a pluripotent cell. In certain embodiments of the invention, the nucleic acid encoding the Cas protein is codon-optimized for expression in a non-pluripotent cell or a pluripotent cell.

[00327] In some embodiments, the nRNA can be introduced into a non-pluripotent cell or a pluripotent cell as an RNA molecule. For example, an nRNA molecule can be transcribed in vitro. In other embodiments, the nRNA can be introduced into a non-pluripotent cell or a pluripotent cell as a DNA molecule encoding an nRNA. For example, a DNA molecule encoding an nRNA can be in a construct and operably linked to a promoter capable of expressing the nRNA in a non-pluripotent cell or a pluripotent cell. In other embodiments, the nRNA can be chemically synthesized.

[00328] In some embodiments, the nRNA can be introduced into a non-pluripotent cell or pluripotent cell as a crRNA-tracrRNA fusion molecule (ie, one transcript). In other embodiments, the nRNA can be introduced into a non-pluripotent cell or a pluripotent cell as separate crRNA and tracrRNA molecules (i.e., separate transcripts). In other embodiments, the nRNA can be introduced into a non-pluripotent cell or a pluripotent cell as separate DNA molecules encoding crRNA and tracrRNA, respectively. For example, individual DNA molecules encoding crRNA and tracrRNA can be in separate constructs and be operably linked to promoters capable of expressing nRNA in a non-pluripotent cell or a pluripotent cell. In any of the above-described embodiments of the invention, any combination of constructs can be in separate nucleic acid molecules or together in a single nucleic acid molecule.

[00329] In some embodiments, the Cas protein and nRNA can be introduced into a non-pluripotent cell or pluripotent cell simultaneously or sequentially. Similarly, crRNA and tracrRNA nRNA can be introduced into a non-pluripotent cell or pluripotent cell simultaneously or sequentially. The ratio between Cas protein (or coding nucleic acid) and nRNA (or coding DNA) and / or the ratio between crRNA and tracrRNA can be approximately stoichiometric so that they can form an RNA-protein complex.

[00330] In certain embodiments of the invention, the Cas protein can be introduced into a non-pluripotent cell or a pluripotent cell as a complex with an nRNA.

[00331] In one embodiment, the pluripotent cell is an induced pluripotent cell (iPS). In one embodiment, the pluripotent cell is a developmentally restricted progenitor cell.

[00332] The presence of a single-stranded or double-stranded break in the recognition site within a selectable marker in various embodiments increases the efficiency and / or frequency of recombination between the targeting vector (such as LTVEC) and the target locus of interest. In one embodiment, the recombination is homologous recombination. In another embodiment, the recombination is insertion due to non-homologous end joining. In various embodiments of the invention, in the case of a single-stranded or double-stranded break, the targeting efficiency of a targeting vector (such as LTVEC) at the target genomic locus is at least about 2 times higher, at least about 3 times higher, at least about 4 times higher than in the absence of a single-stranded or double-stranded break (when using, for example, the same targeting vector and the same homology arms and corresponding target regions in the genomic locus of interest, but without an added nuclease agent that creates a single-stranded or two chain break).

[00333] In one embodiment, the targeted genetic modification at the target locus is biallelic. By "biallelic" is meant that both alleles of a gene contain targeted genetic modification. In each allele, the targeted genetic modification can be the same or different. For example, a biallelic modification can be the result of one modification made in the corresponding alleles in the corresponding homologous chromosomes, or different modifications made in the corresponding alleles in the corresponding homologous chromosomes. Thus, a biallelic modification can lead, for example, to homozygosity for a specific modification at the genomic locus of interest (i.e., a specific modification in both alleles), compound heterozygosity at the genomic locus of interest (i.e., a specific modification in one allele and inactivation or disruption of another allele) or hemizygosity at the genomic locus of interest (e.g., a specific modification in one allele and loss of another allele). In certain embodiments of the invention, the combined use of a targeting vector (including, for example, LTVEC) with a nuclease agent results in a biallelic targeting genetic modification at the genomic locus of interest in the cell as compared to using a targeting vector alone. When a targeting vector is used in combination with a nuclease agent, the effectiveness of biallelic targeting is increased at least two-fold, at least three-fold, at least 4-fold, or more compared with the use of a single targeting vector. In additional embodiments of the invention, the effectiveness of biallelic targeting is at least 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9 %, 1%, 2%, 3%, 4%, or 5% or more.

[00334] Biallelic targeted genetic modification at the target locus can result in a homozygous genetically modified cell. By "homozygous" is meant that both alleles of the target locus (ie, alleles on both homologous chromosomes) have been modified in the same way. In certain embodiments of the invention, the combined use of a targeting vector (including, for example, LTVEC) with a nuclease agent results in a biallelic homozygous targeted genetic modification at the genomic locus of interest in the cell. In one embodiment, a biallelic genetic modification comprises removing an endogenous nucleotide sequence at a genomic locus of interest on two homologous chromosomes (i.e., a pair of first and second homologous chromosomes) and inserting nucleic acid at a genomic locus of interest on two homologous chromosomes (i.e. i.e. a pair of the first and second homologous chromosomes). In some embodiments, a nucleotide insert replaces an endogenous nucleotide sequence at a genomic locus of interest on both homologous chromosomes. In one embodiment, the nucleotide insert is homologous or orthologous to a deleted endogenous nucleotide sequence.

[00335] In one embodiment, targeted genetic modification at a target locus results in a hemizygous genetically modified cell. By "hemizygous" is meant that only one allele is present (ie, an allele on one of the two homologous chromosomes) of the target locus, or only one allele can be expressed and functioned. In other embodiments, targeted genetic modification more generally results in compound heterozygosity. Compound heterozygosity includes situations in which both alleles of the target locus have been modified (i.e., alleles on both homologous chromosomes), but modified in different ways (for example, insertion in one allele and inactivation or disruption of the other allele). In certain embodiments of the invention, the combined use of a targeting vector (including, for example, LTVEC) with a nuclease agent results in a hemispheric targeted genetic modification at the genomic locus of interest in the cell. In certain embodiments of the invention, the combined use of a targeting vector (including, for example, LTVEC) with a nuclease agent results in targeted genetic modifications that create compound heterozygosity at the genomic locus of interest in the cell. In one embodiment, targeted genetic modification at a genomic locus of interest on one chromosome comprises deletion of an endogenous nucleotide sequence and insertion of a nucleic acid. In other embodiments, targeted genetic modification comprises: (1) removing an endogenous nucleotide sequence at a genomic locus of interest on two homologous chromosomes; and (2) inserting a nucleic acid at the genomic locus of interest on the first chromosome and disrupting the genomic locus of interest on the second chromosome. The first chromosome may be the first of two homologous chromosomes, and the second chromosome may be the second of two homologous chromosomes. In other embodiments, targeted modification comprises: (1) removing an endogenous nucleotide sequence at a genomic locus of interest and inserting a nucleic acid at a genomic locus of interest on a first homologous chromosome; and (2) disruption of the genomic locus of interest on the second homologous chromosome. Disruption to an endogenous nucleotide sequence can occur, for example, when a nuclease-generated single-stranded or double-stranded break at a genomic locus of interest is repaired by non-homologous junction-mediated DNA repair (NSCR), resulting in a mutant allele containing an insertion or deletion of a nucleotide sequence in the genomic the genomic locus of interest, and therefore leads to the destruction of the genomic locus of interest. Examples of abnormalities include altering a regulatory element (e.g., a promoter or enhancer) at a genomic locus of interest, missense mutation, truncation mutation, null mutation, or small nucleotide insertion or deletion (e.g., resulting in frameshift mutations). Another example of a disorder is nonsense mutation. The disorder can lead to inactivation (i.e., loss of function) or loss of an allele.

[00336] Homozygous and hemizygous genetic modifications are advantageous because when genetically modified cells containing these mutations are used to produce genetically modified animals, as discussed below, the process of producing genetically modified animals that are non-heterozygous (i.e., homozygous or hemizygous) for the intended targeted genetic modification is more efficient and less time consuming as fewer breeding steps are required. Targeted genetic modifications leading to compound heterozygosity or hemizygosity (i.e., insertion in one allele and inactivation, disruption, or loss of another allele) may be advantageous for the same reason.

[00337] In any of the various methods described above for modifying a genomic locus with a nuclease agent, various cell types can also be used. In specific embodiments of the invention, the cell is a eukaryotic cell, a non-rat eukaryotic cell, a pluripotent cell, a non-pluripotent cell, a non-human pluripotent cell, a human pluripotent cell, a human ES cell, a human adult stem cell, a human progenitor cell with limited development, induced human iPS), mammalian cell, human cell, fibroblast, rodent cell, non-rat rodent cell, mouse cell, hamster cell, or CHO cell.

[00338] Compositions are provided that include the use of a genetically modified non-human animal having a genetic modification at the interleukin-2 gamma receptor locus or at the ApoE locus. Various methods and compositions provided herein allow these modified loci to be transmitted across the germline.

[00339] In certain embodiments, the genetically modified non-human animal or genetically modified pluripotent or non-pluripotent cell comprises a genomic locus containing a targeted genetic modification at an interleukin-2 gamma receptor locus or containing a targeted genetic modification at an ApoE locus, wherein the genomic locus of the receptor an interleukin-2 gamma or ApoE locus comprises: (i) removing at least a portion of the IL-2 gamma receptor locus or at least a portion of the ApoE locus; (ii) insertion of a heterologous nucleotide sequence at the ApoE locus or at the interleukin-2 gamma receptor locus; or (iii) a combination thereof, wherein the genetically modified genomic locus can be transmitted through the germ line.

[00340] In addition, methods are provided that allow the production of such genetically modified non-human animals or such genetically modified pluripotent cells. Such methods include a method of modifying a genomic ApoE locus or an interleukin-2 gamma receptor locus in a pluripotent cell through targeted genetic modification. The method comprises (a) introducing into a pluripotent cell a targeting vector containing a nucleotide insert flanked by a 5 'homology arm at the ApoE locus and a 3' homology arm at the ApoE locus, (b) identifying a genetically modified pluripotent cell containing a targeted genetic modification in the ApoE locus genomic locus ApoE, while targeted genetic modification can be transmitted through the germ line.

[00341] Additional methods include (a) introducing into a pluripotent cell a targeting vector containing a nucleotide insert flanked by a 5 'arm of homology at the IL-2 gamma receptor locus and a 3' arm of homology at the IL-2 gamma receptor locus, (b) genetic identification a modified pluripotent cell containing a targeted genetic modification at the locus of the interleukin-2 gamma receptor, while the targeted genetic modification can be transmitted through the germ line.

iii. Methods for Integrating Multiple Polynucleotides of Interest into a Target Locus

[00342] Various methods and compositions provided herein enable the targeted integration of multiple polynucleotides of interest into a given target locus. You can perform sequential repetition of the various methods described above to provide targeted integration of any number of nucleotide inserts into a given target locus. Thus, various methods involve inserting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleic acids at the target locus. In specific embodiments, such sequencing methods allow the reconstruction of large genomic regions from a eukaryotic cell, for example, a non-rat eukaryotic cell, a mammalian (i.e., human, non-human, non-rat rodent, mouse, monkey, rat, hamster , domestic mammal or farm animal) at the target locus. In such cases, the transfer and reconstruction of genomic regions, which contain both coding and non-coding regions, allows the complexity of a given region to be preserved by at least partially retaining the coding regions, non-coding regions and copy number variations inherent in the native genomic region. Thus, various methods provide, for example, methods of obtaining "heterologous" or "exogenous" genomic regions in any eukaryotic cell, any non-rat eukaryotic cell, any mammalian cell or animal of interest, in particular, in a prokaryotic host cell or in a non-pluripotent cell, pluripotent cell or ES cell. In one non-limiting example, a "humanized" genomic region is obtained in a non-human animal (ie, a rat). This document provides methods for obtaining genomic regions in any cell. In specific embodiments of the invention, the cell is a eukaryotic cell, a non-rat eukaryotic cell, a pluripotent cell, a non-pluripotent cell, a non-human pluripotent cell, a human pluripotent cell, a human ES cell, a human adult stem cell, a human progenitor cell with limited development, induced human iPS), mammalian cell, human cell, fibroblast, rodent cell, non-rat rodent cell, mouse cell, hamster cell, or CHO cell.

3. Humanized genomic locus

[00343] Provided herein are various methods and compositions involving the use of a humanized genomic locus. In the context of this document, a "humanized" genomic locus means a region of the non-human genome containing at least one human nucleotide sequence. A humanized genomic locus may contain a region of DNA from any organism into which a human DNA sequence has been inserted. In certain embodiments, the organism is a eukaryote, a non-rat eukaryote, a non-human mammal, a mammal, a human, a rodent, a non-rat, a rat, a mouse, or a hamster. For example, a "humanized rat locus" contains a region of rat DNA into which a human DNA sequence has been inserted.

[00344] The human DNA sequence may be a naturally occurring human DNA sequence, or may be modified from its native form. In specific embodiments of the invention, the human DNA sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the native human sequence. ... If a human sequence is not a native human sequence, it has greater sequence identity with a native human sequence than with an orthologous non-human sequence. In addition, the human DNA sequence may contain cDNA, a region of human genomic DNA, a non-coding regulatory region, or any portion of a coding, genomic, or regulatory region of human DNA. A human DNA sequence inserted at a non-human locus can contain any of the polynucleotide inserts as described elsewhere in this document. In certain embodiments, the human DNA sequence is orthologous to the non-human target locus, while in other instances, the human DNA sequence is homologous to the non-human target locus.

[00345] In one embodiment, the targeted genetic modification is the insertion or replacement of an endogenous nucleotide sequence with a homologous or orthologous human nucleotide sequence. In one embodiment, targeted genetic modification comprises the insertion or substitution of an endogenous nucleotide sequence with a homologous or orthologous human nucleotide sequence at an endogenous locus that contains the corresponding non-human nucleotide sequence.

[00346] Methods for producing a humanized locus include introducing a human nucleotide sequence into the target locus containing the nucleic acid. In one embodiment, the invention provides a method for producing a humanized non-human animal. Such a method includes (a) modifying the genome of a non-human pluripotent cell or non-pluripotent cell with a targeting vector containing a nucleotide insert that contains a human nucleotide sequence to form a donor cell; (b) introducing a donor cell into a host embryo; and (c) bearing the host embryo by a surrogate mother; while the surrogate mother produces offspring that contain the human nucleotide sequence. In specific embodiments of the invention, the humanized locus can be transmitted through the germ line. In an additional embodiment, the targeting vector comprises a large targeting vector (LTVEC) and the nucleotide insert that contains the human nucleotide sequence is at least 5 kb.

[00347] In other methods, a humanized genomic locus is obtained by modifying a target nucleic acid locus through bacterial homologous recombination (BHR). This method involves introducing into a prokaryotic cell a targeting vector containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, wherein the nucleotide insert contains a human nucleotide sequence, and the prokaryotic cell contains a nucleic acid that is capable of expressing recombinase that mediates BHR in target locus.

[00348] The humanized genomic locus may contain (a) an insert of a homologous or orthologous human nucleotide sequence; (b) replacement of an endogenous nucleotide sequence with a homologous or orthologous human nucleotide sequence; or (c) a combination of the above. In specific embodiments of the invention, a humanized genomic locus can be transmitted via the germline. In other embodiments, a human orthologous sequence replaces a corresponding sequence at a non-human locus.

[00349] Any human nucleotide sequence can be used in the methods and compositions provided herein. Non-limiting examples of human nucleotide sequences that can be used in methods and compositions are discussed in detail elsewhere in this document.

[00350] The human nucleotide sequence to be inserted at the locus of interest can be of any size. In one embodiment, the human nucleotide sequence can be from about 500 nucleotides to about 200 kb, from about 500 nucleotides to about 5 kb, from about 5 kb. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. up to about 30 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kbp, from about 60 kbp. to about 70 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. up to about 190 kbp or from about 190 kbp. up to about 200 kbp In a specific embodiment, the human nucleotide sequence is at least 5 kb.

[00351] In one embodiment, the invention provides a genomic locus in which a homologous or orthologous human nucleotide sequence comprises (a) one or more non-rearranged _{human immunoglobulin heavy chain V H} gene segments, one or more non-rearranged human immunoglobulin heavy chain D gene segments, and one or more non-rearranged _{human immunoglobulin heavy chain J H} gene segments that are operably linked to the nucleotide sequence of a mammalian heavy chain constant region; (b) rearranged nucleotide sequence of the variable region of the heavy chain of a human immunoglobulin, operably linked to the nucleotide sequence of the constant region of the heavy chain of a mammal; (c) one or more non-rearranged human immunoglobulin Vκ or Vλ gene segments and one or more non-rearranged _{human immunoglobulin Jκ or J λ} gene segments, which are operably linked to the nucleotide sequence of the constant λ region or to the light chain of mammalian immunoglobulin; or (d) a rearranged λ variable region or human immunoglobulin light chain nucleotide sequence operably linked to a λ constant region nucleotide sequence or a mammalian immunoglobulin light chain.

[00352] In another embodiment, the invention provides a genomic locus wherein (a) the mammalian heavy chain constant region nucleotide sequence is a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof; or (b) the nucleotide sequence of the λ constant region or the light chain of the mammalian immunoglobulin is a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof.

[00353] In a particular embodiment, the invention provides a genomic locus in which the immunoglobulin heavy chain constant region nucleotide sequence is selected from or comprises CH1, a hinge region, CH2, CH3, and / or a combination thereof.

[00354] In one embodiment, the genomic locus comprises one or more functional human V _H gene segments, including V _H 1-2, V _H 1-3, V _H 1-8, V _H 1-18, V _H 1- 24, V _H 1-45, V _H 1-46, V _H 1-58, V _H 1-69, V _H 2-5, V _H 2-26, V _H 2-70, V _H 3-7, V _H 3-9, V _H 3-1 1, V _H 3-13, V _H 3-15, V _H 3-16, V _H 3-20, V _H 3-21, V _H 3-23, V _H 3-30, V _H 3-30-3, V _H 3-30-5, V _H 3-33, V _H 3-35, V _H 3-38, V _H 3-43, V _H 3-48 , V _H 3-49, V _H 3-53, V _H 3-64, V _H 3-66, V _H 3-72, V _H 3-73, V _H 3-74, V _H 4-4, V _H 4-28, V _H 4-30-1, V _H 4-30-2, V _H 4-30-4, V _H 4-31, V _H 4-34, V _H 4-39, V _H 4 -59, V _H 4-61, V _H 5-51, V _H 6-1, V _H 7-4-1, V _H 7-81, or a combination thereof.

[00355] In one embodiment, a genomic locus comprises one or more functional human D gene segments, including D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2 -15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5 , D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27, or a combination thereof.

[00356] In one embodiment, a genomic locus comprises one or more functional human J _H gene segments, including J _H 1, J _H 2, J _H 3, J _H 4, J _H 5, J _H 6, and / or a combination thereof ... In one embodiment, the nucleotide insert contains one or more human Vκ gene segments, including Vκ4-1, Vκ5-2, Vκ 7-3, Vκ 2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8 , Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6 -21, Vκ1-22, Vκ-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33 , Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40, or a combination thereof.

[00357] In one embodiment, the genomic locus contains one or more human Vκ gene segments, including Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2- 14, Vλ-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, or a combination thereof.

[00358] In one embodiment, the genomic locus comprises one or more human Jκ gene segments, including Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, or a combination thereof.

[00359] In another embodiment, the invention provides a genomic locus comprising a humanized genomic locus containing the nucleotide sequence of a human interleukin-2 receptor (IL2R) or a variant or fragment thereof. In specific embodiments of the invention, the IL2R nucleotide sequence includes the nucleotide sequence of an interleukin-2 alpha receptor, an interleukin-2 beta receptor, or an interleukin-2 gamma receptor, or variants or fragments thereof.

[00360] In additional embodiments, the genomic locus comprises a humanized genomic locus consisting of a portion of the human ApoE locus, the human interleukin-2 gamma receptor locus, the human Rag2 locus, the human Rag1 locus, and / or the human Rag2 / Rag1 locus replacing the corresponding homologous or the orthologous portion of the non-human ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1 locus. In one embodiment, the non-human IL-2Rg ectodomain is replaced by the human IL-2Rg ectodomain and the remainder of the molecule is from a non-human animal.

[00361] In another embodiment, the invention provides a genetically modified non-human animal comprising a humanized genomic locus. Such genetically modified non-human animals comprise (a) an insert of a homologous or orthologous human nucleotide sequence; (b) substitution of a nucleotide sequence with a homologous or orthologous human nucleotide sequence at an endogenous genomic locus; or (c) a combination of the above, wherein the humanized genomic locus can be germ-line transmitted.

[00362] Also provided are genetically modified animals, including non-human animals, containing any of the various humanized genomic loci provided herein and described above.

4. Polynucleotides of interest

[00363] Any polynucleotide of interest can be present in the various nucleotide inserts and therefore integrate into the target locus. The methods disclosed herein provide at least 1, 2, 3, 4, 5, 6 or more polynucleotides of interest for integration into a target genomic locus.

[00364] Integration of a polynucleotide of interest located in a nucleotide insert into a target genomic locus can introduce one or more genetic modifications into a cell. Genetic modification can include the removal of an endogenous nucleotide sequence and / or the addition of an exogenous or heterologous or orthologous polynucleotide at the target genomic locus. In one embodiment, the genetic modification comprises replacing an endogenous nucleotide sequence with an exogenous polynucleotide of interest at a target genomic locus. Thus, the methods provided herein provide a genetic modification including knockout, deletion, insertion, substitution ("knockin"), point mutation, domain change, exon change, intron change, regulatory sequence change, gene change, or a combination thereof. Such modifications can occur after the integration of the first, second, third, fourth, fifth, sixth, seventh, or any subsequent nucleotide insertion into the target genomic locus.

[00365] The polynucleotide of interest, located in the nucleotide insert and / or integrated into the target locus, may contain a sequence that is native to the cell into which it is introduced; the polynucleotide of interest may be heterologous with respect to the cell into which it is introduced; the polynucleotide of interest may be exogenous to the cell into which it is introduced; the polynucleotide of interest may be orthologous to the cell into which it is introduced; or the polynucleotide of interest can be obtained from a species other than the cell into which it is introduced. In the context of this document, the expression "native" as used in relation to a sequence inserted at a target locus means a sequence that is native to the cell containing the target locus, or native to the cell from which the target locus was derived (i.e., from a cell rats). In the context of this document, the expression "heterologous" used in relation to a sequence includes a sequence that belongs to a foreign species, or, if it belongs to the same species, it is significantly different or modified from its native form in the composition and / or genomic locus due to deliberate human intervention. In the context of this document, the expression "exogenous", as used in relation to a sequence, refers to a sequence that belongs to an alien species. The polynucleotide of interest can be from any organism of interest, including, but not limited to, a non-human animal, a rodent, a non-rat rodent, a hamster, a mouse, a rat, a human, a monkey, an agricultural mammal, or a non-agricultural mammal. The polynucleotide of interest may further comprise a coding region, a non-coding region, a regulatory region, or genomic DNA. Thus, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and / or any of the subsequent nucleic acids may contain such sequences.

[00366] In one embodiment, the polynucleotide of interest present in the nucleotide insert and / or integrated into the target locus is native to a mouse nucleotide sequence, human nucleic acid, non-human nucleic acid, eukaryotic nucleic acid, non-rat eukaryotic nucleic acid, a non-human mammalian nucleic acid, a mammalian nucleic acid, a rodent nucleic acid, a non-rat rodent nucleic acid, a rat nucleic acid, a monkey nucleic acid, an agricultural mammal nucleic acid, or a non-agricultural mammal nucleic acid. In additional embodiments, the polynucleotide of interest integrated into the target locus is a genomic nucleic acid fragment. In one embodiment, the genomic nucleic acid is mouse genomic nucleic acid, human genomic nucleic acid, non-human nucleic acid, eukaryotic nucleic acid, non-rat eukaryotic nucleic acid, non-human mammalian nucleic acid, mammalian nucleic acid, nucleic acid, rodent a non-rat rodent nucleic acid, a rat nucleic acid, a monkey nucleic acid, an agricultural mammalian nucleic acid, or a non-agricultural mammal nucleic acid, or combinations thereof.

[00367] In one embodiment, the length of the polynucleotide of interest ranges from about 500 nucleotides to about 200 kb, as described above. The polynucleotide of interest can be from about 500 nucleotides to about 5 kb, from about 5 kb. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. up to about 30 kbp, from about 30 kbp. to about 40 kbp, from about 40 kbp. to about 50 kbp, from about 60 kbp. to about 70 kbp, from about 80 kbp. to about 90 kbp, from about 90 kbp. to about 100 kbp, from about 100 kbp. to about 110 kbp, from about 120 kbp. to about 130 kbp, from about 130 kbp. up to about 140 kbp, from about 140 kbp. to about 150 kbp, from about 150 kbp. to about 160 kbp, from about 160 kbp. to about 170 kbp, from about 170 kbp. to about 180 kbp, from about 180 kbp. up to about 190 kbp or about 190 kbp. to about 200 kbp, from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp

[00368] A polynucleotide of interest, located in a nucleotide insert and / or inserted into a target genomic locus, may encode a polypeptide, may encode siRNA, or may contain any regulatory regions of interest or non-coding regions, including, for example, a regulatory sequence, a promoter sequence, an enhancer sequence, transcriptional repressor-binding sequence, or deletion of a non-protein-coding sequence, but does not contain a deletion of the protein-coding sequence. In addition, the polynucleotide of interest, located in the nucleotide insert and / or inserted into the target genomic locus, can encode a protein expressed in the nervous system, skeletal system, digestive system, circulatory system, muscular system, respiratory system, cardiovascular system, lymphatic system, endocrine system, urinary system, reproductive system, or a combination thereof. In one embodiment, the polynucleotide of interest, present in the nucleotide insert and / or inserted at the target genomic locus, encodes a protein expressed in bone marrow or a bone marrow cell. In one embodiment, the polynucleotide of interest, present in the nucleotide insert and / or inserted at the target locus, encodes a protein expressed in a spleen cell. In additional embodiments, the polynucleotide of interest, located in the nucleotide insert and / or inserted at the target locus, encodes a protein expressed in a B cell, encodes a protein expressed in an immature B cell, or encodes a protein expressed in a mature B cell.

[00369] The polynucleotide of interest within the polynucleotide insert may contain a portion of the ApoE locus, the Il2rg locus, the Rag1 locus, the Rag2 locus, and / or the Rag2 / Rag1 locus. Such portions of these loci are discussed elsewhere in this document, as are various homologous and orthologous regions belonging to any organism of interest that can be used.

[00370] In one embodiment, the polynucleotide of interest, present in the nucleotide insert and / or inserted at the target locus, contains a genomic nucleotide sequence that encodes the amino acid sequence of an immunoglobulin heavy chain variable region. The expressions "heavy chain" or "heavy chain of immunoglobulin" are described elsewhere in this document.

[00371] In one embodiment, the polynucleotide of interest, located in the nucleotide insert and / or integrated into the target locus, contains a genomic nucleotide sequence that encodes the amino acid sequence of the variable region of the heavy chain of a human immunoglobulin.

[00372] In one embodiment, the genomic nucleotide sequence comprises one or more non-rearranged _{human immunoglobulin heavy chain V H} gene segments, one or more non-rearranged human immunoglobulin heavy chain D gene segments, and one or more non-rearranged human immunoglobulin heavy chain _{J H gene segments,} which are operably linked to the nucleotide sequence of the mammalian heavy chain constant region. In one embodiment, the genomic nucleotide sequence comprises a rearranged human immunoglobulin heavy chain variable region nucleotide sequence operably linked to a mammalian heavy chain constant region nucleotide sequence. In one embodiment, the genomic nucleotide sequence comprises one or more non-rearranged human immunoglobulin Vκ or Vλ gene segments and one or more non-rearranged human immunoglobulin Jκ or Jλ gene segments that are operably linked to the nucleotide sequence of a λ constant region or a mammalian immunoglobulin light chain. In one embodiment, the genomic nucleotide sequence comprises a rearranged λ or human immunoglobulin light chain nucleotide sequence operably linked to a λ constant region or mammalian immunoglobulin light chain nucleotide sequence. In one embodiment, the heavy chain constant region nucleotide sequence comprises a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof. In one embodiment, the immunoglobulin λ or κ light chain constant region nucleotide sequence comprises a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof.

[00373] In one embodiment, the nucleotide sequence of an immunoglobulin heavy chain constant region is selected from or comprises CH1, a hinge region, CH2, CH3, and / or a combination thereof. In one embodiment, the heavy chain constant region nucleotide sequence comprises a CH1 hinge region-CH2-CH3.

[00374] In one embodiment, the polynucleotide of interest, located in a nucleotide insert and / or integrated into a target locus, contains a genomic nucleotide sequence that encodes the amino acid sequence of an immunoglobulin light chain variable region. The expression "light chain" includes an immunoglobulin light chain sequence belonging to any organism and is described elsewhere in this document.

[00375] In one embodiment, the polynucleotide of interest, located in a nucleotide insert and / or integrated into a target genomic locus, contains a genomic nucleotide sequence that encodes the amino acid sequence of a human immunoglobulin light chain variable region.

[00376] In one embodiment, the genomic nucleotide sequence comprises one or more non-rearranged _{human immunoglobulin V κ} or V _λ gene segments and one or more non-rearranged _{human immunoglobulin J κ} or J _λ gene segments that are operably linked to the nucleotide sequence of the λ constant region or κ light chain of rodent immunoglobulin. In one embodiment, the genomic nucleotide sequence comprises a rearranged human immunoglobulin λ or κ light chain variable region nucleotide sequence operably linked to a rodent λ or κ light chain constant region nucleotide sequence. In one embodiment, the light chain constant region nucleotide sequence comprises a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof. In one embodiment, the immunoglobulin λ or κ light chain constant region nucleotide sequence comprises a rat constant region nucleotide sequence, a human constant region nucleotide sequence, or a combination thereof.

[00377] A polynucleotide of interest, located in the nucleotide insert and / or integrated into the target locus, can encode an extracellular protein or ligand for the receptor. In specific embodiments of the invention, the encoded ligand is a cytokine. Cytokines of interest include chemokines selected from or including CCL, CXCL, CX3CL and / or XCL. The cytokine can also include tumor necrosis factor (TNF). In other embodiments, the cytokine is interleukin (IL). In one embodiment, the interleukin is selected from or includes IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL- 23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and / or IL-36. In one embodiment, the interleukin is IL-2. In certain embodiments of the invention, such polynucleotides of interest, located in the nucleotide insert and / or integrated into the target genomic locus, are obtained from a person and, in more specific embodiments of the invention, may contain a human genomic sequence.

[00378] A polynucleotide of interest, located in the nucleotide insert and / or integrated into the target genomic locus, can encode apolipoprotein E (ApoE).

[00379] A polynucleotide of interest, located in the nucleotide insert and / or integrated into the target locus, can encode a cytoplasmic protein or membrane protein. In one embodiment, the membrane protein is a receptor, such as a cytokine receptor, an interleukin receptor, an interleukin-2 alpha receptor, an interleukin-2 beta receptor, an interleukin-2 gamma receptor, or a tyrosine kinase receptor. In other cases, the polynucleotide of interest, located in the nucleotide insert and / or integrated into the target locus, may contain an orthologous or homologous region of the target locus.

[00380] A polynucleotide of interest, located in a nucleotide insert and / or integrated into a target locus, may comprise a polynucleotide encoding at least one T cell receptor region, including the alpha T cell receptor. In specific methods, each of the nucleotide inserts contains a genomic region of a T cell receptor locus (ie, alpha T cell receptor locus) such that after serial integration is complete, part or all of the genomic T cell receptor locus is integrated into the target locus. Such nucleotide inserts may contain one or more variable segments or connecting segments of a T cell receptor locus (ie, T cell receptor alpha locus). In additional embodiments of the invention, the polynucleotide of interest encoding a T cell receptor region can be obtained, for example, from a polynucleotide encoding a mutant protein, a non-rat eukaryote polynucleotide, a non-human mammal, a non-rat rodent, a mouse, rat, human, monkey, hamster, agricultural mammal, or domestic mammal.

[00381] In other embodiments, the polynucleotide of interest integrated into the target locus encodes a nuclear protein. In one embodiment, the nuclear protein is a nuclear receptor. In specific embodiments of the invention, such polynucleotides of interest, located in the nucleotide insert and / or integrated into the target locus, are obtained from a person and, in more specific embodiments of the invention, may contain a human genomic sequence.

[00382] The polynucleotide of interest, located in the nucleotide insert and / or integrated into the target genomic locus, may contain a genetic modification in the coding sequence. Such genetic modifications include, but are not limited to, a deletion mutation of a coding sequence, or a fusion of two coding sequences.

[00383] A polynucleotide of interest, located in a nucleotide insert and / or integrated into a target locus, may comprise a polynucleotide encoding a mutant protein, including, for example, a human mutant protein. In one embodiment, the mutant protein has altered binding characteristics, altered localization, altered expression, and / or altered expression profile. In one embodiment, the polynucleotide of interest, located in the nucleotide insert and / or integrated into the target locus, contains at least one disease allele, including, for example, a neurological disease allele, a cardiovascular disease allele, a renal disease allele, a muscle disease allele, an allele a blood disease, an allele of a gene that leads to cancer, or an allele of a disease of the immune system. In such cases, the disease allele may be the dominant allele or the disease allele is the recessive allele. In addition, a disease allele may contain a single nucleotide polymorphism (SNP) allele. A polynucleotide of interest encoding a mutant protein can be obtained from any organism, including, but not limited to, a polynucleotide encoding a mutant protein, a polynucleotide, a non-rat eukaryote, a non-human mammal, a non-rat rodent, a mouse, a rat , human, hamster, monkey, farm mammal, or domestic mammal.

[00384] In one embodiment, genetic modification results in a mutant form of the protein with altered binding characteristics, altered localization, altered expression and / or altered expression profile.

[00385] In one embodiment, the genetic modification results in the removal, addition, substitution, or combination thereof, of a region of the ApoE locus, eg, the rat ApoE locus, wherein the genetic modification at the ApoE locus results in a decrease in ApoE activity. In one embodiment, the APOE is turned off.

[00386] In one embodiment, the genetic modification results in the removal, addition, substitution, or combination thereof, of a region of the Rag1 locus, eg, the rat Rag1 locus, wherein the genetic modification at the Rag1 locus results in decreased Rag1 activity. In one embodiment, Rag1 is turned off. In one embodiment, the genetic modification results in the removal, addition, substitution, or combination thereof, of a region of the Rag2 locus, for example, the rat Rag2 locus, wherein the genetic modification at the Rag2 locus results in decreased Rag2 activity. In one embodiment, the Rag2 is turned off. In one embodiment of the invention, the genetic modification results in the removal, addition, substitution, or combination thereof, of a region of the Rag1 / Rag2 locus, for example, the rat Rag1 / Rag2 locus, wherein genetic modification at the Rag1 / Rag2 locus results in a decrease in Rag1 activity and a decrease in Rag2 activity. In one embodiment, Rag1 / Rag2 is turned off.

[00387] In one embodiment of the invention, the result of genetic modification is the removal, addition, substitution, or combination thereof, of the region of the IL-2 gamma receptor locus, for example, the rat IL-2 gamma receptor locus, wherein the genetic modification at the IL-2 gamma receptor locus results in to reduce the activity of the receptor for interleukin-2 gamma. In one embodiment, the interleukin-2 gamma receptor is turned off.

[00388] As discussed elsewhere in this document, additional proposed embodiments of the invention include modifying one or more of the ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus, and / or the Rag2 / Rag1 locus, for example, the rat ApoE locus, the rat interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus and / or the Rag2 / Rag1 locus by replacing part of the rat ApoE locus, the interleukin-2 gamma receptor locus, the Rag2 locus, the Rag1 locus and / or the Rag2 / Rag1 locus with the corresponding orthologous part of the locus ApoE, interleukin-2 gamma receptor locus, Rag2 locus, Rag1 locus, and / or Rag2 / Rag1 locus from another organism.

[00389] In one embodiment, multiple genetic modifications are created. In one embodiment of the invention, the result of genetic modification is the removal, addition, substitution, or combination thereof, of the region of the IL-2 gamma receptor locus, for example, the rat IL-2 gamma receptor locus, while the genetic modification at the IL-2 gamma receptor locus leads to a decrease in activity receptor for interleukin-2 gamma, and the result of the second genetic modification is the removal, addition, substitution, or their combination of the region of the rat Rag2 locus, while the genetic modification at the Rag2 locus leads to a decrease in Rag2 activity. In one embodiment, the interleukin-2 gamma / Rag2 receptor is turned off. Such a rat has the SCID phenotype.

[00390] In one embodiment, the mammalian nucleic acid comprises a genomic locus that encodes a protein expressed in the nervous system, skeletal system, digestive system, circulatory system, muscular system, respiratory system, cardiovascular system, lymphatic system, endocrine system, urinary system , the reproductive system, or a combination thereof. In one embodiment, the mammalian nucleic acid comprises a genomic locus that encodes a protein expressed in bone marrow or a bone marrow cell. In one embodiment, the nucleic acid comprises a genomic locus that encodes a protein expressed in a spleen cell. In one embodiment, the genomic locus comprises a mouse genomic DNA sequence, a rat genomic DNA sequence, a human genomic DNA sequence, or a combination thereof. In one embodiment, the genomic locus contains, in any order, rat and human genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, murine and human genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, murine and rat genomic DNA sequences. In one embodiment, the genomic locus contains, in any order, rat, murine, and human genomic DNA sequences.

[00391] In one embodiment, the nucleotide insert comprises a genetic modification in the coding sequence of a gene. In one embodiment, the genetic modification comprises a deletion mutation in a coding sequence. In one embodiment, the genetic modification comprises the fusion of two endogenous coding sequences.

[00392] In one embodiment, the genetic modification comprises deletion of a non-protein coding sequence, but does not include deletion of a protein coding sequence. In one embodiment, the deletion of a non-protein coding sequence comprises deletion of a regulatory element. In one embodiment, the genetic modification comprises the addition of a promoter. In one embodiment, the genetic modification comprises substitution of a promoter or regulatory element. In one embodiment, the regulatory element is an enhancer. In one embodiment, the regulatory element is a transcriptional repressor binding element.

[00393] In one embodiment, the genetic modification comprises the substitution of a human nucleotide sequence encoding a mutant human protein. In one embodiment, the genetic modification comprises at least one human disease allele from a human gene. In one embodiment, the human disease is a neurological disease. In one embodiment, the human disease is a cardiovascular disease. In one embodiment, the human disease is kidney disease. In one embodiment, the human disease is a muscle disease. In one embodiment, the human disease is a blood disorder. In one embodiment, the human disease is cancer. In one embodiment, the human disease is a disease of the immune system. In one embodiment, the human disease allele is the dominant allele. In one embodiment, the human disease allele is a recessive allele. In one embodiment, the human disease allele comprises a single nucleotide polymorphism (SNP) allele.

[00394] A polynucleotide of interest, located in a nucleotide insert and / or integrated into a target locus, can also contain a regulatory sequence, including, for example, a promoter sequence, an enhancer sequence, or a transcriptional repressor binding sequence. In certain embodiments of the invention, the polynucleotide of interest, located in the nucleotide insert and / or integrated into the target genomic locus, contains a polynucleotide containing a deletion of a non-protein coding sequence, but does not contain a deletion of a protein coding sequence. In one embodiment, the deletion of a non-protein coding sequence comprises deletion of a regulatory sequence. In another embodiment of the invention, removal of a regulatory element comprises removal of a promoter sequence. In one embodiment of the invention, removal of a regulatory element comprises removal of an enhancer sequence. Such a polynucleotide of interest can be obtained from any organism, including, but not limited to, a mutant protein encoding a polynucleotide of a eukaryote, non-rat eukaryote, non-human mammal, mammal, non-rat rodent, mouse, rat, human, monkey , an agricultural mammal or a domestic mammal.

5. Methods for introducing sequences and obtaining transgenic animals

[00395] As indicated above, provided herein are methods and compositions for targeted integration of one or more polynucleotides of interest into a target locus. In such systems, many components are used and for convenience, the term "targeted integration system" as used herein generally includes all components necessary for integration (i.e., as non-limiting examples, these are various nuclease agents, recognition sites, polynucleotide DNA inserts , targeting vectors, genomic target locus and / or polynucleotides of interest).

[00396] Methods provided herein include introducing into a cell one or more polynucleotide or polypeptide constructs containing various components of a targeted genomic integration system. "Introduction" means the transfer of a sequence (polypeptide or polynucleotide) to a cell such that the sequence gains access to the interior of the cell. The methods provided herein are independent of the particular method for introducing any component of the directed genomic integration system into a cell, but only that the polynucleotide gains access to the interior of at least one cell. Methods for introducing polynucleotides into various cell types are known in the art and include, but are not limited to, stable transfection methods, transient transfection methods, and virus-mediated methods.

[00397] Any cells from any organism can be used in the methods provided herein. In specific embodiments, the cells are derived from a non-rat eukaryote, non-human mammal, human, non-rat, mouse, or hamster eukaryote. In certain embodiments, the cells are a eukaryotic cell, a non-rat eukaryotic cell, a pluripotent cell, a non-pluripotent cell, a non-human pluripotent cell, a non-human mammalian cell, a human pluripotent cell, a human ES cell, a human adult stem cell, a human progenitor developmental, human induced pluripotent (iPS) cell, mammalian cell, human cell, fibroblast, rodent cell, non-rat rodent cell, rat cell, mouse cell, hamster cell, or CHO cell.

[00398] In some embodiments, the cells used in the methods and compositions contain a DNA construct stably incorporated into their genome. "Stably incorporated" or "stably introduced" means the introduction of a polynucleotide into a cell in such a way that the nucleotide sequence is integrated into the genome of the cell and is able to be inherited by its offspring. Any protocol can be used to stably introduce DNA constructs or various components of a directed genomic integration system.

[00399] Transfection protocols, as well as protocols for the introduction of polypeptides or polynucleotide sequences, can vary. Non-limiting methods of transfection include chemical methods of transfection, including the use of 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); dendrimers; or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation; sonoporation; and optical transfection. Particle-based transfection involves the use of a gene gun, magnetic transfection (Bertram, J. (2006) Current Pharmaceutical Biotechnology 7, 277 28). Viral methods can also be used for transfection.

[00400] In one embodiment, the introduction of one or more polynucleotides into a cell is mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection ™ mediated.

[00401] In one embodiment, introducing one or more polynucleotides into a cell further comprises: introducing an expression construct comprising a nucleotide sequence of interest operably linked to a promoter. In one embodiment, the promoter is a constitutively active promoter. In one embodiment, the promoter is an inducible promoter. In one embodiment, the promoter is active in a stem cell, eg, an embryonic stem cell.

[00402] In one embodiment, the expression construct is co-administered with the LTVEC. In one embodiment, the expression construct is applied separately from the LTVEC over time.

[00403] In one embodiment of the invention, the introduction of one or more polynucleotides into a cell can be performed many times over a period of time. In one embodiment of the invention, the introduction of one or more polynucleotides into the cell is carried out at least twice over a period of time, at least three times over a period of time, at least four times over a period of time, at least five times over a period of for some time, at least six times for some time, at least seven times for some time, at least eight times for some time, at least nine times for some time, at least ten times for for some time, at least eleven times, at least twelve times for some time, at least thirteen times for some time, at least fourteen times for some time, at least fifteen times for some time, at least sixteen times for some time, at least seventeen times for some about a time, at least eighteen times over a period of time, at least nineteen times over a period of time, or at least twenty times over a period of time.

[00404] In one embodiment, the nuclease agent is introduced into the cell simultaneously with a targeting vector or large targeting vector (LTVEC). Alternatively, the nuclease agent is administered separately from the targeting vector or LTVEC over time. In one embodiment of the invention, the nuclease agent is administered prior to the insertion of the targeting vector or LTVEC, while in other embodiments of the invention, the nuclease agent is administered after the insertion of the targeting vector or LTVEC.

[00405] In one embodiment, the screening step includes quantitative analysis to assess allele modification (MOA) of the parent chromosome. In one embodiment of the invention, quantitative analysis is performed by quantitative PCR. In one embodiment, the quantitative PCR is real-time PCR (qPCR). In one embodiment, real-time PCR comprises using a first primer set that recognizes a target locus and a second primer set that recognizes a non-target control locus. In one embodiment, the primer group contains a fluorescent probe that recognizes the amplified sequence. In one embodiment, quantitative analysis is performed by fluorescence in situ hybridization (FISH). In one embodiment, the assay is performed by comparative genomic hybridization. In one embodiment of the invention, quantitative analysis is performed by isothermal DNA amplification. In one embodiment of the invention, quantitative analysis is performed by isothermal DNA amplification. In one embodiment, the assay is performed by quantitative hybridization with the immobilized probe (s). In one embodiment of the invention, quantitation is performed using Invader Probes®. In one embodiment of the invention, quantitation is performed using MMP assays®. In one embodiment, the quantitation is performed using a TaqMan® Molecular Beacon. In one embodiment, the quantitation is performed using the Eclipse ™ probe method. (See, for example, US2005 / 0144655, which is incorporated herein by reference in its entirety).

[00406] Additionally, there is provided a method for producing a humanized non-human animal, comprising: (a) modifying the genome of a pluripotent cell with a targeting vector containing a nucleotide insert that contains a human nucleotide sequence for forming a donor cell; (b) introducing a donor cell into a host embryo; and (c) bearing the host embryo by a surrogate mother; while the surrogate mother produces offspring that contain the human nucleotide sequence. In one embodiment, the donor cell is introduced into a host embryo that is at the blastocyst or pre-morula stage (i.e., 4-cell stage or 8-cell stage). In addition, step (a) can also be performed with a large targeting vector (LTVEC) and / or a human nucleotide sequence of at least 5 kb in length. In other embodiments of the invention, the genetic modification can be transmitted through the germ line.

[00407] Genetically modified non-human animals can be obtained using various methods disclosed herein. Such methods include (1) integrating one or more polynucleotides of interest at a target locus of a pluripotent cell to obtain a genetically modified pluripotent cell containing a nucleotide insert at the target genomic locus using the methods disclosed herein; (2) selecting a genetically modified pluripotent cell containing one or more polynucleotides of interest at the target genomic locus; (3) introduction of a genetically modified pluripotent cell into a host embryo; and (4) implanting a host embryo containing a genetically modified pluripotent cell into a surrogate mother. Get offspring from a genetically modified pluripotent cell. In one embodiment, the donor cell is introduced into a host embryo that is at the blastocyst or pre-morula stage (i.e., 4-cell stage or 8-cell stage). The offspring are obtained that can transmit the genetic modification through the germ line. The pluripotent cell can be an ES cell, as discussed elsewhere in this document.

[00408] Nuclear transfer techniques can also be used to produce genetically modified non-human animals. Briefly, the nuclear transfer method includes the steps of: (1) removing the nucleus from the oocyte; (2) isolating a donor cell or nucleus for introduction into an oocyte; (3) introducing a cell or nucleus into a non-nuclear oocyte to form a reconstituted cell; (4) implanting the reconstituted cell into the uterus of the animal to form an embryo; and (5) enabling the embryo to develop. In such methods, oocytes are generally obtained from dead animals, although they can also be isolated from the fallopian tubes and / or ovaries of living animals. Prior to removal of the nucleus, oocytes can mature in a variety of media known to those skilled in the art. Removal of a nucleus from an oocyte can be accomplished in a variety of ways well known to those skilled in the art. The introduction of a donor cell or nucleus into a nuclear-free oocyte to form a reconstituted cell is usually accomplished by microinjection of the donor cell under the zona pellucida prior to fusion. Fusion can be induced by passing a constant electrical pulse through the contact / fusion plane (electrofusion), treating cells with fusion-stimulating chemicals such as polyethylene glycol, or by means of an inactivated virus such as Sendai virus. The reconstituted cell is typically activated electrically and / or non-electrically before, during and / or after the fusion of the donor nucleus and the recipient oocyte. Methods of activation include electrical impulses, chemically induced shock, sperm penetration, increased levels of divalent cations in the oocyte, and decreased phosphorylation of cellular proteins (eg, through kinase inhibitors) in the oocyte. The activated reconstituted cells or embryos are typically cultured in a medium well known to those skilled in the art and then transferred to the uterus of the animal. See, for example, US 20080092249, WO / 1999/005266 A2, US 20040177390, WO / 2008/017234 A1, and US patent No. 7612250, all of which are incorporated herein by reference.

[00409] In one aspect, there is provided a method of producing a humanized non-human animal, comprising modifying a genomic locus of interest in a pluripotent cell using endonuclease-mediated gene targeting to modify the genomic locus of interest to obtain a modified pluripotent cell, maintaining the modified pluripotent cell under conditions sufficient to preserve pluripotency, the use of a modified pluripotent cell as a donor cell in a host embryo and bearing a host embryo containing a modified pluripotent cell by a surrogate mother, while the host embryo is carried by a surrogate mother and genetically modified offspring are born.

[00410] In one embodiment, the target sequence is located in an intron. In one embodiment, the target sequence is located in an exon. In one embodiment, the target sequence is located in a promoter. In one embodiment, the target sequence is located in the regulatory region of a promoter. In one embodiment, the target sequence is located in an enhancer region.

[00411] In one embodiment, the application step is performed many times over a period of time using a plurality of endonucleases that recognize different target sequences. In one embodiment, the step is performed at least twice over a period of time using a plurality of endonucleases that recognize different target sequences at least three times over a period of time using a plurality of endonucleases that recognize different target sequences at least four times over a period of time using a plurality of endonucleases that recognize different target sequences at least five times over a period of time using a plurality of endonucleases that recognize different target sequences at least six times over a period of time using a plurality of endonucleases that recognize different target sequences at least seven times over a period of time using a plurality of endonucleases that recognize different target sequences at least eight times over a period of time using a plurality of endonucleases that recognize t different target sequences at least nine times over a period of time using a plurality of endonucleases that recognize different target sequences at least ten times over a period of time using a plurality of endonucleases that recognize different target sequences at least eleven times over a period of time using a plurality of endonucleases that recognize different target sequences at least twelve times over a period of time using a plurality of endonucleases that recognize different target sequences at least thirteen times over a period of time using a plurality of endonucleases that recognize different target sequences at least fourteen times over a period of time using a plurality of endonucleases that recognize different target sequences at least fifteen times over a period of time using a plurality of endonucleases that recognize different target sequences at least sixteen times over a period of time using a plurality of endonucleases that recognize different target sequences at least seventeen times over a period of time using a plurality of endonucleases that recognize different target sequences at least eighteen times over a period of time using a plurality of endonucleases that recognize different target sequences at least nineteen times over a period of time using a plurality of endonucleases that recognize different target sequences or at least twenty times over a period of time using a plurality of endonucleases that recognize different target sequences.

[00412] In one embodiment, the application step is mediated by electroporation, intracytoplasmic injection, adenovirus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection ™ mediated.

[00413] In one embodiment, the method further comprises introducing an exogenous nucleic acid into a genetically modified pluripotent cell. In one embodiment, the exogenous nucleic acid is a transgene. In one embodiment, the exogenous nucleic acid is introduced into an endogenous locus. In one embodiment, the exogenous nucleic acid is introduced ectopically (eg, at a locus other than its endogenous locus).

[00414] In one aspect, there is provided a method for producing a humanized non-human animal, comprising modifying a genomic locus of interest in a pluripotent cell using RNA-driven genetic engineering to modify the genomic locus of interest to produce a modified pluripotent cell, keeping the modified pluripotent cell in conditions sufficient to preserve pluripotency, the use of a modified pluripotent cell as a donor cell in a host embryo and bearing a host embryo containing a modified pluripotent cell by a surrogate mother, while the host embryo is carried by a surrogate mother and genetically modified offspring are born.

[00415] In one embodiment, the targeting level for this method ranges from about 2% to about 80%.

[00416] In one embodiment, the method includes co-inserting multiple second expression constructs containing different target genomic sequences for multiple editing of different genomic loci. In one embodiment, the method comprises introducing a plurality of second expression constructs containing different target genomic sequences for multiple editing of different genomic loci over a period of time.

[00417] In one embodiment, the application step is performed multiple times over a period of time. In one embodiment, application step (b) is performed at least twice over a period of time, at least three times over a period of time, at least four times over a period of time, at least five times over a period of time at least six times over a period of time, at least seven times over a period of time, at least eight times over a period of time, at least nine times over a period of time, at least ten times over a period of time at least eleven times over a period of time, at least twelve times over a period of time, at least thirteen times over a period of time, at least fourteen times over a period of time, at least fifteen times over a period of time at least sixteen times during a period of time, at least seventeen times during a period of time, at least eighteen times during e period of time, at least nineteen times during a period of time, at least twenty times during a period of time.

[00418] In one embodiment, the first expression construct and the second expression construct are expressed from a single plasmid.

[00419] In one embodiment, the application step is mediated by electroporation, intracytoplasmic injection, adenovirus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection ™ mediated.

[00420] In one embodiment, the method further comprises introducing an exogenous nucleic acid into a pluripotent cell containing the mutant allele.

[00421] In one embodiment, the exogenous nucleic acid is a transgene. In one embodiment, the exogenous nucleic acid is introduced into an endogenous locus. In one embodiment, the exogenous nucleic acid is introduced ectopically (eg, at a locus other than its endogenous locus).

[00422] In one embodiment, the method further comprises introducing an exogenous nucleic acid into a genetically modified pluripotent cell. In one embodiment, the exogenous nucleic acid is a transgene. In one embodiment, the exogenous nucleic acid is introduced into an endogenous locus. In one embodiment, the exogenous nucleic acid is introduced ectopically (eg, at a locus other than its endogenous locus).

[00423] In one aspect, there is provided a method of producing a humanized non-human animal, comprising modifying a pluripotent cell with an LTVEC containing an insert that contains a human sequence of at least 5 kb, and using the pluripotent cell as a donor cell , the introduction of a donor cell into the host embryo and the gestation of the host embryo by a surrogate mother, while the surrogate mother produces offspring that contains humanization.

[00424] Other methods are provided for producing a genetically modified non-human animal containing in the germ line one or more genetic modifications described herein, including: (a) modifying a target locus contained in a prokaryotic cell using various methods described herein ; (b) selecting a modified prokaryotic cell containing a genetic modification at the target locus; (c) isolating the genetically modified targeting vector from the genome of the modified prokaryotic cell; (d) introducing a genetically modified targeting vector into a pluripotent cell to obtain a genetically modified pluripotent cell containing a nucleotide insert at the target genomic locus; (f) selection of a genetically modified pluripotent cell; (f) introducing a genetically modified pluripotent cell into a host embryo at the premorula stage; and (g) implanting a host embryo containing a genetically modified pluripotent cell into a surrogate mother to obtain an F0 generation derived from a genetically modified pluripotent cell. In such methods, the targeting vector may include a large targeting vector. The pluripotent cell can be an ES cell. In additional methods, the isolation step (c) further comprises (c1) linearizing the genetically modified targeting vector (i.e., the genetically modified LTVEC). In additional embodiments of the invention, the step of introducing (d) further comprises (d1) introducing a nuclease agent described herein into a pluripotent cell. In one embodiment, selection steps (b) and / or (e) are performed by applying a selective agent as described herein to a prokaryotic cell or pluripotent cell. In one embodiment, selection steps (b) and / or (e) are performed by allele modification analysis (MOA) as described herein.

[00425] Additional methods are proposed for modifying a target genomic locus of a mammalian cell by means of bacterial homologous recombination (BHR) in a prokaryotic cell, including: (a) obtaining a prokaryotic cell containing a target locus containing nucleic acid, (b) introducing a targeting vector into the prokaryotic cell, containing a nucleotide insert flanked by a 5 'homology arm and a 3' homology arm, wherein the nucleotide insert contains a mammalian region (including, for example, a human DNA insert), and (c) selection of a targeted prokaryotic cell containing a nucleotide insert at the target locus, while a prokaryotic cell is capable of expressing recombinase, which mediates BHR. Step (a1) may include obtaining a prokaryotic cell containing a target locus containing a nucleic acid containing a first polynucleotide containing a first recognition site for a first nuclease agent, and step (b1) may further include expressing in a prokaryotic cell a nuclease agent that creates a single-stranded or double strand break at or near the first recognition site. Steps (a) - (c) can be repeated serially, as described herein, to make it possible to introduce multiple nucleotide insertions at a target locus in a prokaryotic cell. After “insertion” of the target genomic locus into the prokaryotic cell, a targeting vector containing the modified target locus can be isolated from the prokaryotic cell and introduced into the target genomic locus in the pluripotent cell. Pluripotent cells (ie, ES cells) containing the modified genomic locus can then be transformed into genetically modified non-human animals.

[00426] In some embodiments, the various genetic modifications of targeted genomic loci described herein can be performed by a series of homologous recombination reactions (HGR) in bacterial cells using LTVEC derived from bacterial homologous chromosome (BHC) DNA using genetic engineering VELOCIGENE® (see, for example, U.S. Patent No. 6,586,251 and Valenzuela, D. M. et al. (2003), High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotechnology 21 (6): 652- 659, which are hereby incorporated by reference in their entirety).

[00427] In some embodiments of the invention, targeted ES cells containing various genetic modifications described herein are used as insertion ES cells and introduced into an embryo at the pre-morula stage of the corresponding organism, for example, a mouse embryo at an 8-cell stage, when using the VELOCIMOUSE® method (see, for example, US 7576259, US 7659442, US 7294754 and US 2008-0078000 A1, all of which are incorporated herein by reference in their entirety). An embryo containing genetically modified ES cells is incubated to the blastocyst stage and then implanted into a surrogate mother's body to obtain F0. Animals carrying a modified genomic locus can be identified by allele modification analysis (MOA) as described herein. The resulting non-human animal of the F0 generation, obtained from genetically modified ES cells, is crossed with a non-human wild-type animal to obtain the offspring of the F1 generation. After genotyping with specific primers and / or probes, non-human F1 animals that are heterozygous for the genetically modified genomic locus are crossed with each other to produce animals that are homozygous for the genetically modified genomic locus. Alternatively, a cross between a female F0 of a non-human animal and a male F0 of a non-human animal that have a genetic modification can be performed to produce a non-human F1 animal homozygous for the genetic modification.

[00428] In one aspect, there is provided, for example, a genetically modified rat genome comprising directed modification of an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence from another organism.

[00429] In one embodiment, the length of the homologous or orthologous nucleotide sequence is from about 5 kb. up to about 200 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 5 kb. up to about 10 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 10 kb. up to about 20 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 20 kb. up to about 30 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 30 kb. up to about 40 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 40 kb. up to about 50 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 50 kb. up to about 60 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 60 kbp. up to about 70 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 70 kbp. up to about 80 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 80 kb. up to about 90 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 90 kb. up to about 100 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 100 kb. up to about 110 kbp. In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 110 kb. up to about 120 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 120 kb. up to about 130 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 140 kb. up to about 150 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 150 kb. up to about 160 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 160 kb. up to about 170 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 170 kb. up to about 180 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 180 kb. up to about 190 kbp In one embodiment, the homologous or orthologous non-rat nucleotide sequence ranges from about 190 kb. up to about 200 kbp Various polynucleotides of interest that can be used in a nucleotide insert are described elsewhere in this document.

[00430] Additional methods for targeted modification of the genome of a non-human animal are provided. Such methods may include (a) modifying a genomic locus of interest in a non-human pluripotent cell in accordance with any of the methods provided herein for modifying a genomic locus of interest to thereby obtain a genetically modified non-human pluripotent cell containing targeted genome modification; (b) introducing a modified non-human pluripotent cell from step (a) into a non-human host embryo; and (c) bearing a non-human host embryo containing a modified pluripotent cell by a surrogate mother, wherein the surrogate mother produces F0 offspring containing targeted genome modification, and the targeted genome modification can be transmitted through the germ line.

[00431] In some embodiments, targeted genome modification comprises the simultaneous removal of an endogenous nucleotide sequence at a genomic locus of interest and insertion of an exogenous nucleic acid at a genomic locus of interest (ie, deletion and insertion in a single step). In some embodiments, the targeted genome modification comprises a biallelic genetic modification. Biallelic genetic modification may involve the removal of an endogenous nucleotide sequence and the insertion of an exogenous nucleic acid at a genomic locus of interest on two homologous chromosomes (ie, a pair of first and second homologous chromosomes).

[00432] In other embodiments, targeted genome modification results in a modified pluripotent cell that is compound heterozygous at the genomic locus of interest. In other embodiments, targeted genome modification results in a modified pluripotent cell that is hemizygous at the genomic locus of interest. In some embodiments, targeted genome modification at a genomic locus of interest on a single chromosome comprises deletion of an endogenous nucleotide sequence and insertion of an exogenous nucleic acid. For example, targeted genome modification can include: (1) removing an endogenous nucleotide sequence at a genomic locus of interest on two homologous chromosomes; and (2) inserting an exogenous nucleic acid at the genomic locus of interest on the first chromosome and disrupting the genomic locus of interest on the second chromosome. The first chromosome may be the first of two homologous chromosomes, and the second chromosome may be the second of two homologous chromosomes.

6. Cells

[00433] Various methods and compositions described herein employ a system for targeting a genomic locus in a cell. In one embodiment, the cell is a pluripotent cell. In one embodiment, the cell is a non-pluripotent cell. In one embodiment, the pluripotent cell is a non-human pluripotent cell. In one embodiment, the non-human pluripotent cell is a mammalian pluripotent cell. In one embodiment, the pluripotent cell is a human induced pluripotent stem (iPS) cell.

[00434] In other embodiments, the cell is a eukaryotic cell, a non-rat eukaryotic cell, a human pluripotent cell, a human ES cell, a human adult stem cell, a restricted human progenitor cell, a non-human mammalian cell, a mammalian cell, a human cell, fibroblast, rodent cell, non-rat rodent cell, rat cell, mouse cell, hamster cell, or CHO cell.

[00435] In one embodiment, the eukaryotic cell is the primary cell. Primary cells include cells or cell cultures that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. They include any cell obtained from an organism, organ, or tissue that has not previously been passaged in tissue culture or that has previously been passaged in tissue culture, but which cannot be passaged indefinitely in tissue culture. Such cells can be isolated by conventional methods and include, for example, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, adipocytes, preadipocytes, neurons, glial cells, hepatocytes, and skeletal myoblasts. smooth muscles. In some embodiments, the primary cells are derived from connective tissue, muscle tissue, nervous system tissue, or epithelial tissue.

[00436] In another embodiment, the eukaryotic cell is an immortalized cell. Immortalized cells include cells from a multicellular organism, which normally could not proliferate indefinitely, but due to mutation or change have escaped the process of cellular senescence and continue to divide instead. Such mutations or changes can occur naturally or can be intentionally induced. Examples of immortalized cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (eg, HEK 293 cells), and mouse embryonic fibroblasts (eg, 3T3 cells). Many types of immortalized cells are known in the art.

[00437] In some embodiments, the immortalized cells are derived from cancer cells. In another embodiment, the primary or immortalized cell is one that is typically used for culture or for expression of recombinant genes or proteins.

[00438] In other embodiments of the invention, a pluripotent cell is capable of retaining its pluripotency after at least one directed genetic modification of its genome and is capable of transmitting directed germline modification of the F1 generation.

[00439] In one embodiment, the pluripotent cell is a non-human fertilized egg at the unicellular stage. In one embodiment, the non-human fertilized egg is a fertilized mammalian egg. In one embodiment of the invention, the fertilized mammalian egg is a fertilized rodent egg at the unicellular stage. In one embodiment, the fertilized mammalian egg is a single-celled fertilized mouse or rat egg.

[00440] Various cells used in the methods and compositions described herein can also include prokaryotic cells, such as bacterial cells, including E. coli. In specific embodiments of the invention, the prokaryotic cell is a recombination-competent E. coli strain. In one embodiment, a prokaryotic cell contains a nucleic acid that encodes a recombinase, while in other cases, a prokaryotic cell does not contain a nucleic acid that encodes a recombinase, and the nucleic acid that encodes a recombinase is introduced into the prokaryotic cell. In one embodiment, the nucleic acid encoding the recombinase comprises DNA or mRNA. In some embodiments, the nucleic acid encoding the recombinase is pABG. In one embodiment, the recombinase is expressed under the control of an inducible promoter. In one embodiment, the expression of the recombinase is driven by arabinose.

A. Medium with low osmolarity for the production and maintenance of human induced pluripotent cells

[00441] Provides a cell culture medium for use in the methods and compositions of the invention. In one embodiment, the medium is suitable for generating a population of human iPS cells. In another embodiment, the medium is suitable for maintaining human iPS cells in culture. In some embodiments of the invention, human IPSs are naive or naive-like.

[00442] A medium provided herein contains at least a basic medium, a leukemia inhibiting factor (LIF) polypeptide, a glycogen synthase kinase 3 (GSK3) inhibitor, and a MEK inhibitor.

[00443] This medium is a low osmolarity medium. In one example, the osmolarity is about 175-280 mOsm / kg. In additional examples, the osmolarity of the medium is about 180-270 mOsm / kg, about 200-250 mOsm / kg, about 220-240 mOsm / kg, or about 225-235 mOsm. In a particular embodiment, the osmolarity of the medium is about 233 mOsm / kg.

[00444] The basic medium of the invention is a low osmolarity basic medium to which additional components are added. This basic medium differs from the basic media typically used to maintain human iPS cells in culture, which include Dulbecco's Modified Eagle's Medium (DMEM) in various forms (eg, DMEM from Invitrogen, Cat. # 1 1971-025) and Low salt DMEM available commercially under the name KO-DMEM ™ (Invitrogen Cat # 10829-018).

[00445] The basic medium of the invention is a basic medium with low osmolarity, but has characteristics that are not limited to low osmolarity. For example, the DMEM formulation shown in Table A can be made suitable for the purposes of the invention by varying the concentrations of sodium chloride and / or sodium bicarbonate as suggested herein, which will result in a change in osmolarity compared to standard DMEM basic medium or DMEM basic medium with low salt content (KO-DMEM) shown in Table A.

[00446]

[00447] This basic medium may contain an alkali metal salt and a halide such as sodium chloride (NaCl). Typical NaCl concentrations in basic media include 50 ± 5 mM, or about 3 mg / ml.

[00448] In another embodiment of the invention, some concentration of carbon dioxide is observed in the basic environment. The carbonic acid salt can be sodium salt. In such an example, the sodium salt may be sodium bicarbonate. In a specific embodiment, sodium bicarbonate is present in the basic medium at a concentration of about 26 ± 5 mM, or about 2.2 mg / ml.

[00449] In another embodiment, the basic medium is a low osmolarity basic medium. The osmolarity of the base medium can be in the range of about 175-280 mOsm / kg, about 180-250 mOsm / kg, about 190-225 mOsm / kg, or about 195-205 mOsm / kg. Typical osmolarity of the basic medium can be 200, 214, 216, or 218 mOsm / kg. In a specific example, the osmolarity of the basic medium is 200 mOsm / kg. Osmolarity can be determined when cells are cultured with different CO2 concentrations. In some examples, cells are cultured in 3% CO ₂ or 5% CO ₂ .

[00450] In a preferred embodiment, the basic medium contains NaCl at a concentration of 3.0 mg / ml, sodium bicarbonate at a concentration of about 2.2 mg / ml and has an osmolarity of 200 mOsm / kg.

[00451] The additives included in the basic medium of the invention are suitable for obtaining, maintaining, or enriching the human iPS cells disclosed herein. In the present description, such additives are referred to as "additives" or "+ additives". The term "additives" or the expression "+ additives" includes one or more additional elements added to the components of the basic medium shown in Table A. For example, additives can include, without limitation, F-12® medium (Gibco), N2® additive ( Gibco; 100X solution), NEUROB A SAL® medium (Gibco), B-27® supplement (Gibco; 5 0X solution), L glutamine, glucose, 2 mercaptoethanol, leukemia inhibiting factor (LIF) polypeptide, glycogen synthase 3 kinase inhibitor ( GSK3), MEK inhibitor, or any combination thereof.

[00452] In a specific embodiment, the LIF polypeptide is a human LIF polypeptide (hLIF). In some examples, the LIII polypeptide is used at a concentration of about 1-1000 units / ml, about 20-800 units / ml, about 50-500 units / ml, about 75-250 units / ml, or about 100 units / ml.

[00453] In another specific embodiment, the GSK3 inhibitor comprises CHIR99021. In some examples, CHFR99021 is used at a concentration of about 0.1 to 10 μM, about 1-5 μM, about 2-4 μM, or about 3 μM.

[00454] In another specific embodiment, the MEK inhibitor comprises PD0325901. In some examples, PD0325901 is used at a concentration of about 0.1-5 μM, about 0.2-1 μM, about 0.3-0.7 μM, or about 0.5 μM.

[00455] A typical medium contains about 24.75% (v / v) of the basic low osmolarity medium described herein, about 24.75% (v / v) of F-12 medium, about 0.5% (v / v ) N2 supplements, about 49% (v / v) NEUROBASAL medium, about 1% (v / v) B-27 supplements, about 2 mM L-glutamine, about 0.1 mM 2 mercaptoethanol, about 100 units / ml hLIF, about 3 μM CHIR99021 and about 0.5 mkMPD0325901.

[00456] In another specific embodiment, the medium may or may not contain basic fibroblast growth factor (bFGF, also known KaKFGF2 or FGF-p). Preferably, this medium does not contain bFGF.

B. Human induced pluripotent stem cells

[00457] Provided herein are methods and compositions for generating a population of human iPS cells. Additionally, methods and compositions are provided for maintaining human iPS cells in culture. Also proposed are human iPS cells that are obtained or maintained in culture.

[00458] The term "pluripotent cell" or "pluripotent stem cell" includes an undifferentiated cell that has the ability to develop into more than one type of differentiated cell. Such pluripotent cells can be, for example, mammalian embryonic stem cells (ES cells) or mammalian induced pluripotent stem cells (iPS cells). Examples of pluripotent cells include human iPS cells.

[00459] The term "embryonic stem cell" or "ES cell" means an embryo-derived totipotent or pluripotent stem cell obtained from the inner cell mass of a blastocyst that can be maintained in vitro under suitable conditions. ES cells are capable of differentiating into cells of any of the three germ layers of vertebrates, for example, endoderm, ectoderm, or mesoderm. ES cells are also characterized by their ability to multiply indefinitely under suitable in vitro culture conditions. See, for example, Thomson et al. (Science (1998) Vol. 282 (5391), pp. 1145-1147).

[00460] The term "induced pluripotent stem cell" or "iPS cell" includes a pluripotent stem cell that can be obtained directly from a differentiated adult cell. Human iPS cells can be obtained by introducing special groups of reprogramming factors into a non-pluripotent cell, which can include, for example, Oct3 / 4, transcription factors of the Sox family (for example, Sox1, Sox2, Sox3, Sox 15), transcription factors of the Myc family (for example, with -Mus, 1-Myc, n-Myc), the Kruppel-like (KLF) family of transcription factors (e.g., KLF1, KLF2, KLF4, KLF5) and / or related transcription factors such as NANOG, LIN28 and / or Glisl. Human iPS cells can also be obtained, for example, by using miRNAs, small molecules that mimic the actions of transcription factors, or lineage specifiers. Human iPS cells are characterized by their ability to differentiate into any cells of the three germ layers of vertebrates, for example, endoderm, ectoderm, or mesoderm. Human iPS cells are also characterized by their ability to multiply indefinitely under suitable in vitro culture conditions. See, for example, Takahashi and Yamanaka (Cell (2006) Vol. 126 (4), pp. 663-676).

[00461] The terms "naive" or "primed" indicate a different state of pluripotency of human iPS cells. The term "naive-like" refers to a cell expressing a pluripotent state that exhibits one or more of the characteristics of a naive pluripotent cell. Naive-like human iPS cells may also be referred to as "conditionally naive" human iPS cells. In some embodiments of the invention, naive-like human iPS cells exhibit one or more morphological characteristics of naive human iPS cells, for example, a morphology characterized by compact hemispherical colonies. In some embodiments, naive-like human iPS cells express one or more of the pluripotency markers described herein. In some embodiments, the naive or naive-like human iPS cells are naive human iPS cells. In other embodiments, naive or naive-like human iPS cells are naive-like human iPS cells.

[00462] The characteristics of naive and primed iPS cells are described in the art. See, for example, Nichols and Smith (Cell Stem Cell (2009) Vol. 4 (6), pp. 487-492). Naive human iPS cells exhibit a state of pluripotency similar to ES cells of the inner cell mass of a preimplantation embryo. Such naive cells are not primed in terms of the specification and determination of differentiation. Female naive iPS cells are characterized by the presence of two active X chromosomes. Self-renewal of naive human iPS cells in culture is dependent on leukemia inhibitory factor (LIF) and other inhibitors. Cultured naive human iPS cells exhibit a clonal morphology characterized by hemispherical colonies and lack of apico-basal polarity. Cultured naive cells can also exhibit one or more pluripotency markers described elsewhere in this document. Under suitable conditions, the doubling time of naive human iPS cells in culture can be between 16 and 24 hours.

[00463] Primed human iPS cells express a state of pluripotency similar to postimplantation epiblast cells. Such cells are primed for the specification and determination of differentiation. Female iPS-primed cells are characterized by the presence of one active X chromosome and one inactive X chromosome. Self-renewal of primed human iPS cells in culture is dependent on fibroblast growth factor (FGF) and activin. Cultured primed human iPS cells exhibit a clonal morphology characterized by an epithelial monolayer and exhibit apico-basal polarity. Under suitable conditions, the doubling time of primed human iPS cells in culture can be 24 hours or more.

[00464] In one embodiment, human iPS cells can be obtained from non-pluripotent cells transformed to express a pluripotent state. Such transformed cells include, for example, cells that have been transformed to express reprogramming genes that induce pluripotency. A pluripotent state can include, for example, the expression of one or more pluripotency markers described herein. Such cells (such as human foreskin fibroblasts) can be transformed to express reprogramming genes, or any additional genes of interest, by any means known in the art. See, for example, Takahashi and Yamanaka (Cell (2006) Vol. 126 (4), pp. 663-676). For example, they can be introduced into cells using one or more elements from plasmids, lentiviral vectors, or retroviral vectors. In some cases, vectors are integrated into the genome and can be removed after reprogramming is complete. In specific embodiments of the invention, non-pluripotent cells are transformed with reprogramming genes, including Oct4, Sox2, Klf4, Myc, or any combination thereof. In some examples, the transformed cells include primed human iPS cells.

[00465] In some embodiments, human iPS cells cultured in a low osmolarity medium described herein express one or more phenotypes, gene expression profiles, or markers characteristic of the naive state. In one example, human iPS cells express one or more pluripotency markers characteristic of the naive state. Such pluripotency markers may include alkaline phosphatase, NANOG, 5T4, ABCG2, Activin RIB / ALK-4, Activin RIIB, E-cadherin, Cbx2, CD9, CD30 / TNFRSF8, CD117 / c-kit, CDX2, CHD1, Cripto, DNMT3B, DPPA2, DPPA4, DPPA5 / ESG1, EpCAM / TROP1, ERR beta / NR3B2, ESGP, protein F-box 15 / FBXO15, FGF-4, FGF-5, FoxD3, GBX2, GCNF / NR6A1, GDF-3, Gi24 / VISTA / B7-H5, integrin alpha 6 / CD49f, integrin alpha 6 beta 1, integrin alpha 6 beta 4, integrin beta 1 / CD29, KLF4, KLF5, L1TD1, Lefty, Lefty-1, Lefty-A, LIN-28A, LIN -28B, LIN-41, cMaf, cMyc, Oct-3/4, Oct-4A, podocalyxin, Rex-1 / ZFP42, Smad2, Smad2 / 3, SOX2, SSEA-1, SSEA-3, SSEA-4, STAT3 , Stella / Dppa3, SUZ12, TBX2, TBX3, TBX5, TERT, TEX 19, TEX19.1, THAP11, TRA-1-60 (R), TROP-2, UTF1 and / or ZIC3. In a specific example, the expressed pluripotency marker is alkaline phosphatase, NANOG, or both.

[00466] In another embodiment, human iPS cells cultured in a low osmolarity medium described herein exhibit morphological characteristics characteristic of the naive state. Typical morphology is characterized by cells forming compact hemispherical colonies in culture.

[00467] In another embodiment, human iPS cells cultured in a low osmolarity medium described herein can be mechanically or enzymatically separated to a single cell suspension, passaged and / or subcultured. In one example, enzymatic separation can be performed using trypsin. When cultured in the presence of low osmolarity media, human iPS cells can provide greater transformation efficiency due to improved dissociation into a single cell suspension. For other types of media (eg mTeSR ™ media or 2i media) commonly used to maintain human iPS cells in culture, the separation of human iPS cells must be performed mechanically or using enzymes such as collagenase that are less rigid than trypsin. Accordingly, cells do not dissociate as efficiently or as completely. In contrast, in the presence of low osmolarity media, trypsin can be used to separate cells, and improved dissociation leads to increased transformation efficiency. In addition, unlike other types of media commonly used to maintain human iPS cells in culture (e.g., mTeSR ™ media or medium 2i), enzymatic separation of human iPS cells cultured in the presence of low osmolarity media (preferably low osmolarity media, not containing bFGF) can be carried out in the absence of one or more inhibitors, which are generally required for passage of such cells. A typical inhibitor that can be neglected is the Rho-associated protein kinase (ROCK) inhibitor. A ROCK inhibitor is generally required in the passage of human iPS cells to inhibit the activation of proapoptotic pathways.

[00468] In a further embodiment, subcultured human iPS cells cultured in a low osmolarity medium described herein may maintain a naive or naive-like state after enzymatic separation and subculturing. In some examples, subcultured human iPS cells may continue to exhibit a morphology characterized by compact hemispherical colonies. Subcultured human iPS cells can also continue to express one or more of the pluripotency markers described herein.

C. Methods for Generating and Maintaining a Population of Human Induced Pluripotent Stem Cells

[00469] Methods and compositions for the production of human iPS cells in in vitro culture are provided. Additionally, methods and compositions are provided for maintaining human iPS cells in in vitro culture.

[00470] The term "obtaining" includes culturing non-pluripotent cells, transformed to express one or more reprogramming factors described herein, under suitable conditions to induce a change in cellular phenotype, gene expression, or both, such that the cells exhibit naive or similar naive state, i.e. express one or more characteristics of human iPS cells. A naive or naive-like state can be expressed in response to specific culture conditions, for example, culture in a low osmolarity medium described herein. In some examples, the proportion of cells expressing a naive or naive-like state is at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and up to 100% of the cells in culture.

[00471] In one embodiment, the method enriches an in vitro culture with a population of naive or naive-like human iPS cells. In such an embodiment, naive or naive-like human iPS cells can preferentially proliferate in culture as compared to cells that do not express the naive or naive-like state. In another embodiment, naive or naive-like human iPS cells can be selected from culture, enzymatically separated, and subcultured to produce an enriched population of naive or naive-like human iPS cells.

[00472] In one embodiment, non-pluripotent cells transformed to express a pluripotent state are cultured in vitro in a medium provided herein that is suitable for inducing expression of a naive or naive-like state for a period of at least 1, 2, 5, 7, 10, 14, 21, or 28 days, or any period of time sufficient to induce the expression of a naive or naive-like state in culture. The transformed cells can be cultured in the presence of medium for at least 1, 2, 3 or 4 weeks. Sometimes the transformed cells are cultured for 1-4 weeks. Expression of a naive or naive-like state can be determined by observing morphological characteristics or expression of pluripotency markers, which are described elsewhere in this document.

[00473] In one embodiment, non-pluripotent cells transformed to express a pluripotent state are cultured in the presence of a low osmolarity medium until they begin to express characteristics of a naive or naive-like state. The cells can then be cultured in a medium to maintain a naive or naive-like state. In another embodiment, the non-pluripotent cells transformed to express the pluripotent state are first cultured in a high osmolarity medium prior to culture in the presence of a low osmolarity medium. Such a high osmolarity medium has a higher osmolarity than the proposed low osmolarity medium and may contain bFGF. Some high osmolarity media contain one or more of bovine serum albumin, bFGF, transforming growth factor β (TGFβ), lithium chloride, pipicolic acid, and gamma-aminobutyric acid (GABA). Examples of high osmolarity media include mTeSR ™ media (Stemcell Technologies).

[00474] In some embodiments, non-pluripotent cells transformed to express a pluripotent state may first be cultured in a high osmolarity medium containing bFGF until they begin to express characteristics of a naive or naive-like state, and from that point on the cells are cultured in the presence of a low osmolarity medium. In one example, it can be cultured in a high osmolarity medium containing bFGF for a period of at least 1, 2, 5, 10, 30, 60, or 90 days, a period of 1, 2, 4, 8, or 12 weeks, or a period from 1 day to 3 months. A typical culture period in high osmolarity medium containing bFGF is 2 months.

[00475] In other embodiments, non-pluripotent cells transformed to express a pluripotent state may first be cultured in a high osmolarity medium containing bFGF until they show a morphology characterized by three-dimensional cell clumps, and from that point on the cells are cultured in the presence of a low osmolarity medium. In such embodiments, cells exhibiting three-dimensional clumps can be selected, separated (eg, using trypsin) and transferred to new culture in a low osmolarity medium described herein.

[00476] The terms "maintain" and "maintenance" include the retention of at least one or more characteristics or phenotypes described herein, human iPS cells. Such characteristics may include the retention of pluripotency, gene expression profiles and / or functional characteristics of naive cells. The terms "support" and "maintenance" can also include cell proliferation and / or increasing the number of cultured naive cells. These terms include culture conditions that prevent cells from being converted to a primed or non-pluripotent state. These terms further include culture conditions that allow cells to remain pluripotent and / or naive, while cells may or may not continue to divide and grow in number.

[00477] In one embodiment, human iPS cells are cultured in vitro in an environment as provided herein that is suitable for maintaining such cells in a naive or naive-like state. In a specific example, human iPS cells can be cultured in a suitable medium for a period of 1, 2, 5, 7, 10, 14, 21, or 28 days, or a period of about 2 weeks, about 3 weeks, about 4 weeks or more. as long as the cultured cells remain in a naive or naive-like state. The cells can be cultured for at least 1, 2, 3, or 4 weeks. Sometimes the cells are cultured for 1-4 weeks. Human iPS cells can be maintained, for example, for any period of time sufficient for the cells to multiply in culture, genetically modify the cells, and / or subculture the cells.

[00478] In another embodiment, human iPS cells or non-pluripotent cells transformed to express a pluripotent state can be cultured on a substrate or feeder cell layer suitable for in vitro culture. In a specific example, cells are cultured on MATRIGEL ™ (BD Biosciences). In another example, cells are cultured on neonatal human foreskin fibroblast feeder cells (NuFF). In another example, cells are cultured on GELTREX ™ (Life Technologies).

[00479] In a further embodiment, the doubling time of human iPS cells cultured in the presence of low osmolarity medium is reduced compared to primed human iPS cells or non-pluripotent cells transformed to express the pluripotent state. In a specific example, the doubling time of these human iPS cells is about 16-24 hours.

7. Identity of sequences

[00480] The methods and compositions provided herein employ many different components of the targeted genomic integration system (ie, nuclease agents, recognition sites, nucleotide inserts, polynucleotides of interest, targeting vectors, selectable markers, and other components). From the description it is clear that some components of the targeted genomic integration system may have active variants and fragments. Such components include, for example, nuclease agents (ie, engineered nuclease agents), nuclease recognition sites, polynucleotides of interest, target sites, and corresponding targeting vector homology arms. The biological activities of each of these components are described elsewhere in this document.

[00481] As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences refers to residues in two sequences that are the same when aligned for maximum match in a particular comparison window. When percent sequence identity is used with respect to proteins, it should be understood that residue positions that are not identical often differ in conservative amino acid substitutions where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore not lead to a change in the functional properties of the molecule. When sequences differ in conservative substitutions, the percentage of sequence identity can be increased given the conservative nature of the substitution. Sequences that differ in such conservative substitutions are said to have "sequence similarity" or "similarity". Methods for making such adjustments are well known to those skilled in the art. Typically, a conservative substitution is taken into account as a partial rather than a complete mismatch, thereby increasing the percentage of sequence identity. Thus, for example, if an identical amino acid is assigned a value of 1, and a non-conservative substitution is assigned a value of zero, a conservative substitution is assigned a value between zero and 1. The values of conservative substitutions are calculated, for example, as in the PC / GENE program (Intelligenetics, Mountain View, California) ...

[00482] In the context of this document, "percent sequence identity" means a value determined by comparing two optimally aligned sequences in a comparison window, where a portion of a polynucleotide sequence in a comparison window may contain additions or deletions (i.e. gaps) compared to a control sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which both sequences contain identical nucleotide bases or amino acid residues to obtain the number of matches, dividing the number of matches by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage sequence identity.

[00483] Unless otherwise indicated, the sequence identity / similarity values given herein refer to values obtained using GAP version 10 using the following parameters: in case of% identity and% similarity for nucleotide sequence using a gap penalty of 50 and penalty for continuation 3, as well as substitution matrix nwsgapdna.cmp; in the case of% identity and% similarity for the amino acid sequence using a gap penalty of 8 and a continuation penalty of 2, as well as the BLOSUM62 substitution matrix; or any other equivalent program. "Equivalent program" means any sequence comparison program that, for any two sequences being compared, creates an alignment giving an identical nucleotide or amino acid residue match as well as the same percentage of sequence identity when compared to the corresponding alignment generated by GAP version 10.

[00484] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. While any methods and materials similar or equivalent to those described herein can also be used to practice or test the disclosed invention, preferred methods and materials are described in the text. All publications mentioned in this document are incorporated by reference to disclose and describe the methods and / or materials in connection with which these publications are referred.

[00485] It should be noted that as used in the text of this document and the accompanying claims, the singular form the singular includes the plural, unless otherwise clearly indicated by the context. All technical and scientific terms used in this document have the same meanings.

[00486] Discussed in this document publications are given solely in relation to their content prior to the filing date of this application. Nothing in this document should be construed as an admission that the described invention does not give the right to date such publication retroactively based on the previous invention. In addition, the dates of publication provided may differ from the actual dates of publication, which may require independent confirmation.

[00487] The implementation of the described invention can be carried out in other specific forms without departing from its essence or essential features, and, accordingly, reference should be made not to the above description, but to the attached claims as defining the scope of the invention.

[00488] Non-limiting embodiments of the invention include:

[00489] 1. A method of directed genetic modification of a genomic locus of interest in a pluripotent rat cell, comprising (a) introducing into a pluripotent rat cell a large targeting vector (LTVEC) containing a nucleotide insert flanked by a 5 'rat shoulder of homology and a 3' rat shoulder of homology wherein the 5 'and 3' homology arms total at least 10 kb, but less than 150 kb; and (b) identifying a genetically modified pluripotent rat cell containing targeted genetic modification at the genomic locus of interest, wherein the targeted genetic modification can be germline-transmitted.

[00490] 2. A method according to embodiment 1, wherein the targeted genetic modification is biallelic.

[00491] 3. The method of embodiment 1 or 2, wherein the pluripotent rat cell is a rat embryonic stem (ES) cell.

[00492] 4. A method according to embodiment 1, 2 or 3, wherein the pluripotent rat cell is derived from a DA strain or an ACI strain.

[00493] 5. A method according to any one of embodiments 1-4, characterized in that the pluripotent rat cell is characterized by the expression of at least one pluripotency marker, including Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1 , LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof.

[00494] 6. A method according to any one of embodiments 1-4, wherein the pluripotent rat cell has one or more of the following characteristics:

(a) lack of expression of one or more pluripotency markers, including c-Myc, Ecatl and / or Rexo1; (b) lack of expression of mesodermal markers, including brachiuria and / or Bmpr2; (c) lack of expression of one or more endodermal markers, including Gata6, Sox 17 and / or Sox7; or (d) lack of expression of one or more neural markers, including Nestin and / or Pax6.

[00495] 7. A method as in any one of embodiments 1-6, wherein the 5 'and 3' arms of LTVEC homology total from about 10 kbp. to about 30 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to 150 kbp

[00496] 8. The method of any one of embodiments 1-6, wherein the 5 'and 3' arms of LTVEC homology total from about 16 kbp. up to about 150 kbp

[00497] 9. A method according to any one of embodiments 1-8, wherein the targeted genetic modification comprises: (a) replacing an endogenous rat nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) removal of an endogenous rat nucleotide sequence; (c) deletion of an endogenous rat nucleotide sequence, the deletion range being from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (d) an exogenous nucleotide sequence ranging from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp; (e) an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (f) a chimeric nucleotide sequence comprising a human and rat nucleotide sequence; (g) a conditioning allele flanked by target sequences of a site-specific recombinase; or (h) a reporter gene operably linked to a promoter active in a rat cell.

[00498] 10. A method according to any one of embodiments 1-9, wherein the genomic locus of interest comprises (i) a first nucleotide sequence that is complementary to the 5 'rat arm of homology; and (ii) a second nucleotide sequence that is complementary to the 3 'rat arm of homology.

[00499] 11. The method according to embodiment 10, wherein the first and second nucleotide sequences are separated by at least 5 kb, but less than 3 pb.

[00500] 12. The method of embodiment 10, wherein the first and second nucleotide sequences are separated by at least 5 kb, but less than 10 kb, at least 10 kb. bp, but less than 20 kb, at least 20 kb, but less than 40 kb, at least 40 kb, but less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb but less than 100 kb bp, at least 100 kbp but less than 150 kbp, or at least 150 kbp, but less than 200 kbp, at least about 200 kb but less than about 300 kb, at least about 300 kb but less than about 400 kb, at least at least about 400 kb but less than about 500 kb, at least about 500 kb but less than about 1 ppm, at least about 1 mpo, but less than about 1.5 mpo, at least about 1.5 mpo, but less than about 2 mpo, at least at least about 2 m.p., but less than about 2.5 m po, or at least about 2.5 ppm, but less than about 3 ppm.

[00501] 13. The method according to any one of embodiments 1-12, characterized in that the introduction step (a) further comprises the introduction of a second nucleic acid encoding a nuclease agent that stimulates homologous recombination between the targeting construct and the genomic locus of interest in the pluripotent rat cage.

[00502] 14. A method according to embodiment 13, wherein the nuclease agent comprises (a) a chimeric protein comprising a zinc finger DNA binding domain fused to a FokI endonuclease; or (b) a chimeric protein comprising a transcription activator-like effector nuclease (TALEN) fused to a FokI endonuclease.

[00503] 15. A method according to any one of embodiments 1-12, characterized in that the step of introducing (a) further comprises introducing into the pluripotent rat cell: (i) a first expression construct comprising a first promoter operably linked to the first nucleotide sequence coding for a protein (Cas) associated with short palindromic repeats, regularly spaced groups (CRISPR), (ii) a second expression construct containing a second promoter operably linked to a target genomic sequence linked to a guide RNA (nRNA), while the target genomic the sequence is immediately flanked at the 3 'end by a protospacer-adjacent motif sequence (PAM).

[00504] 16. The method of embodiment 15, wherein the genomic locus of interest comprises the nucleotide sequence of SEQ ID No. 1.

[00505] 17. The method of embodiment 15 or 16, wherein the nRNA comprises a third nucleotide sequence encoding RNA of short palindromic repeats in regular clusters (CRISPR) (crRNA) and trans-activating CRISPR RNA (tracrRNA).

[00506] 18. The method of embodiment 15, 16, or 17, wherein the Cas protein is Cas9.

[00507] 19. A method according to embodiment 15, 16, 17 or 18, wherein the nRNA comprises: (a) a chimeric RNA from the nucleotide sequence of SEQ ID No. 2; or (b) chimeric RNA from the nucleotide sequence of SEQ ID No. 3.

[00508] 20. The method according to embodiment 17, wherein the crRNA comprises SEQ ID No. 4; SEQ ID No. 5; or SEQ ID No. 6.

[00509] 21. A method according to embodiment 17, wherein the tracrRNA comprises SEQ ID No. 7 or SEQ ID No. 8.

[00510] 22. A modified rat genomic locus comprising: (i) an insert of a homologous or orthologous human nucleotide sequence; (ii) replacement of an endogenous rat nucleotide sequence with a homologous or orthologous human nucleotide sequence; or (iii) a combination thereof, wherein the modified rat genomic locus can be germline transmitted.

[00511] 23. The modified rat genomic locus of embodiment 22, wherein the size of the insert or substitution is from about 5 kbp. up to about 400 kbp

[00512] 24. A rat genomic locus according to embodiment 22, wherein the size of the insert or substitution is from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp

[00513] 25. A method of producing a humanized rat, comprising: (a) targeting a genomic locus of interest in a pluripotent rat cell with a targeting construct containing human nucleic acid to form a genetically modified pluripotent rat cell; (b) introducing a genetically modified pluripotent rat cell into a rat host embryo; and (c) bearing a rat host embryo by a surrogate mother, the surrogate mother producing rat offspring containing a modified genomic locus that contains: (i) an insert of a human nucleotide sequence; (ii) substitution of a rat nucleotide sequence at the genomic locus of interest with a homologous or orthologous human nucleotide sequence; (iii) a chimeric nucleotide sequence containing a human and rat nucleotide sequence; or (iv) a combination thereof, wherein the modified genomic locus can be transmitted through the germ line.

[00514] 26. The method of embodiment 25, wherein the targeting construct is a large targeting vector (LTVEC) and the 5 ′ and 3 ′ arms of LTVEC homology total at least 10 kbp. , but less than 150 kbp.

[00515] 27. A method according to embodiment 26, wherein the 5 'and 3' homology arms of the guide structure total from about 10 kbp. to about 30 kb, from about 20 kb. to 40 kbp, from about 40 kbp to about 60 kbp, from about 60 kbp. up to about 80 kbp or from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to 150 kbp

[00516] 28. The method of embodiment 25, 26, or 27, wherein the human nucleotide sequence is at least 5 kb but less than 400 kb.

[00517] 29. A method according to embodiment 25, 26, or 27, wherein the human nucleotide sequence is at least 5 kb, but less than 10 kb, at least 10 kbp, but less than 20 kbp, at least 20 kbp, but less than 40 kbp, at least 40 kbp. but less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb but less than 100 kbp, at least 100 kbp but less than 150 kbp, at least 150 kbp, but less than 200 kbp. at least 200 kb but less than 250 kb, at least 250 kb but less than 300 kb, at least 300 kb bp, but less than 350 kbp, or at least 350 kbp, but less than 400 kbp.

[00518] 30. A method according to any one of embodiments 25-29, wherein the pluripotent rat cell is a rat embryonic stem (ES) cell.

[00519] 31. A method according to any one of embodiments 25-30, wherein the pluripotent rat cell is derived from a DA strain or an ACI strain.

[00520] 32. A method according to any one of embodiments 25-31, characterized in that the pluripotent rat cell is characterized by the expression of at least one pluripotency marker, including Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1 , LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof.

[00521] 33. A method according to any one of embodiments 25-31, wherein the pluripotent rat cell has one or more of the following characteristics: (a) lack of expression of one or more pluripotency markers, including c-Myc, Ecat1, and / or Rexo1; (b) lack of expression of one or more mesodermal markers, including brachiuria and / or Bmpr2; (c) lack of expression of one or more endodermal markers, including Gata6, Sox17 and / or Sox7; or (d) lack of expression of one or more neural markers, including Nestin and / or Pax6.

[00522] 34. A modified rat containing a humanized genomic locus, the humanized genomic locus comprising: (i) an insert of a homologous or orthologous human nucleotide sequence; (ii) replacement of a rat nucleotide sequence at an endogenous genomic locus with a homologous or orthologous human nucleotide sequence; (iii) a chimeric nucleotide sequence containing a human and a rat nucleotide sequence, or (iv) a combination thereof, wherein the modified genomic locus can be transmitted through the germline.

[00523] 35. A rat or rat cell containing a targeted genetic modification at its genomic locus, wherein the genomic locus is an interleukin-2 gamma receptor locus, ApoE locus, Rag1 locus, Rag2 locus, or Rag2 / Rag1 locus, wherein the targeted genetic the modification includes: (a) deletion of the endogenous rat nucleotide sequence at the genomic locus; (b) insertion of a homologous nucleic acid, orthologous nucleic acid, or chimeric nucleic acid containing a human and a rat nucleotide sequence, or (c) a combination thereof, wherein the targeted genetic modification can be transmitted through a rat or rat germ line raised from a rat cell.

[00524] 36. A rat or rat cell of embodiment 35, wherein (a) the removal of endogenous rat nucleic acid at the genomic locus is at least about 10 kb; or (b) the removal of endogenous rat nucleic acid at the genomic locus is from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (c) the insertion of an exogenous nucleotide sequence at the genomic locus is at least about 5 kb; or (d) the insertion of an exogenous nucleotide sequence at the genomic locus is from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp

[00525] 37. A rat or rat cage according to embodiment 35 or 36, wherein (a) targeted genetic modification at an interleukin-2 gamma receptor locus results in a decrease or absence of interleukin-2 gamma receptor protein activity; (b) targeted genetic modification at the ApoE locus results in a decrease or absence of the protein activity of ApoE; (c) targeted genetic modification at the Rag1 locus results in a decrease or absence of Rag1 protein activity; (d) targeted genetic modification at the Rag2 locus results in a decrease or absence of Rag2 protein activity; or (e) targeted genetic modification at the Rag2 / Rag1 locus results in a decrease or absence of Rag2 protein activity and Rag1 activity.

35, 36 or 37, characterized in that the directed genetic modification of the locus of the interleukin-2 gamma receptor comprises: (a) removing the entire coding region of the rat interleukin-2 gamma receptor or part thereof; (b) replacing the entire coding region of the rat interleukin-2 gamma receptor, or a portion thereof, with the coding region of the human interleukin-2 gamma receptor, or a portion thereof; (c) replacing the ectodomain of the coding region of the rat interleukin-2 gamma receptor with an ectodomain of the human interleukin-2 gamma receptor; or (d) removal of at least 3 kb at the interleukin-2 gamma receptor locus.

[00527] 39. A rat or rat cell according to any one of embodiments 35-37, wherein the targeted genetic modification of the ApoE locus comprises: (a) removing the entire coding region of ApoE or a portion thereof; or (b) deletion at an ApoE locus containing an ApoE coding region of at least 1.8 kb.

[00528] 40. A rat or rat cage according to any one of embodiments 35-37, wherein the targeted genetic modification of the Rag2 locus comprises: (a) deleting the entire coding region of Rag2 or a portion thereof; (b) deletion at the Rag2 locus containing the Rag2 coding region of at least 5.7 kb.

[00529] 41. A rat or rat cage according to any one of embodiments 35-37, wherein the targeted genetic modification of the Rag2 / Rag1 locus comprises: (a) removing the entire Rag2 coding region or a portion thereof and removing the entire Rag1 coding region, or parts of it; or (b) deletion at the Rag2 / Rag1 locus containing the Rag2 coding region of at least 16 kb.

[00530] 42. A rat or rat cell according to any one of embodiments 35-41, wherein the targeted genetic modification comprises inserting an expression cassette containing a selectable marker at the interleukin-2 gamma receptor locus, ApoE locus, Rag1 locus, Rag locus 2 or the Rag2 / Rag1 locus.

[00531] 43. A rat or rat cell according to any one of embodiments 42, wherein the expression cassette comprises a lacZ gene operably linked to an endogenous promoter at the genomic locus and a human ubiquitin promoter operably linked to a selectable marker.

[00532] 44. A rat or rat cell according to any one of embodiments 35-43, wherein the targeted genetic modification at the interleukin-2 gamma receptor locus, the ApoE locus, the Rag1 locus, the Rag2 locus, or the Rag2 / Rag1 locus comprises a self-destructive insert selection cassette.

[00533] 45. A rat or rat cell according to embodiment 44, wherein the self-destructive selection cassette contains a selectable marker gene operably linked to a promoter active in a rat cell and a recombinase gene operably linked to a specific male germ cell a promoter, while the self-destructing cassette is flanked by recombination recognition sites recognized by the recombinase.

[00534] 46. A rat or rat cell according to embodiment 45, wherein (a) the male germ cell specific promoter is a protamine-1 promoter; or (b) the recombinase gene encodes Cre and the recombination recognition regions are loxP regions.

[00535] 47. A rat or rat cell according to any one of embodiments 35-46, characterized in that the insertion of an exogenous nucleotide sequence at the genomic locus comprises a reporter nucleic acid operably linked to the endogenous promoter of the interleukin-2 gamma receptor, the endogenous promoter of ApoE, endogenous Rag1 promoter or endogenous Rag2 promoter.

[00536] 48. A rat or rat cell according to embodiment 47, wherein the reporter nucleic acid encodes a reporter, including (3-galactosidase, mPlum, mCherry, tdTomato, Strawberry, J-Red, DsRed, mOrange, mKO, mCitrine , Venus, YPet, enhanced yellow fluorescent protein (uLPP), Emerald, enhanced green fluorescent protein (uZPP), CyPet, blue fluorescent protein (GBP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

[00537] 49. A rat cell according to any one of embodiments 35-48, wherein the rat cell is a pluripotent rat cell or a rat embryonic stem (ES) cell.

[00538] 50. A rat cell according to embodiment 49, wherein the pluripotent rat cell or rat embryonic stem (ES) cell (a) is obtained from a DA strain or an ACI strain; (b) is characterized by expression of at least one pluripotency marker, including Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof; or (c) is characterized by one or more of the following characteristics: (i) lack of expression of one or more pluripotency markers, including c-Myc, Ecat1 and / or Rexo1; (ii) lack of expression of mesodermal markers, including brachiuria and / or Bmpr2; (iii) lack of expression of one or more endodermal markers, including Gata6, Sox17 and / or Sox7; or (iv) lack of expression of one or more neural markers, including Nestin and / or Pax6.

[00539] 51. A method for modifying a target genomic locus at an interleukin-2 gamma receptor locus, ApoE locus, Rag1 locus, Rag2 locus, or Rag2 / Rag1 locus in a pluripotent rat cell, comprising: (a) introducing a targeting vector into a pluripotent rat cell a nucleotide insert flanked by 5 'and 3' rat arms of homology homologous to the target genomic locus, (b) identification of a genetically modified pluripotent rat cell containing a targeted genetic modification at the target genomic locus, while the targeted genetic modification can be transmitted through the germ line of a rat from a pluripotent rat cell.

[00540] 52. The method of embodiment 51, wherein the targeting vector is a large targeting vector (LTVEC), the 5 'and 3' rat shoulders of homology totaling at least about 10 kbp. about., but less than about 150 kbp.

[00541] 53. A method according to embodiment 51 or 52, wherein the introduction of a targeting vector into a pluripotent rat cell results in: (i) removal of an endogenous rat nucleotide sequence at the target genomic locus; (ii) insertion of an exogenous nucleotide sequence at the target genomic locus; or (iii) a combination thereof.

[00542] 54. The method of embodiment 53, wherein (a) the removal of endogenous rat nucleic acid at the genomic locus is at least about 10 kb; or (b) the removal of endogenous rat nucleic acid at the genomic locus is from about 5 kbp. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. to ABOUT 1 MPO, from about 1 MPO up to ABOUT 1.5 MPO, from about 1.5 MPO up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (c) the insertion of an exogenous nucleotide sequence at the genomic locus is at least about 5 kb; or (d) the insertion of an exogenous nucleotide sequence at the genomic locus is from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp

[00543] 55. The method according to any one of embodiments 51-54, characterized in that (a) targeted genetic modification at the locus of the interleukin-2 gamma receptor results in a decrease or absence of the protein activity of the interleukin-2 gamma receptor; (b) targeted genetic modification at the ApoE locus results in a decrease or absence of the protein activity of ApoE; (c) targeted genetic modification at the Rag1 locus results in a decrease or absence of Rag1 protein activity; (d) targeted genetic modification at the Rag2 locus results in a decrease or absence of Rag2 protein activity; or (e) targeted genetic modification at the Rag2 / Rag1 locus results in a decrease or absence of Rag2 protein activity and Rag1 protein activity.

[00544] 56. A method according to any one of embodiments 51-54, characterized in that targeted genetic modification of the IL-2 gamma receptor locus comprises: (a) removing the entire coding region of the rat IL-2 gamma receptor or a portion thereof; (b) replacing the entire coding region of the rat interleukin-2 gamma receptor, or a portion thereof, with the coding region of the human interleukin-2 gamma receptor, or a portion thereof; (c) replacing the ectodomain of the coding region of the rat interleukin-2 gamma receptor with an ectodomain of the human interleukin-2 gamma receptor; or (d) deletion at an interleukin-2 gamma receptor locus containing an interleukin-2 gamma receptor coding region of at least 3 kb.

[00545] 57. A method according to any one of embodiments 51-55, characterized in that targeted genetic modification of the ApoE locus comprises: (a) removing the entire coding region of ApoE or a portion thereof; or (b) deletion at an ApoE locus containing an ApoE coding region of at least 1.8 kb.

[00546] 58. A method according to any one of embodiments 51-55, characterized in that the targeted genetic modification of the Rag2 locus comprises: (a) removing the entire coding region of Rag2 or a portion thereof; or (b) deletion at the Rag2 locus containing the Rag2 coding region of at least 5.7 kb.

[00547] 59. A method according to any one of embodiments 51-55, characterized in that directed genetic modification of the Rag1 / Rag2 locus comprises: (a) removing the entire coding region of Rag2 or a portion thereof and removing the entire coding region of Rag1 or a portion thereof; or (b) a deletion at the Rag2 / Rag1 locus containing the Rag2 and Rag1 coding regions of at least 16 kb.

[00548] 60. A method according to any one of embodiments 51-59, wherein the nucleotide insert comprises an expression cassette containing a polynucleotide encoding a selectable marker.

[00549] 61. The method of embodiment 60, wherein the expression cassette comprises a lacZ gene operably linked to an endogenous promoter at a genomic locus and a human ubiquitin promoter operably linked to a selectable marker.

[00550] 62. A method according to any one of embodiments 51-60, wherein the nucleotide insert comprises a self-destructive selection cassette.

[00551] 63. The method of embodiment 62, wherein the self-destructive selection cassette contains a selectable marker operably linked to a promoter active in a rat pluripotent cell and a polynucleotide encoding a recombinase operably linked to a male germ cell-specific promoter and the self-destructing cassette is flanked by recombination recognition sites recognized by the recombinase.

[00552] 64. The method of embodiment 63, wherein (a) the male germ cell specific promoter is a protamine-1 promoter; or (b) the recombinase gene encodes Cre and the recombination recognition regions are 1xP regions.

[00553] 65. A method according to embodiment 53, wherein the insertion of an exogenous nucleotide sequence at the genomic locus comprises a reporter nucleotide sequence operably linked to an endogenous interleukin-2 gamma receptor promoter, an endogenous ApoE promoter, an endogenous Rag1 promoter, or an endogenous Rag2 promoter ...

[00553] 66. The method of embodiment 65, wherein the reporter nucleotide sequence encodes a reporter including β-galactosidase, mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet , enhanced yellow fluorescent protein (uZPP), Emerald, enhanced green fluorescent protein (uZPP), CyPet, blue fluorescent protein (GBP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

[00555] 67. A method according to any one of embodiments 51-66, wherein the puripotent rat cell is a rat embryonic stem (ES) cell.

[00556] 68. A method according to any one of embodiments 51-67, characterized in that the pluripotent rat cell (a) is obtained from a DA strain or an ACI strain; or (b) is characterized by the expression of a pluripotency marker, including Oct-4, Sox-2, alkaline phosphatase, or a combination thereof; or (c) is characterized by one or more of the following characteristics: (i) lack of expression of one or more pluripotency markers, including c-Myc, Ecat1 and / or Rexo1; (ii) lack of expression of mesodermal markers, including brachiuria and / or Bmpr2; (iii) lack of expression of one or more endodermal markers, including Gata6, Sox 17 and / or Sox7; or (iv) lack of expression of one or more neural markers, including Nestin and / or Pax6.

[00557] 69. The method of any one of embodiments 51-68, further comprising detecting targeted genetic modification at the target genomic locus, wherein the detection step uses quantitative analysis to assess allele modification (MOA) at the target genomic locus.

[00558] 70. A method according to any one of embodiments 51-69, characterized in that the introduction step (a) further comprises the introduction of a second nucleic acid encoding a nuclease agent that stimulates homologous recombination between the targeting vector and the target genomic locus in a pluripotent rat cage.

[00559] 71. The method of embodiment 70, wherein the nuclease agent comprises a chimeric protein comprising a zinc finger DNA binding domain fused to a FokI endonuclease.

[00560] 72. A method according to embodiment 71, wherein said method results in a biallelic modification of the target genomic locus.

[00561] 73. A method according to any one of embodiments 51-70, wherein the step of introducing (a) further comprises introducing into a pluripotent rat cell: (i) a first expression construct comprising a first promoter operably linked to a first nucleotide sequence coding for a protein (Cas) associated with short palindromic repeats, regularly spaced groups (CRISPR), (ii) a second expression construct containing a second promoter operably linked to a target genomic sequence linked to a guide RNA (nRNA), while the target genomic the sequence is immediately flanked at the 3 'end by a protospacer-adjacent motif sequence (PAM).

[00562] 74. The method of embodiment 73, wherein the genomic locus of interest comprises the nucleotide sequence of SEQ ID No. 1.

[00563] 75. The method of embodiment 73 or 74, wherein the nRNA comprises a third nucleotide sequence encoding RNA of short palindromic repeats in regular clusters (CRISPR) (crRNA) and trans-activating CRISPR RNA (tracrRNA).

[00564] 76. The method of embodiment 73, wherein the Cas protein is Cas9.

[00565] 77. A method according to embodiment 73, 74, or 75, wherein the nRNA comprises: (a) a chimeric RNA from the nucleotide sequence of SEQ ID No. 2; or (b) chimeric RNA from the nucleotide sequence of SEQ ID No. 3.

[00566] 78. The method of embodiment 75, wherein the crRNA comprises SEQ ID No. 4; SEQ ID No. 5; or SEQ ID No. 6.

[00567] 79. The method of embodiment 75, wherein the tracrRNA comprises SEQ ID No. 7 or SEQ ID No. 8.

[00568] 80. A rat or rat cell according to any one of embodiments 35-50, wherein the rat or rat cell contains targeted genetic modifications at the interleukin-2 gamma receptor locus, ApoE locus, Rag1 locus, Rag2 locus and / or locus Rag2 / Rag1.

[00569] 81. A rat or rat cell of embodiment 80, wherein the rat or rat cell contains targeted genetic modifications at the interleukin-2 gamma receptor locus and the Rag2 / Rag1 locus.

[00570] Additional non-limiting embodiments of the invention include:

[00571] 1. A method for modifying a genomic locus of interest in a eukaryotic cell, comprising: (a) introducing into a eukaryotic cell: (i) a large targeting vector (LTVEC) containing a first nucleic acid flanked by a 5 'homology arm and a 3' homology arm , wherein the LTVEC contains at least 10 kb; (ii) a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein, (iii) a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding a guide RNA (nRNA) containing a nucleotide sequence , which hybridizes with the target sequence, and trans-activating RNA CRISPR (tracrRNA), while the first and second promoters are active in a eukaryotic cell; and (b) identifying a modified eukaryotic cell containing targeted genetic modification at the genomic locus of interest.

[00572] 2. A method according to embodiment 1, wherein the targeted genetic modification is a biallelic genetic modification.

[00573] 3. The method of embodiment 1, wherein the LTVEC is at least 15 kb, at least 20 kb, at least 30 kb. at least 40 kbp, at least 50 kbp, at least 60 kbp, at least 70 kbp, at least 80 kbp .on. or at least 90 kb.

[00574] 4. The method of embodiment 1, wherein the LTVEC is at least 100 kb, at least 150 kb. or at least 200 kbp.

[00575] 5. A method according to embodiment 1, wherein the eukaryotic cell is a mammalian cell.

[00576] 6. A method according to embodiment 5, wherein the mammalian cell is a fibroblast.

[00577] 7. A method according to embodiment 1, wherein the eukaryotic cell is a pluripotent cell.

[00578] 8. The method of embodiment 7, wherein the pluripotent cell is a human pluripotent cell.

[00579] 9. The method of embodiment 8, wherein the human pluripotent cell is a human embryonic stem (ES) cell or a human adult stem cell.

[00580] 10. The method of embodiment 8, wherein the human pluripotent cell is a developmentally limited human progenitor cell.

[00581] 11. The method of embodiment 8, wherein the human pluripotent cell is a human induced pluripotent stem (iPS) cell.

[00582] 12. The method of embodiment 1, wherein the Cas protein is Cas9.

[00583] 13. The method of embodiment 1, wherein the target sequence is directly flanked at the 3 'end by a protospacer adjacent motif sequence (PAM).

[00584] 14. The method of embodiment 1, wherein the 5 'and 3' homology arms total between about 10 kbp. up to about 150 kbp

[00585] 15. The method of embodiment 1, wherein the 5 'and 3' arms of LTVEC homology total from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to 150 kbp

[00586] 16. A method according to embodiment 1, wherein the targeted genetic modification comprises: (a) replacing an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) removal of the endogenous nucleotide sequence; (c) deletion of an endogenous nucleotide sequence, the deletion range being from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (d) insertion of an exogenous nucleotide sequence; (e) insertion of an exogenous nucleotide sequence in the range of about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp; (f) insertion of an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (g) insertion of a chimeric nucleotide sequence containing a human and non-human nucleotide sequence; (h) insertion of a conditioning allele flanked by target sequences of site-specific recombinase; (i) insertion of a selectable marker or reporter gene operably linked to a promoter active in the pluripotent cell; or (j) a combination of both.

[00587] 17. The method of embodiment 1, wherein the genomic locus of interest comprises (i) a 5 'target sequence that is homologous to the 5' arm of homology; and (ii) a 3 'target sequence that is homologous to the 3' homology arm.

[00588] 18. The method of embodiment 17, wherein the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 3 ppm.

[00589] 19. The method of embodiment 17, wherein the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 10 kb, by at least at least 10 kb, but less than 20 kb, at least 20 kb, but less than 40 kb, at least 40 kb. but less than 60 kb, at least 60 kb, but less than 80 kb, at least about 80 kb, but less than 100 kb, at least 100 kb but less than 150 kb, or at least 150 kb but less than 200 kb at least about 200 kb but less than about 300 kb, at least about 300 kb but less than about 400 kb ., at least about 400 kbp, but less than about 500 kbp, at least about 500 kbp, but less than about 1 ppm, at least about 1 ppm but less than about 1.5 ppm, at least about 1.5 ppm but less than about 2 ppm ., at least about 2 p.o., but less than h it is about 2.5 mpo, or at least about 2.5 mpo, but less than about 3 mpo.

[00590] 20. The method of embodiment 1, wherein the genomic locus of interest comprises an interleukin-2 gamma receptor locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or both Rag1 and Rag2.

[00591] 21. A method according to embodiment 1, wherein the first and second expression constructs are in the same nucleic acid molecule.

[00592] 22. A method for modifying a genome, comprising exposing the genome to the Cas protein and PLC CRISPR in the presence of a large targeting vector (LTVEC) containing a nucleotide sequence of at least 10 kb, while after exposure to the Cas protein, RNA CRISPR and LTVEC modifies the genome so that it contains a nucleotide sequence of at least 10 kb.

[00593] 23. The method of embodiment 22, wherein the LTVEC comprises a nucleotide sequence of at least 20 kb, at least 30 kb, at least 40 kb .about., at least 50 kbp, at least 60 kbp, at least 70 kbp, at least 80 kbp. or at least 90 kb.

[00594] 24. The method of embodiment 22, wherein the LTVEC comprises a nucleotide sequence of at least 100 kb, at least 150 kb. or at least 200 kbp.

[00595] 25. A method for modifying a genome comprising contacting the genome with a Cas protein, CRISPR RNA that hybridizes to a target sequence, and tracrRNA in the presence of a large targeting vector (LTVEC), the LTVEC being at least 10 kb. O. and contains the first nucleic acid flanked by a 5 'homology arm and a 3' homology arm, and upon contact with Cas protein, CRISPR RNA and tracrRNA in the presence of LTVEC, the genome is modified at the genomic locus of interest to contain the first nucleic acid.

[00596] 26. A method according to embodiment 25, wherein the genome is in a eukaryotic cell and the Cas protein, CRISPR RNA, tracrRNA, and LTVEC are introduced into the eukaryotic cell.

[00597] 27. The method of embodiment 26, further comprising detecting a modified eukaryotic cell containing a targeted genetic modification at a genomic locus of interest.

[00598] 28. A method according to embodiment 26 or 27, wherein CRISPR RNA and tracrRNA are introduced together in the form of a single guide RNA (nRNA).

[00599] 29. A method according to embodiment 26 or 27, wherein CRISPR RNA and tracrRNA are introduced separately.

[00600] 30. A method according to any one of embodiments 26-29, characterized in that: (a) the Cas protein is introduced into the eukaryotic cell in the form of a protein, messenger RNA (mRNA) encoding the Cas protein, or DNA encoding the Cas protein ; (b) CRISPR RNA is introduced into a eukaryotic cell in the form of RNA or DNA encoding CRISPR RNA; and (c) tracrRNA is introduced into a eukaryotic cell in the form of RNA or DNA encoding tracrRNA.

[00601] 31. A method according to embodiment 30, wherein the Cas protein, CRISPR RNA, and tracrRNA are introduced into a eukaryotic cell as a protein-RNA complex.

[00602] 32. A method according to embodiment 30, wherein: (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein; (b) the DNA encoding CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding CRISPR RNA; and (c) the DNA encoding tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a fourth nucleic acid encoding tracrRNA, the first, second and third promoters being active in a eukaryotic cell.

[00603] 33. A method according to embodiment 32, wherein the first, second and / or third expression constructs are in the same nucleic acid molecule.

[00604] 34. The method according to embodiment 32, characterized in that: (a) the DNA encoding the C as protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein; and (b) DNA encoding CRISPR RNA and DNA encoding tracrRNA are in the form of a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding an nRNA comprising CRISPR RNA and tracrRNA; the first and second promoters are active in the eukaryotic cell.

[00605] 35. A method according to embodiment 34, wherein the first and second expression constructs are in the same nucleic acid molecule.

[00606] 36. A method according to any one of embodiments 27-35, wherein the targeted genetic modification comprises simultaneously removing an endogenous nucleotide sequence at a genomic locus of interest and inserting a first nucleic acid at a genomic locus of interest.

[00607] 37. A method according to any one of embodiments 27-36, wherein the targeted genetic modification is a biallelic genetic modification.

[00608] 38. A method according to embodiment 37, wherein the biallelic genetic modification comprises removing an endogenous nucleotide sequence and inserting a first nucleic acid at a genomic locus of interest on two homologous chromosomes

[00609] 39. A method according to any one of embodiments 27-36, wherein the modified eukaryotic cell is hemizygous at the genomic locus of interest.

[00610] 40. The method of embodiment 39, wherein targeted genetic modification at a genomic locus of interest on one chromosome comprises removing an endogenous nucleotide sequence and inserting a first nucleic acid.

[00611] 41. The method of embodiment 39, wherein the targeted genetic modification comprises: (1) removing an endogenous nucleotide sequence at a genomic locus of interest on two homologous chromosomes; and (2) inserting the first nucleic acid at the genomic locus of interest on the first chromosome and disrupting the genomic locus of interest on the second chromosome.

[00612] 42. A method according to any one of embodiments 25-41, wherein the LTVEC is at least 15 kb, at least 20 kb, at least 30 kb. bp, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kbp. or at least 90 kb.

[00613] 43. A method according to any one of embodiments 25-42, wherein the LTVEC is at least 100 kb, at least 150 kb. or at least 200 kbp.

[00614] 44. A method according to any one of embodiments 25-43, wherein the first nucleic acid is at least 20 kb, at least 30 kb, at least 40 kbp, at least 50 kbp, at least 60 kbp, at least 70 kbp, at least 80 kbp at least 90 kbp, at least 100 kbp, at least 150 kbp, at least 200 kbp, at least 250 tonnes .on. or at least 300 kbp.

[00615] 45. A method according to any one of embodiments 26-44, wherein the eukaryotic cell is a mammalian cell.

[00616] 46. A method according to embodiment 45, wherein the mammalian cell is a fibroblast.

[00617] 47. A method according to any one of embodiments 26-43, wherein the eukaryotic cell is a pluripotent cell.

[00618] 48. The method of embodiment 47, wherein the pluripotent cell is a non-human pluripotent cell.

[00619] 49. The method of embodiment 48, wherein the non-human pluripotent cell is a rodent pluripotent cell.

[00620] 50. The method of embodiment 49, wherein the rodent pluripotent cell is a mouse or rat embryonic stem (ES) cell.

[00621] 51. The method of embodiment 47, wherein the pluripotent cell is a human pluripotent cell.

[00622] 52. The method of embodiment 51, wherein the human pluripotent cell is a human embryonic stem (ES) cell or a human adult stem cell.

[00623] 53. The method of embodiment 51, wherein the human pluripotent cell is a developmentally restricted human progenitor cell.

[00624] 54. The method of embodiment 51, wherein the human pluripotent cell is a human induced pluripotent stem (iPS) cell.

[00625] 55. The method of any one of embodiments 25-54, wherein the Cas protein is Cas9.

[00626] 56. A method according to any one of embodiments 25-55, characterized in that the target sequence is directly flanked by the sequence of a protospacer contiguous motif (PAM).

[00627] 57. A method according to any one of embodiments 25-56, wherein the 5 'and 3' arms of LTVEC homology total from about 10 kbp. up to about 150 kbp

[00628] 58. The method of any one of embodiments 25-57, wherein the 5 'and 3' arms of LTVEC homology total from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 120 kbp or from about 120 kb. up to 150 kbp

[00629] 59. A method according to any one of embodiments 27-58, wherein the targeted genetic modification comprises: (a) replacing an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) removal of the endogenous nucleotide sequence; (c) deletion of an endogenous nucleotide sequence, the deletion range being from about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. up to about 150 kbp or from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 300 kbp, from about 300 kbp. to about 400 kbp, from about 400 kbp. to about 500 kbp, from about 500 kbp. up to about 1 m.p., from about 1 m.p. up to about 1.5 mpo, from about 1.5 mpo. up to about 2 m.p., from about 2 m.p. up to about 2.5 m.p. or from about 2.5 m.p. up to about 3 m.p .; (d) insertion of an exogenous nucleotide sequence; (e) insertion of an exogenous nucleotide sequence in the range of about 5 kb. to about 10 kbp, from about 10 kbp. to about 20 kb, from about 20 kb. to about 40 kbp, from about 40 kbp. to about 60 kbp, from about 60 kbp. to about 80 kbp, from about 80 kbp. to about 100 kbp, from about 100 kbp. to about 150 kbp, from about 150 kbp. to about 200 kbp, from about 200 kbp. to about 250 kbp, from about 250 kbp. to about 300 kbp, from about 300 kbp. up to about 350 kbp or from about 350 kbp. up to about 400 kbp; (f) insertion of an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (g) insertion of a chimeric nucleotide sequence containing a human and non-human nucleotide sequence; (h) insertion of a conditioning allele flanked by target sequences of site-specific recombinase; (i) insertion of a selectable marker or reporter gene operably linked to a promoter active in the pluripotent cell; or (j) a combination of both.

[00630] 60. A method according to any one of embodiments 25-59, wherein the genomic locus of interest comprises (i) a 5 'target sequence that is homologous to the 5' arm of homology; and (ii) a 3 'target sequence that is homologous to the 3' homology arm.

[00631] 61. The method of embodiment 60, wherein the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 3 ppm.

[00632] 62. The method of embodiment 60, wherein the 5 'target sequence and 3' target sequence are separated by at least 5 kb, but less than 10 kb, by at least at least 10 kb, but less than 20 kb, at least 20 kb, but less than 40 kb, at least 40 kb. but less than 60 kb, at least 60 kb, but less than 80 kb, at least about 80 kb, but less than 100 kb, at least 100 kb but less than 150 kb, or at least 150 kb but less than 200 kb at least about 200 kb but less than about 300 kb, at least about 300 kb but less than about 400 kb ., at least about 400 kbp, but less than about 500 kbp, at least about 500 kbp, but less than about 1 ppm, at least about 1 ppm but less than about 1.5 ppm, at least about 1.5 ppm but less than about 2 ppm ., at least about 2 p.o., but less than h it is about 2.5 mpo, or at least about 2.5 mpo, but less than about 3 mpo.

[00633] 63. The method of embodiment 60, wherein the 5 'target sequence and 3' target sequence are separated by at least 20 kb, at least 30 kb, at least at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb so 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 kbp, at least 190 kbp. or at least 200 kbp.

[00634] 64. A method according to any one of embodiments 25-63, wherein the genomic locus of interest comprises an interleukin-2 gamma receptor locus, ApoE locus, Rag1 locus, Rag2 locus, or both a Rag1 and Rag2 locus.

[00635] 65. A method according to any one of embodiments 25-63,

characterized in that the genomic locus of interest comprises an Adamts5 locus, a Trpa1 locus, a Folh1 locus or an Erbb4 locus.

[00636] 66. A method according to any one of embodiments 25-63, wherein the genomic locus of interest comprises an Lrp5 locus.

[00637] 67. A method of obtaining a non-human animal of the F0 generation, which contains a targeted genetic modification at a genomic locus of interest, comprising: (a) contacting a genome in a non-human ES cell with a Cas protein, CRISPR RNA and tracrRNA in the presence of a large targeting vector (LTVEC) for the formation of a modified non-human ES cell, while the LTVEC consists of at least 10 kb. and contains a first nucleic acid flanked by a 5 'homology arm and a 3' homology arm; (b) identifying a modified non-human ES cell containing targeted genetic modification at the genomic locus of interest; (c) introducing a modified non-human ES cell into a non-human host embryo; and (d) bearing a non-human host embryo by a surrogate mother, wherein the surrogate mother produces a non-human F0 generation animal that contains a targeted genetic modification at the genomic locus of interest.

[00638] 68. A method according to embodiment 67, wherein CRISPR RNA and tracrRNA are introduced together in the form of a single guide RNA (nRNA).

[00639] 69. A method according to embodiment 67, wherein CRISPR RNA and tracrRNA are added separately.

[00640] 70. A method according to any one of embodiments 67-69, characterized in that: (a) the Cas protein is introduced into a non-human ES cell in the form of a protein, messenger RNA (mRNA) encoding a Cas protein, or DNA encoding a protein Cas; (b) CRISPR RNA is introduced into a non-human ES cell in the form of RNA or DNA encoding CRISPR RNA; and (c) tracrRNA is introduced into a non-human ES cell in the form of RNA or DNA encoding tracrRNA.

[00641] 71. A method according to embodiment 70, wherein the Cas protein, CRISPR RNA, and tracrRNA are introduced into a non-human ES cell as a protein-RNA complex.

[00642] 72. A method according to embodiment 70, wherein: (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein; (b) the DNA encoding CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding CRISPR RNA; and (c) the DNA encoding tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a fourth nucleic acid encoding tracrRNA, the first, second and third promoters being active in a non-human ES cell.

[00643] 73. A method according to embodiment 72, wherein the first, second and / or third expression constructs are in the same nucleic acid molecule.

[00644] 74. A method according to embodiment 70, wherein: (a) the DNA encoding the Cas protein is in the form of a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein; and (b) DNA encoding CRISPR RNA and DNA encoding tracrRNA are in the form of a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding an nRNA comprising CRISPR RNA and tracrRNA; the first and second promoters are active in the non-human ES cell.

[00645] 75. The method of embodiment 74, wherein the first and second expression constructs are in the same nucleic acid molecule.

[00646] 76. A method according to any one of embodiments 67-75, wherein the targeted genetic modification comprises simultaneously removing an endogenous nucleotide sequence at a genomic locus of interest and inserting a first nucleic acid at a genomic locus of interest.

[00647] 77. A method according to any one of embodiments 67-76, wherein the targeted genetic modification is a biallelic genetic modification.

[00648] 78. The method of embodiment 77, wherein the biallelic genetic modification comprises removing an endogenous nucleotide sequence and inserting a first nucleic acid at a genomic locus of interest on two homologous chromosomes.

[00649] 79. A method according to any one of embodiments 67-76, wherein the modified non-human ES cell is hemizygous at the genomic locus of interest.

[00650] 80. A method according to embodiment 79, wherein targeted genetic modification at a genomic locus of interest on one chromosome comprises removing an endogenous nucleotide sequence and inserting a first nucleic acid.

[00651] 81. The method of embodiment 79, wherein the targeted genetic modification comprises: (1) removing an endogenous nucleotide sequence at a genomic locus of interest on two homologous chromosomes; and (2) inserting the first nucleic acid at the genomic locus of interest on the first chromosome and disrupting the genomic locus of interest on the second chromosome.

[00652] 82. A method according to any one of embodiments 67-81, wherein the Cas protein is Cas9.

EXAMPLES

[00653] The following examples are provided to provide those skilled in the art with a complete disclosure and description of the implementation and application of the present invention, and are not intended to limit the scope of what the inventors claim as their invention, nor to state that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantity, temperature, etc.), but the possibility of some experimental bias and bias should be considered. Unless otherwise indicated, fractions are by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1. Obtaining and characterization of rat ES cells

1.1. Characteristics of rat ES cells

[00654] As shown in Figure 1, rat ESCs grow in compact spherical colonies that usually detach and float in the dish (close-up, Figure 8). Rat ESCs express pluripotency markers, including Oct-4 (Figure 2A) and Sox2 (Figure 2B), and express high levels of alkaline phosphatase (Figure 3). The karyotype for line DA.2B is 42X, Y (Figure 4). Rat ESCs often become tetraploid; therefore, a preliminary screening of lines was carried out by counting metaphase chromosomal preparations; the lines with the most normal results were then formally karyotyped.

[00655] ACI blastocysts were obtained from commercially purchased superovulated females. DA blastocysts were cultured from commercially purchased frozen 8-cell embryos. The glitter was removed using Tyrode's acid; and blastocysts were plated on mitotically inactivated PEM. The enlarged blastocysts were harvested and propagated by standard methods. All blastocysts were seeded, cultured and propagated using 2i medium (Li et al. (2008) Germline competent embryonic stem cells derived from rat blastocysts, Cell 135: 1299-1310; incorporated herein by reference in its entirety).

1.2 .: Obtaining rats

[00656] Chimeric rats were obtained by injection into blastocysts and genome transfer of rat ESCs. Chimeras obtained by microinjection into blastocysts using parental ESCs of ACI.G1 rats are shown in Figure 9. Agouti F1 pups with albino litter pups obtained from the ACI / SD chimera marked with an asterisk (*) in Figure 9 are shown in Figure 10 ...

Germline transmission of parental rat ESCs.

[00657] Three euploid rat ESC lines were evaluated for pluripotency by microinjection into SD albino blastocysts. Chimeras were identified by the color of the agouti skin, which indicates the effect of rat ESCs (see Figure 10). In each line, for the majority of chimeras, the genome of cESCs was transferred to F1 descendants (Table 2).

1.3 .: Obtaining rat embryonic stem cells

[00658] Superovulation Protocol, rats

[00659] Day 0: Fertile mare serum was injected: WB, 20 U (0.4 ml).

[00660] Day 1: no action

[00661] Day 2: (after 46 hours): hCG, WB, 50 U (1 ml) were injected.

[00662] - mating of single females.

[00663] Day 3: copulatory plugs were checked. The females had plugs. This corresponds to day 0.5.

[00664] Day 6 (ie 3.5): Females were sacrificed and the embryos were washed away.

[00665] Protocol for obtaining ES cells (superovulation)

[00666] Day 0:

[00667] 1) Killed the female rat with CO ₂ .

[00668] 2) Wipe the abdomen with 70% ethanol; the abdominal wall was opened with scissors to gain access to the internal organs.

[00669] 3) The fallopian tubes and uterine horns were excised and placed in a tissue culture dish containing warm N2B27 medium. As far as possible, the blood was washed out and transferred to a new plate with N2B27.

[00670] 4) Using a 1 ml syringe and a 27g blunt needle, passed the medium through the uterine horns and fallopian tubes to extract the blastocysts into the medium.

[00671] 5) Blastocysts were collected using a pipette and transferred to an embryo culture dish containing KSOM + 2i (1 μM PD0325901, 3 μM CHIR99021). KSOM is a culture medium manufactured by Millipore. Catalog number MR-106-D.

[00672] 6) Cultured overnight at 37 °; 7.5% CO ₂ .

[00673] Protocol for obtaining ES cells (frozen embryos)

[00674] Day 0:

[00675] 1) Thaw frozen 8-cell embryos (commercially purchased) in M2 medium. It was cultivated for 10 min at room temperature.

[00676] 2) Transferred to KSOM + 2i and cultured overnight.

[00677] Protocol for obtaining ES cells (the same for both cases)

[00678] Day1:

[00679] 1) Cavitated embryos were transferred to medium 2i and cultured overnight.

[00680] 2) Continued cultivation of non-cavitated embryos in KSOM + 2i

[00681] Day 2:

[00682] 1) Transfer all the remaining embryos to medium 2i (regardless of whether they were cavitated).

[00683] 2) Cultivated overnight; continued cultivation of earlier embryos in 2i medium.

[00684] Day 3:

[00685] 1) Transfer the embryos for 30-60 seconds in Tyrode's acid to remove the zona pellucida.

[00686] 2) The embryos were washed 3x in 2i medium to remove Tyrode's acid.

[00687] 3) Place each embryo in a separate well of a 96-well feeding plate (the wells contained a monolayer of mitotically inactivated mouse embryonic fibroblasts (FEM)).

[00688] 4) Cultivated overnight in medium 2i.

[00689] Day 4-5:

[00690] 1) The seeded embryos were monitored for the presence of embryos (amorphous undifferentiated cell mass). The embryos are ready for transfer when they are approximately twice the size of the inoculated embryo.

[00691] 2) Every day: the spent medium was removed with a micropipette and replaced with fresh medium 2i.

[00692] 3) Transfer the embryos to new feeding wells:

[00693] a. The spent medium was removed and the well was carefully washed with PBS.

[00694] b. PBS was removed and 30 μl of 0.05% trypsin was added; incubated for 10 minutes.

[00695] c. The trypsin reaction was stopped by adding 30 μl of 2i + 10% PBS.

[00696] d Cells were carefully separated using a micropipettor and the entire contents of the well were transferred to a new well in a 24-well feeding plate. This corresponded to passage 1 (P1).

[00697] e. Cultivated overnight in medium 2i.

[00698] Day 5-8: (time depends on how much each line reproduces)

[00699] 1) The medium (medium 2i) was changed every day and monitored for the presence of colonies with ESC morphology.

[00700] 2) After the appearance of colonies, the culture was continued until the colonies reached ~ 50% confluence.

[00701] 3) Colonies were trypsinized and passaged as before; were plated on feeding layers, 1 well per line, in a 6-well plate. This corresponded to passage 2 (P2).

[00702] Current Activities:

[00703] 1) Continue feeding and monitoring each line to approximately 50% confluence.

[00704] 2) Treated with trypsin as usual.

[00705] 3) Stop the trypsin reaction with 2i + 10% PBS; the cells were precipitated by centrifugation (5 ', 1200 rpm in a Beckman-Coulter tabletop centrifuge).

[00706] 4) The supernatant was aspirated and the cells were carefully resuspended in 400 μl of freezing medium (70% 2i, 20% PBS, 10% DMSO).

[00707] 5) Spread the cells into two tubes and frozen at -80 °. This corresponded to passage 3 (P3).

[00708] 6) For long-term storage, the tubes were placed in liquid N ₂ .

[00709] Medium 2i was prepared as shown in Table 3.

[00710] Materials: Serum gonadotropin of a colt mare (SGFA)

[00711] Chorionic gonadotropin of urine of a pregnant woman (hCG)

[00712] Female rats (5-12 weeks old)

[00713] Male rats (12 weeks to 8 months), one per cage

[00714] Syringes / needles

[00715] Vivarium with lights on 6: 00-18: 00

[00716] Procedure:

[00717] Day 1: 8: 00-10: 00 am

[00718] Injection of females with 20 IU FGFA (0.4 ml), WB

[00719] Removal of unused FGFA.

[00720] Day 3: 8: 00-10: 00 am (48 hours after FGFA injection)

[00721] Injection of females with 50 IU hCG (1 ml), WB

[00722] Placing one female per male in a mating cage.

[00723] Removal of unused hCG.

[00724] Day 4: 8: 00-10: 00 am (24 hours after HCG injection)

[00725] Checking females for copulatory plugs.

[00726] Hormone Suppliers

[00727] FFA: Sigma # G-4877 (1000 ME). Re-suspended in PBS to a final concentration of [] 50 IU / ml. Store at -20 ° in 1 ml aliquots.

[00728] hCG: Sigma # CG-5 (5000 ME). Re-suspended in PBS to a final concentration of [] 50 IU / ml. Store at -20 ° in 1 ml aliquots.

1.4 .: Karyotyping of rat embryonic stem cell lines

[00729] The rat ES cell lines obtained herein were karyotyped and the results are summarized in Tables 4-7.

[00730]

[00731]

[00732]

[00733]

1.5 .: Electroporation of a vector into a rat embryonic stem cell

[00734] 1. Rat ES cells were passaged 24-48 hours before electroporation.

[00735] 2. The medium was changed to RVG2i + ROCKi (10 μM Y-27632) 24 hours before electroporation

[00736] 3. Change the medium 30 minutes before trypsin treatment.

[00737] 4. DNA was aliquoted for electroporation.

[00738] 5. Allow the DNA to warm at RT for> 10 min.

[00739] 6. Heat the DNA for 5 min at 62 ° C. Placed DNA on ice.

[00740] 7. Trypsinized cells:

[00741] a. Collected floating colonies. Washed the plate to collect as many floating cells as possible.

[00742] b. Colonies were besieged: 3 min at 750 rpm.

[00743] c. Washed precipitate 1x 5-10 ml PBS and re-centrifuged / precipitated

[00744] d. The supernatant was aspirated; 500 μl of trypsin, 0.05% + 1% chicken serum was added.

[00745] i. Do not collect more than 1 10 cm of the colony plate in the test tube. If there are too many colonies at the bottom of the tube during trypsinization, they will aggregate and most cells will be lost.

[00746] e. 4 min at 37 °. Colonies were pipetted several times to minimize aggregation.

[00747] f. Steps 1–2 X were repeated: 4 min at 37 °.

[00748] g. The trypsin reaction was stopped with 500 μl RVG2i + 10% PBS.

[00749] 8. The cells were besieged: 5 minutes at 1200 rpm.

[00750] 9. Re-suspend cells in 10 ml PBS. Two 20 μl aliquots were counted to determine the total number of cells.

[00751] 10. Seed cells (5 min / 1200 rpm); the total number of cells and the total volume of resuspension were counted to obtain the correct cell concentration (target # / 75 μl of EP buffer).

[00752] 11. Re-suspend in the minimum volume of EP buffer; the total volume was determined and adjusted to the target volume with EP buffer. Electroporation buffer is sold by Millipore. The catalog number corresponds to ES-003-D. See Valenzuela et al. (2003) Nature Biotechnology 21: 652-659, which is incorporated herein by reference.

[00753] 12. Add 75 μl of cells in 50 μl of DNA; transferred 125 μl of cell / DNA solution into one well of a 48-well BTX cuvette.

[00754] a. Empty wells in the same column were filled with 125 μl EP buffer.

[00755] 13. Once the cuvette was irradiated in the VTX electroporator:

[00756] a. Settings: 400 V; Ω; 100 μF (settings may vary)

[00757] 14. Place the cuvette on ice for 15 minutes to recover.

[00758] 15. Transfer the cells into 5 ml RVG2i + 10 μM ROCKi.

[00759] 16. Added to a 15 cm plate with 20 ml RVG2i + 10 μM ROCKi. The tablet contained 2x neoR FEM (or other FEM depending on the project). The neoR selectable marker is the neomycin phospho transferase (neo) gene according to Beck et al. (1982) Gene, 19: 327-36 or US patent No. 7205148 or 6596541, which are all incorporated herein by reference.

[00760] 17. Incubated at 37 °. Sampling started after 48 h.

[00761] The ROCK inhibitor was Y-27632.

1.6: Selection of targeted genetic modification in rat embryonic stem cells.

[00762] 1. Cells were passaged 24-48 hours before electroporation.

[00763] 2. The medium was changed to RVG2i + ROCKi (10 μM Y-27632) 24 hours before electroporation

[00764] 3. Change the medium 30 minutes before trypsin treatment.

[00765] 4. DNA was aliquoted for electroporation.

[00766] 5. Allow the DNA to warm at RT for> 10 min.

[00767] 6. Heat the DNA for 5 min at 62 ° C. Placed DNA on ice.

[00768] 7. Trypsinized cells:

[00769] a. Collected floating colonies. Washed the plate to collect as many floating cells as possible.

[00770] b. Colonies were besieged: 3 min at 750 rpm.

[00771] c. Washed precipitate 1x 5-10 ml PBS and re-centrifuged / precipitated

[00772] d. The supernatant was aspirated; 500 μl of trypsin, 0.05% + 1% chicken serum was added.

[00773] i. Do not collect more than 1 10 cm of the colony plate in the test tube. If there are too many colonies at the bottom of the tube during trypsinization, they will aggregate and most cells will be lost.

[00774] e. 4 min at 37 °. Pipette colonies multiple times to minimize aggregation

[00775] f. Repeated 1-2 X: 4 min at 37 °.

[00776] g. The trypsin reaction was stopped with 500 μl RVG2i + 10% PBS.

[00777] 8. The cells were besieged: 5 minutes at 1200 rpm.

[00778] 9. Re-suspend cells in 10 ml PBS. Two 20 μl aliquots were counted to determine the total number of cells.

[00779] 10. Cells were pelleted (5 min / 1200 rpm); the total number of cells and the total volume of resuspension were counted to obtain the correct cell concentration (target # / 75 μl of EP buffer).

[00780] 11. Re-suspend in the minimum volume of EP buffer; the total volume was determined and adjusted to the target volume with EP buffer.

[00781] 12. Add 75 μl of cells in 50 μl of DNA; transferred 125 μl of cell / DNA solution into one well of a 48-well BTX cuvette.

[00782] a. Empty wells in the same column were filled with 125 μl EP buffer.

[00783] 13. Once the cuvette was irradiated in the VTX electroporator:

[00784] a. Settings: 400 V; 100 μF (settings may vary)

[00785] 14. Place the cuvette on ice for 15 minutes to recover.

[00786] 15. Transfer the cells into 5 ml RVG2i + 10 μM ROCKi.

[00787] 16. Added to a 15 cm plate with 20 ml RVG2i + 10 μM ROCKi. The tablet contained 2x neoR FEM (or other FEM depending on the project).

[00788] 17. Incubated at 37 °. Sampling started after 48 h.

[00789] 18. The G418 selection protocol was as follows:

[00790] a. Day 2 (day 2 after EP): cells were incubated in 2i + G418 medium, 75 μg / ml.

[00791] b. Day 3: cells were incubated in medium 2i without G418

[00792] c. Day 4: cells were incubated in 2i + G418 medium, 75 μg / ml.

[00793] d. Day 5: cells were incubated in medium 2i without G418

[00794] e. Day 6: cells were incubated in 2i + G418 medium, 75 μg / ml.

[00795] f. Day 7: cells were incubated in medium 2i without G418

[00796] g. Day 8: The cells were incubated in 2i + G418 medium, 75 μg / ml.

[00797] h. Day 9: cells were incubated in medium 2i without G418

[00798] i. Day 10: cells were incubated in 2i + G418 medium, 75 μg / ml.

[00799] j. Day 11: cells were incubated in medium 2i without G418

[00800] k. Day 12: Colonies were harvested for propagation for screening. Each colony was split in 0.05% trypsin + 1% chicken serum for 10 minutes and then plated into 1 well of a 96-well feeding plate.

[00801] 19. Colonies were propagated for 3 days in medium 2i.

[00802] 20. Clones were passaged 1: 1 into new 96-well feeding plates.

[00803] 21. Propagated clones for 3 days in medium 2i.

[00804] 22. Colony separation in trypsin was performed for each clone.

2/3 of each clone was frozen and stored at -80 °; plated the remaining 1/3 into laminin plates (96-well plates coated with 10 μg / ml laminin).

[00805] 23. When laminin plates reached confluency, they were transferred to a screening laboratory for clone genotyping.

1.7. Molecular portrait of rat embryonic stem cells

[00806] The genes listed in Table 8 were expressed 20 times less in rat ES cells than the corresponding genes in mouse ES cells. The genes listed in Table 9 were expressed at a level 20 times greater in rat ES cells than the corresponding genes in murine ES cells.

[00807] The microarray data in Tables 8 and 9 were obtained as follows. Rat ES cells (ACI.G2 and DA.2B) and mouse ES cells (F1H4) were cultured in medium 2i for 3 passages until confluence was achieved. F1H4 cells were cultured on gelatin-coated plates in the absence of feeding. ES cells of F1H4 mice were obtained from heterozygous embryos 129S6 / SvEvTac and C57BL / 6NTac (see, for example, US patent No. 7294754 and Poueymirou, WT, Auerbach, W., Frendewey, D., Hickey, JF, Escaravage, JM, Esau, L., Dore, AT, Stevens, S., Adams, NC, Dominguez, MG, Gale, NW, Yancopoulos, GD, DeChiara, TM, Valenzuela, DM (2007), incorporated herein by reference in their entirety).

[00808] The following protocol was used for sample preparation: 1.5 ml Eppendorf tubes were labeled with sample IDs. Cells grown on the plate were washed in 37 ° C phosphate buffered saline (PBS). The PBS was removed and 300 μl Trizol® was added. A scraper was used to disrupt cells in Trizol® (Life Technology). Lysed cells were harvested in Trizol® in a 1.5 ml Eppendorf tube. The cells grown in suspension were washed in 37 ° C PBS and collected in a 1.5 ml tube. The cells were centrifuged; FSB was removed; and added to the cells 300 μl of Trizol®. Cell membranes were disrupted by pipetting. The samples were sorted by FACS from 10 to 105 cells, the volume was reduced by concentration to less than 100 μl. Added 4 volumes of RNA lysis buffer and mixed by pipetting. To obtain a sample, 320 μl of RNA lysis buffer was added to 80 μl of the sample. The samples were stored at 20 ° C.

[00809] RNA sequencing was used to determine the level of expression of murine and rat genes. The sequencing results were mapped to the mouse and rat control genome using Tophat and the RPKM (fragments per thousand base pairs of exon per million mapped fragments) was calculated for the mouse and rat genes. Homologous genes were selected based on the gene symbol, and then a t-test was applied to compare the expression level of each gene in mice and rats. miR-32 was one of the 10 most expressed components in rat ESCs, but was not expressed in mouse ES cells. Although there are no comparative data on miR-632, based on the level of its expression compared to other genes expressed in rat ESCs and its known function in embryonic development, miR-632 was chosen as a marker for rat ES cells.

[00812]

[00813] An additional molecular portrait was also developed using pluripotency markers / genes for rat ES cells. Table 11 lists the genomes and their degree of expression from the RNA profiling data. mRNA was isolated from rat ES cells and the expression levels of various markers were compared with respect to each other. The term "degree" refers to the comparative levels of expression of individual genes: the higher the degree (the highest is the 1st), the higher the expression. For example, the degree Oct4 13 means that among all analyzed genes it was expressed at a higher level than 12 genes. The background value in this experiment was any expression value below 30; 6107 genes had an expression level of 30 and higher.

[00814]

Example 2: Inactivation of genomic loci in rats

2.1: Inactivation of endogenous genomic loci by an endonuclease agent

[00815] To introduce a mutant allele into an endogenous rat genomic locus, the rat ES cells described herein are electroporated with expression vectors (or mRNA) that express ZFN 1 and 2 (or TALEN 1 and 2). These proteins bind their target sequences on opposite strands separated by about 6 bp. up to about 40 p. A double-stranded break is formed at the target locus, which the cells try to repair through non-homologous end joining (NHSC). In many cases, IHSC creates a deletion that often disrupts the function of the gene (most often due to the creation of a frameshift mutation). To identify a mutant allele containing a positive clone, electroporated cells are plated at low density, as drug susceptibility screening is not performed. Colonies are harvested and target site analysis is performed to determine if a mutation has been created (eg, by allele modification analysis (MOA) described above). The selected ES cells containing the mutant allele are then introduced into a rat host embryo, for example, a pre-morula or blastocyst rat embryo, and implanted into the uterus of a surrogate mother to obtain a founder rat (F0 rat). Thereafter, the founding rat is crossed with the wild-type rat to produce F1 offspring heterozygous for the mutant allele. By mating heterozygous F1 rats, progeny homozygous for the mutant allele can be obtained.

2.2 .: Targeting rat ESCs to inactivate the rat apolipoprotein E (ApoE) gene using zinc finger nucleases

[00816] Zinc finger nucleases use sequence-specific modular DNA-binding domains to direct endonuclease activity to a specific target sequence in the genome. ZFNs are designed as a pair of monomers. Each monomer contains a nonspecific FokI endonuclease cleavage domain fused to 3 or more zinc finger DNA binding domains. Each zinc finger binds a 3 bp subsite, and specificity is achieved by combining the target sites of both monomers. ZFNs create double strand breaks (DSBs) in DNA, and mutations (insertions or deletions) often occur during non-homologous end-joining (NHSC). Figure 15 illustrates the mechanism by which genome-editing endonucleases such as ZFN and TALEN introduce double-stranded breaks in the target genomic sequence and activate NGSCs in the cell. DSBs are often stimulated by homology-directed repair (HGR) by homologous recombination if the donor sequence contains ZFN.

[00817] Such ZFNs have been used in combination with various methods and compositions described herein to improve targeting efficacy. Targeting of the rat apolipoprotein E (ApoE) locus was performed as described in Example 3.2 (a) (i), except that expression vectors that express ZFN 1 and 2 were also introduced into rat ES cells. See Figure 11 for a schematic of targeting ApoE in combination with rTZFN1P and rTZFN2P. The targeting efficacy was determined as discussed below in Example 5, and the results are shown in Table 12. For screening for heterozygous targeting, homozygous targeting and "mixed" pairs (eg, compound-heterozygous targeting), specific primers and probes were used to determine ... Unexpectedly, targeting efficiency increased 8-10 times.

[00818]

[00819] A plasmid guide vector was constructed with a self-destructive drug sensitivity selection cassette and the lacZ gene as a reporter gene (see Figure 14 illustrating the homologous and non-homologous recombination that can occur after electroporation of the guide vector containing the selection cassette). Good targeting efficiency was achieved and high% chimeras were obtained. Zinc finger nucleases (ZFN) were also investigated in combination with targeting vectors to study their effect on improving targeting efficiency (see Figure 16 illustrating a gene targeting method using ZFN or TALEN to improve the efficiency of homologous targeting vector recombination). The targeting vector was co-expressed with 2-pair ZFN expression vectors that cut the ApoE locus. Rat ESC clones electroporated with both the targeting vector and the ZFN group showed targeting efficiency 8-10 times higher than the rat ESC clones electroporated with the targeting vector alone. In addition, biallelic homozygous targeting was detected in approximately 2% of clones. High% chimeras were generated from these two targeted clones.

[00820] Clones of rat ESCs with targeted ApoE (using ZFN) were microinjected into SD blastocysts, which were then transferred into pseudopregnant SD female recipients using standard methods. Chimeras were identified by skin color (see Figure 17 illustrating ApoE-ZFN-AB5 chimeras (i.e. ApoE ^{- / -} chimeras); male F0 chimeras were crossed with SD females. F1 germline pups were genotyped for the presence of the targeted ApoE allele (Table 13) High% chimeras were generated from these two targeted clones.

[00821]

[00822] The APOE disabled rat is a tool for studying various types of disorders and diseases. In humans, apolipoprotein is found in chylomicron, HDL, LDL, and VLDL. ApoE is essential for the normal catabolism of triglyceride-rich constituents of lipoproteins. Defects in APOE result in numerous disease conditions including, for example, familial hypercholesterolemia, hyperlipidemia, betalipoproteinemia, familial dysbetalipoproteinemia, type III hyperlipoproteinemia (HLP III), risk of coronary artery disease. One isoform (ApoE4) is associated with familial late-onset Alzheimer's and sporadic Alzheimer's, and possibly MS (multiple sclerosis).

[00823] In mice, ApoE is mainly found in HDL; tolerates cholesterol, as in humans. Mice lacking ApoE (2 independent shutdowns) have 5 times normal blood cholesterol levels; Formation of foamy, cell-rich deposits in the proximal aorta at 3 months of age (comparable to human syndrome).

[00824] Turning off ApoE in rats provides an animal model for studying endothelial function, including, but not limited to, plaque formation, transcriptional changes (RNA sequencing), ex vivo function. In addition, the larger size of the rats facilitates these analyzes and potentially improves the quality of the RNA sequencing data.

2.3. Inactivation of the rat interleukin-2 gamma receptor locus (IL2r-γ) using zinc finger nucleases

[00825] The targeting of the rat interleukin-2 gamma receptor locus (IL2r-γ or Il2rg) was performed as described in Example 3.3 (a), except that expression vectors that express ZFN U (ZFN upstream) were also introduced into rat ES cells and ZFN D (ZFN underlying). Figure 18 shows a schematic representation of targeting IL2r-γ in combination with ZFN U and ZFN D. The sequence of the IL2r-γ locus that these zinc fingers bind is indicated in Figure 18 within SEQ ID No. 93. The targeting efficacy was determined as described below in Example 3.3 (a), and the results are shown in Table 14. Briefly, homozygous targeted clones were confirmed by PCR. For the ZFN1 pair: 173 mutant clones out of 192 examined (90%), and for the ZFN2 pair: 162 clones out of 192 investigated (84%).

[00826]

[00827] Clones of rat ESCs with targeted IL2r-γ (using ZFN) were microinjected into SD blastocysts, which were then transferred into pseudopregnant SD female recipients using standard methods. Chimeras were identified by skin color; F0 chimera males were crossed with SD females. F1 germline pups were genotyped for the presence of the targeted IL2r-γ allele.

2.4 .: Inactivation of the rat interleukin-2 gamma (IL2r-γ) receptor locus by CRISPR / Cas9

[00828] Targeting of the rat TL2r-γ locus was performed as described in Example 3.3 (a), except that the CRISPR / Cas9 system was also introduced into the rat ES cells to improve targeting efficiency. SBI: System Biosciences Cas9 “SmartNuclease” all-in-one vectors were used and Cas9 expression was driven by the CAG, EF1a, PGK or CMV promoter. The special nRNA was ligated into a vector and expressed using the H1 promoter. 4 nRNAs were constructed against Il2rg. Regions of the rat IL2r-γ locus targeted by nRNAs 1-4 are shown in Figure 19. Specific genotypes and probes were used to screen for targeting (eg, heterozygous targeting, homozygous targeting, and compound heterozygous targeting). The targeting results using different guide RNAs are shown in Table 15. “Strong” and “weak” refer to the aggregate of screening data that the colony has the target modification.

[00829]

2.5 .: Inactivation of the murine hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene by CRISPR / Cas9

[00830] The mouse Hprt locus was targeted in mouse ES cells using LTVEC alone or in combination with CRISPR / Cas9. The entire 32.9 kb Hprt coding sequence was targeted. to create a deletion and replacement with the pCAGG-Puro puromycin resistance selection cassette, which also expresses uZPB. The start and stop codons were the extreme points of the deletion. The guide RNA sequence used corresponded to 5'-GACCCGCAGUCCCAGCGUCG-3 '(SEQ ID No. 84) and was targeted to exon 1 of the murine Hprt gene. The predicted target cleavage site was 22 bp from the 5 'end of the deletion. The efficiency of Cas9 / nRNA cleavage in the target region, observed in ES cells, was ≥ 93%. The results are summarized in Table 16. Application of CRISPR / Cas9 to aid in targeting the full 32.9 kb Hprt locus. resulted in a fivefold increase in targeting compared to using LTVEC alone.

[00831]

Example 3: Targeted Modification of Rat Genomic Loci

3.1: Targeting rat ESCs: The rat Rosa26 locus.

[00832] The rat Rosa26 locus is located between the Setd5 and Thumpd3 genes, as in mice, at the same intervals. The rat Rosa26 locus (Figure 12, panel B) differs from the murine Rosa26 locus (Figure 12, panel A). Murine Rosa26 transcripts are composed of 2 or 3 exons. The rat locus contains the 2nd exon 1 (Ex1b) in addition to the homologous exon to the murine exon 1 (Ex1a). No exon 3 was detected in rats. The targeting of the rat Rosa26 allele is illustrated in Figure 12C, where the 5 kb homology arms. each were cloned by PCR using genomic DNA from ESCs from DA rats. The targeted allele contains the SA (splicing acceptor) cassette -lacZ-hUb-neo replacing the 117 bp deletion. in nitrone of rat Rosa26.

[00833] The targeting efficiency of the rat Rosa26 locus was determined (Table 17). The linearized vector was electroporated into ESCs from DA or ACI rats, and the transfected colonies were cultured in 2i media + G418 using standard methods. Individual colonies were harvested and screened by Lost Allele Analysis (LOA) (Valenzuela, D. et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21: 652-660, included in this document by reference).

[00834] Obtaining chimeras and germline transmission using Rosa26-targeted clones of rat ESCs. Reconfirmed Rosa26-targeted clones of rat ESCs were microinjected into SD blastocysts, which were then transferred into pseudopregnant SD female recipients using standard methods. Chimeras were identified by skin color; F0 chimera males were crossed with SD females. F1 germline pups (agouti) were genotyped for the presence of the targeted Rosa26 allele; nine of the 22 agouti pups were genotyped as heterozygous for the Rosa26 locus (Table 18).

[00835] To confirm that the genetically modified allele at the Rosa26 locus was transmitted via the germline, lacZ expression was confirmed by staining with X-gal in heterozygous Rosa26-targeted rats. X-gal staining of the brain, heart, thymus and lungs of 14-week-old heterozygous rats with targeted Rosa26 showed the presence of lacZ expression (Figure 13B, D and F, respectively), while control wild-type animals of the same age showed low levels background staining with X-gal (Figure 13A, C and E, respectively). X-gal staining in E12.5 and E 14.5 heterozygous rat embryos targeting Rosa26 showed ubiquitous lacZ expression (Figure 13G and I, respectively), while control rat embryos showed low levels of background X-gal staining (Figure 13H and J , respectively).

3.2. (A) (i): Targeting the rat apolipoprotein E (ApoE) locus.

[00836] The rat apolipoprotein E (ApoE) locus was targeted to block the function of ApoE. The targeting of the ApoE locus was performed using a targeting vector containing a lacZ-hUb-neo cassette flanked by 5 'and 3' homology arms homologous to the ApoE locus. Figure 20 illustrates a genetically modified rat ApoE locus that was destroyed by 1.8 kb. deletions and insertions of the lacZ-hUb-neo cassette, which additionally contains a self-destructive Cre cassette containing the Crei gene under the control of a protamine promoter. The electroporation conditions were as follows: 6 μg DNA; 2.05 x 10 ⁶ cells; 400 V; 200 μF: 342 V, 593 μs; sown at 15 cm with 2x density neoR PEM in 2i + 10 μM ROCKi.

[00837] The targeting efficiency at the ApoE locus was determined and the results are shown in Table 19. The linearized vector was electroporated in DA.2B rat ESCs obtained from the DA strain, and the transfected colonies were cultured using standard methods. Collected individual colonies and screened by lost allele analysis (LOA).

[00838] Carried out the production of chimeras and transmission of the germ line using clones of rat ESCs with targeted ApoE. Clones of rat ESCs with targeted ApoE were microinjected into SD blastocysts, which were then transferred into the body of pseudopregnant female SD recipients using standard methods. Chimeras were identified by skin color; F0 chimera males were crossed with SD females. Germline transmission has been achieved. F1 pups were genotyped for the presence of the targeted ApoE allele (Table 20).

[00839] Confirmation of LacZ expression under the control of the endogenous ApoE promoter was carried out by X-gal staining of the brain, blood vessels and liver of 12 month old female rats with ApoE ^+/- (Figures 43-45, respectively). Figures 43-45 illustrate the expression profile of lacZ, which mimics the expression profile of endogenous ApoE. Wild-type control animals of the same age showed low background X-gal staining.

[00840] Additionally studied the phenotypes of rats with removed ApoE -. Continuous serum chemistry tests were performed to determine cholesterol, LDL, HDL, and triglyceride levels at three week intervals. Figure 46A-D illustrates serum cholesterol, LDL, HDL and triglyceride levels in homozygous targeted, heterozygous targeted and wild type rats at 6 weeks of age, 9 weeks, 12 weeks, and 15 weeks. Blood sampling from the eyes was carried out for the corresponding age group, consisting of 2 wild-type rats, 7 heterozygous and 8 homozygous rats. No significant difference was observed between males and females. Homozygous rats with removed ApoE showed increased levels of cholesterol and LDL cholesterol and decreased levels of HDL. Unlike APOE ^{- / -} mice, no significant increase in triglyceride levels was observed in rats with removed APOE.

[00841] Additional phenotypic analyzes performed included histological / vivo visualization of aortic arch plaque formation, in vivo visualization of aortic arch plaque formation, and transcriptional changes (shotgun sequencing of the complete transcriptome (RNA sequencing)) in the aortic arch endothelium. The timing of these tests depends on the duration of the plaque formation. Plaques become detectable in ApoE ^{- / -} mice at 24 weeks.

[00842] Additional data on targeting ApoE are shown in Table 22.

3.2. (A) (ii). Targeting ApoE in rats with a targeting vector

[00843] Figure 20 is a schematic representation of a rat ApoE locus and a targeting plasmid. The top diagram in Figure 20 shows the genomic structure of the rat ApoE locus and the genomic regions corresponding to the 5 'and 3' homology arms (5 kb and 5.4 kb, respectively; dark gray rectangles) ... Exon 1 ApoE is non-coding and is shown as an unstained rectangle closest to the 5 'homology arm. The three introns in the ApoE gene are indicated as lines, and exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle.

[00844] The lower diagram of Figure 20 shows a direction vector. The 5 'and 3' homology arms (5 kb and 5.4 kb, respectively) are indicated by dark gray rectangles. The targeting vector contains a reporter gene (lacZ) and a self-destructive cassette flanked by loxP regions (unstained arrows). The self-destructive cassette contains the Crei gene operably linked to the murine Prm1 promoter and a selection cassette containing the neomycin resistance gene operably linked to the human ubiquitin promoter.

[00845] The Crei gene contains two exons encoding Cre recombinase, which are separated by an intron (Crei) to prevent its expression in a prokaryotic cell. See, for example, US patent 8697851 and US published patent application 2013-0312129, which describe in detail the self-destructive cassette, and which are fully incorporated herein by reference. The self-destructive cassette can be removed using the Prm1 promoter, in particular in male F0 rat germ cells. The targeting vector was electroporated into the rat ES cells obtained in Example 1, and cells were seeded at 15 cm with 2x density of neomycin resistant PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured, selected and maintained as described in Example 1.

[00846] As shown in Table 44, 384 colonies were screened and 23 targeted clones were obtained. The targeting efficiency was 5.99%. 3 clones were injected into blastocysts as described in Example 1. 3 clones producing chimeras were obtained and 1 of the clones transmitted germline targeting.

3.2. (A) (iii). Targeting ApoE in rats with a targeting vector in combination with zinc finger nucleases

[00847] The targeting vector used in Example 3.2 (a) (ii) was used in combination with zinc finger nucleases to target the rat ApoE locus. Table 21 summarizes the genomic structure of the rat ApoE locus. The positions shown in Table 21 were taken from the rat genome standard sequence 5.0 (ENSMBL) construct. ApoE is located on chromosome 1 on the (-) chain.

[00848]

[00849] Figure 11 is a schematic representation of the rat ApoE locus, and the gray rectangles represent the cutting sites ZFN1 and ZFN2. The ZFN1 cleavage site is in exon 3, and the ZNF2 cleavage site is in intron 3. The exact positions of both ZFN sites are shown in Table 21. Genomic regions corresponding to the 5 'and 3' homology arms (5 kb and 5.4 etc., respectively) are indicated by dark gray rectangles. Exon 1 ApoE is non-coding and is shown as an unstained rectangle closest to the 5 'homology arm. The three introns in the ApoE gene are indicated as lines, and exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle.

[00850] The targeting vector used was the same as in Example 3.2 (a) (ii) and is shown in Figure 20, and Figure 21A shows a diagram of targeting the ApoE locus in rat ES cells using zinc finger nucleases and a targeting vector illustrated in Figure 20. ZFN was introduced as two expression plasmids, one for each half of the ZFN pair. Used 20 μg of plasmids for ZFN1 and 20 μg of plasmids for ZFN2. ZFN were acquired from Sigma. The expression of each ZFN was driven by the CMV promoter.

[00851] The targeting vector was electroporated into the rat ES cells obtained in Example 1, and cells were seeded at 15 cm with 2x density neoR PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured, selected and maintained as described in Example 1.

[00852] As shown in Table 22 and Table 44, 384 colonies were screened and 290 targeted clones were obtained. The targeting efficiency was 75.52%. Two clones were injected into blastocysts as described in Example 1. Two clones producing chimeras were obtained and one of the clones transmitted targeting via the germline.

[00853] In addition, the use of ZFN1 and ZFN2 resulted in 8 biallelic targeted clones with an efficiency of 2.08%.

[00854]

3.2. (B) (i): Targeted modification of the rat apolipoprotein E (ApoE) locus using a large targeting vector (LTC).

[00855] Targeting the ApoE locus is performed with a large targeting vector (LTVEC) containing a lacZ murine Prm1-Crei cassette flanked by a 5 'arm of homology to the ApoE locus of about 45 kbp. and a 3 'arm homology to the ApoE locus of about 23 kb. Figure 22 illustrates the rat ApoE locus in which the ApoE locus was disrupted due to 1.83 kb. deletions and insertions of the lacZ gene and a self-destructive cassette containing the mPrm11-Crei cassette and the hUb-neo selection cassette. The methods used in 3.2 (a) (i) can be used to introduce this vector into rat ES cells.

Example 3.2. (B) (ii). Targeting the rat ApoE locus with a large targeting vector (LTVEC)

[00856] Figure 22 is a schematic representation of the rat ApoE locus and large targeting vector (LTVEC). The upper diagram in Figure 22 shows the genomic structure of the rat ApoE locus and the genomic regions corresponding to the 5 'and 3' arms of homology (45 kb and 23 kb, respectively; dark gray rectangles). Exon 1 ApoE is non-coding and is shown as an unstained rectangle closest to the 5 'homology arm. The three introns in the ApoE gene are indicated as lines, and exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle.

[00857] The lower diagram of Figure 22 shows the LTVEC. The 5 'and 3' homology arms (45 kb and 23 kb, respectively) are indicated by dark gray rectangles. The targeting vector contains a reporter gene (lacZ) and a self-destructive cassette flanked by 1oxP regions (open arrows), which contains the Crei gene functionally linked to the murine Prml promoter, and a drug susceptibility selection cassette containing the neomycin resistance gene, functionally linked to a human ubiquitin promoter. Crei contains two exons encoding Cre recombinase, which are separated by an intron (Crei) to prevent its expression in the prokaryotic cell. See, for example, US patent 8697851 and US published patent application 2013-0312129, which describe in detail the self-destructive cassette, and which are fully incorporated herein by reference. The self-destructive cassette can be removed using the murine Prml promoter, in particular in F0 male germ cells of rats.

[00858] LTVECs were electroporated into rat ES cells obtained in Example 1, and cells were seeded at 15 cm with 2x density neoR PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured, selected and maintained as described in Example 1.

[00859] As shown in Table 44, 288 colonies were screened and 8 targeted clones were obtained. The targeting efficiency was 2.78%. 3 clones were injected into the host embryo at the blastocyst stage as described in Example 2 to obtain chimeric rats (F0). In addition, one biallelic targeted clone was obtained with a biallelic efficiency of 0.35%.

3.2. (B) (iii). Targeting ApoE in rats with a large targeting vector (LTVEC) in combination with zinc finger nucleases

[00860] The LTVEC used in Example 3.2 (b) (ii) was used in combination with zinc finger nucleases to target the rat ApoE locus. Table 21 summarizes the genomic structure of the rat ApoE locus, and the positions shown were taken from construct 5.0 of the rat genome standard sequence (ENSMBL).

[00861] Figure 23 is a schematic representation of the rat ApoE locus and the gray rectangles represent the cutting sites ZFN1 and ZFN2. The ZFN1 cut is in exon 3, and the ZNF2 cut is in intron 3. The exact positions of both ZFN sites are shown in Table 21. The 5 'and 3' homology arms (45 kb and 23 kb) , respectively) are indicated by dark gray rectangles. Exon 1 in the ApoE gene is non-coding and is shown as an unstained rectangle closest to the 5 'homology arm. The three introns in the ApoE gene are indicated as lines. Exons 2 and 3 contain coding regions and are shown as dashed gray rectangles. Exon 4 contains both coding and non-coding sequences, indicated by a gray dotted line and an uncolored rectangle.

[00862] The LTVEC used was the same as in Example 3.2 (b) (ii) and is shown in Figure 22. ZFN was introduced as two expression plasmids, one for each half of the ZFN pair. Used 20 μg of plasmids for ZFN1 and 20 μg of plasmids for ZFN2. ZFN were acquired from Sigma. ZFN were acquired from Sigma. The expression of each ZFN was driven by the CMV promoter.

[00863] The targeting vector was electroporated into the rat ES cells obtained in Example 1, and cells were seeded at 15 cm with 2x density neoR PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured, selected and maintained as described in Example 1.

[00864] As shown in Table 44, 288 colonies were screened and 16 targeted clones were obtained. The targeting efficiency was 5.56%. One clone was injected into blastocysts as described in Example 2.

[00865] In addition, the use of ZFN1 and ZFN2 resulted in one biallelic targeted clone with an efficiency of 0.35%.

3.2. (B) (iv). ApoE targeting in rats with a large targeting vector (LTVEC) in combination with CRISPR / Cas9 [00866] LTVEC used in Example 3.2. (B) (ii) was used in combination with CRISPR / Cas9 to target the rat ApoE locus. Table 23 compares the results of experiments in which ApoE LTVEC was used alone to target the rat ApoE locus or used in combination with the CRISPR / Cas9 nuclease to target the rat ApoE locus. In each experiment, electroporated cells were plated at high density and subjected to drug selection to find drug resistant colonies. Drug resistant colonies were harvested and screened for targeted modification by allele modification assay (MOA) as described herein. In particular, 4 × 10 ⁶ cells were electroporated with 2 μg ApoE LTVEC at a voltage of 400 V, a capacitance of 100 μF, and a resistance of 0. In the last experiment, 6 μg of the Cas9 expression plasmid and 3 μg of mRNA2 ApoE or 3 μg of nRNA3 ApoE were also electroporated. Selection was performed using 75 μg / ml G418. nRNA2 ApoE has the sequence GCAGGCCCTGAACCGCTTCTTGG (SEQ ID No. 87) and targets a 67 bp region. 3 'from the start of exon 3 of rat ApoE. nRNA3 ApoE has the sequence CCTGCGCTGGGTGCAGACGCTTT (SEQ ID NO. 88) and targets the 97 bp region. 3 'from the start of exon 3 of rat ApoE (see Figure 47). As shown in Table 23, when Cas9 and any of the nRNAs were introduced into the cell along with the APOE LTVEC, the targeting efficiency increased (from 43% to 53% or 47%). Biallelic targeting was observed in five colonies targeted by APOE LTVEC in combination with nRNA2 or 3 APOE, but no biallelic modification was observed for APOE LTVEC alone.

[00867]

3.3 (a): Targeting the rat interleukin-2 gamma receptor locus (IL2r-γ)

[00868] The rat interleukin-2 gamma receptor locus (IL2r-γ or Il2rg) was targeted to block IL2r-γ function. IL2r-γ plays an important role in signaling IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, and mutations in IL2r-γ are associated with severe disorders in the development of T-, B- and ΝΚ-cells. [00869] The targeting of the IL2r-γ locus was performed using a targeting vector containing an eGFP-hUb-neo cassette flanked by the 5 'and 3' homology arms homologous to the IL2r-γ locus, as illustrated in Figure 24. Figure 25 illustrates the genomic structure of the rat IL2r-γ, in which the IL2r-γ locus was disrupted due to 3.2 kb. deletions. The targeted IL2r-γ locus also contains the uZPB gene and a self-destructive cassette containing Crei operably linked to the murine protamine 1 promoter, and a drug susceptibility selection cassette containing the hUb promoter operably linked to the neomycin resistance gene.

[00870] The targeting efficiency at the IL2r-γ locus was determined, and the results are shown in Table 24. The linearized vector was electroporated into the ESCs of DA.2B rats and the transfected colonies were cultured using standard methods. Collected individual colonies and screened by lost allele analysis (LOA).

[00871] Chimera production and germline transmission were performed using clones of rat ESCs with targeted IL2r-y. Clones of rat ESCs with targeted IL2r-y were microinjected into SD blastocysts, which were then transferred into the body of pseudopregnant female SD recipients using standard methods. Chimeras were identified by skin color; F0 chimera males were crossed with SD females. The F1 germline pups were genotyped for the presence of the targeted IL2r-γ allele (Table 25). In another microinjection experiment with clone Il2rg-CG12, germline transmission was also confirmed by skin color and genotyping.

[00872]

[00873] In addition, the phenotype of the Il2rg-γ # 3 chimera was studied.

Peripheral blood mononuclear cells (PBMCs) were stained with antibodies that recognize antigens in several lymphoid lineages. PBB-positive PBMCs were detected in 2 chimeras, as shown in Figure 30. In addition, PBB + cells were negative for the T-cell marker CD3 (Figure 29A) and were mostly negative for the B-cell marker B220 and marker ΝΚ -cells CD161a (Figure 29B and C, respectively). PBMCs obtained from wild-type rats were used as a negative control for the expression of PBS. See Figure 29D-F. Small, double-positive populations are consistent with published phenotypic data for Il2rg-disabled mice. These data were obtained from a chimeric rat that contains positive for the IL2 receptor gamma cells, and this may complicate the analysis of the phenotype. It is also possible to perform flow cytometric analysis with populations of cells from the bone marrow and spleen to detect the corresponding diseases in a large number of lymphocytes. See Mashimo et al. (2010) PLoS One 5 (l): e8870.

3.3 (b): Targeted modification of the rat interleukin-2 gamma receptor locus (IL2r-γ)

[00874] The rat interleukin-2 gamma receptor locus (IL2r-γ) was targeted to block IL2r-γ function in rats. Figure 25 illustrates the genomic structure of the rat Il2rg locus (upper panel in Figure 25) and a targeting vector introduced into the locus (lower panel in Figure 25). UZFB was chosen as a reporter so that the immunophenotype of genetically modified rats could be determined by the FACS method. The self-destructive cassette (hUb-Neo; PrmJ-Cre) was used to remove the drug susceptibility selection cassette and the Cre gene, particularly in male F0 rat germ cells. In addition, the targeting vector was designed to remove the entire coding region (about 3.2 kb) of the rat Il2rg gene.

[00875] The size of the deletion in rat ESCs was confirmed by PCR using primers specific to the rat Il2rg locus. After microinjection of targeted clones into host embryos at the blastocyst stage, chimeras with a high percentage were obtained. These chimeras were selected for crossbreeding. To determine if targeting is performing as expected, peripheral blood was taken from chimeras prior to crossing and the phenotype of immune cells in peripheral blood was analyzed by FACS. As shown in Figure 30, ZFB-positive cells were detected in the blood of 2 of 3 chimeras tested, and chimeric rats contained less than 1% T cells, less than 1% B cells, and less than 1% ΝΚ-cells, which were positive for PBS (i.e. KO Il2rg cells) (Figure 29A-C).

3.4 (a) (i). Targeting the rat Rag2 locus using a large targeting vector (LTVEC)

[00876] Table 26 summarizes the genomic structure of the rat Rag2 locus, and the positions shown were taken from construct 5.0 of the rat genome standard sequence (ENSMBL). Rag2 is located on chromosome 3 on the (+) strand.

[00877]

Figure 26 is a schematic representation of the rat Rag2 locus and large targeting vector (LTVEC). LTVEC is 140 kbp. and is aimed at approximately 5.7 kbp. part of the rat Rag2 locus to create a deletion. The top diagram in Figure 26 shows the genomic structure of the rat locus

[00878] and genomic regions corresponding to the 5 'and 3' arms of homology (48 kb and 84 kb, respectively; dark gray rectangles). Rag2 contains one exon, indicated by dashed gray shading.

[00879] The lower diagram of Figure 26 shows LTVEC. The 5 'and 3' homology arms (48 kb and 84 kb, respectively) are indicated by dark gray rectangles. LTVEC contains a reporter gene (lacZ) and a self-destructive cassette flanked by 1oxP regions (uncolored arrows). The self-destructive cassette contains a murine Prml promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing a human ubiquitin promoter operably linked to the neomycin resistance gene. A different version of LTVEC was created, in which the neomycin resistance gene was replaced with the hygromycin resistance gene to allow re-targeting of Il2rg-targeted rat ES cells. Crei contains two exons encoding Cre recombinase, which are separated by an intron (Crei) to prevent its expression in the prokaryotic cell. See, for example, US patent 8697851 and US published patent application 2013-0312129, which describe in detail the self-destructive cassette, and which are fully incorporated herein by reference. The self-destructive cassette can be removed using the murine Prml promoter, in particular in F0 male germ cells of rats.

[00880] LTVECs were electroporated into rat ES cells obtained in Example 1 and cells were seeded at 15 cm with 2x density neoR PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured and maintained as described in Example 1.

[00881] Colonies were screened as described elsewhere in this document and targeted clones were obtained. The targeted clones were injected into the host embryo as described elsewhere in this document to produce F0 rats.

3.4 (a) (ii). Targeting the Rag2 locus in rats using a large targeting vector (LTVEC) and CRISPR / Cas9

[00882] Table 27 compares the results of experiments in which Rag2 LTVEC containing the hygromycin resistance gene (see Figure 48) was used alone to target the rat Rag2 locus or used in combination with CRISPR / Cas9 nuclease to target the rat Rag2 locus. In each experiment, electroporated cells were plated at high density and subjected to drug selection to find drug resistant colonies. Drug resistant colonies were harvested and screened for targeted modification by allele modification assay (MOA) as described herein. In particular, 4 × 10 ⁶ cells were electroporated with 2 μg of Rag2 LTVEC at a voltage of 400 V, a capacitance of 100 μF and a resistance of 0. In the last experiment, 6 μg of the Cas9 expression plasmid and 3 μg of hRNK1 Rag2 or 3 μg of Rag2 nRNA4 were also electroporated. Selection was performed using 75 μg / ml G418. hRHK1 Rag2 has the sequence CCAGCTACTTGCTCGTACAA (SEQ ID No. 89) and targets a 219 bp region. 3 'from the start of the rat Rag2 start codon (ATG). nRNA4 Rag2 has the sequence CCCCTCAGATTCACGTGCGT (SEQ ID NO. 90) and targets a 12 bp region. 3 'from the rat Rag2 stop codon (TAG) (see Figure 48). As shown in Table 27, if Cas9 and any of the nRNAs were added to the cell along with Rag2 LTVEC, the targeting efficiency increased (from 0 to 10% or 38%). Biallelic targeting was observed in one colony.

[00883]

3.4. (b) (i): Targeting the Rag1 and Rag 2 locus in rats

[00884] Figure 27 shows the genomic structure of the rat Rag1 / Rag2 locus. CDS stands for coding sequence and gray boxes represent exons. Rag2 is located on the plus strand with the right transcriptional direction. Rag1 is located on the minus strand with a left transcriptional direction. M.p. = one million base pairs.

[00885] Table 28 summarizes the genomic structure of the rat Rag2 locus and the rat Rag1 locus, and the positions shown were taken from construct 5.0 of the rat genome standard sequence (ENSMBL). Rag1 is located on chromosome 3 on the (-) chain.

[00886]

[00887] Figure 28 is a schematic representation of the rat Rag2 and Rag1 locus and large targeting vector (LTVEC). LTVEC is about 70 kbp. and is aimed at approximately 16.6 kbp. a rat genomic locus containing the Rag1 and Rag2 loci to create a deletion. The upper diagram in Figure 28 shows the genomic structure of the Rag1 and Rag2 loci and the genomic regions corresponding to the 5 'and 3' arms of homology (48 kb and 15 kb, respectively; dark gray rectangles). Rag1 and Rag2 each contain one exon, indicated by gray dotted shading. The lower diagram in Figure 28 shows LTVEC. The 5 'and 3' homology arms (48 kb and 15 kb, respectively) are indicated by dark gray rectangles. LTVEC contains a reporter gene (lacZ) and a self-destructive cassette flanked by 1oxP regions (uncolored arrows). The self-destructive cassette contains a rat Prml promoter operably linked to the Crei gene and a drug susceptibility selection cassette containing a human ubiquitin promoter operably linked to a neomycin resistance gene. A different version of LTVEC was created, in which the neomycin resistance gene was replaced with the hygromycin resistance gene to allow re-targeting of Il2rg-targeted rat ES cells. Crei contains two exons encoding Cre recombinase, which are separated by a nitrone (Crei) to prevent its expression in the prokaryotic cell. See, for example, US patent 8697851 and US published patent application 2013-0312129, which describe in detail the self-destructive cassette, and which are fully incorporated herein by reference. The self-destructive cassette can be removed from male F0 rat germ cells using the rat Prml promoter, which drives the expression of Crei, in particular in male germ cells. [00888] LTVECs were electroporated into rat ES cells obtained in Example 1, and cells were seeded at 15 cm with 2x density neoR PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured and maintained as described in Example 1.

[00889] Colonies were screened as described elsewhere in this document and targeted clones were obtained. The targeted clones were injected into the host embryo as described elsewhere in this document to produce F0 rats.

3.4. (B) (ii): Re-targeting of the Rag1 and Rag2 locus in rat ES cells with an already targeted Il2rg locus

[00890] Received LTVEC according to Figure 50 to target the Rag1 and Rag2 loci to create a deletion. The total length of the LTVEC was 72 kbp. LTVECs were electroporated into rat ES cells that had already been targeted to remove the Il2rg locus as described in Example 3.3. In particular, the rat ES cells belonged to clone Il2rg-CG12, for which germline transmission was confirmed in Example 3.3 (a). Transformed rat ES cells were cultured and maintained as described in Example 1. Dual targeting clones were screened as described elsewhere in this document and targeted clones were obtained. The Il2rg-CG12 cells were re-targeted with an efficiency of 85%, while the Il2rg mutations were still present in the targeted clones. Electroporation was performed as described elsewhere in this document, and antibiotic susceptibility screening was performed using 1.5 μg / ml puromycin. Then, the targeted clones were injected into the host embryo, as described elsewhere in this document, to obtain F0 rats. Re-targeting has the advantage that it takes less time than mating Rag1 / Rag2-targeted rats with Il2rg-targeted rats.

Example 4. Humanization

4.1. Humanization of rat genomic loci

[00891] Humanization of rat genomic loci is carried out using rat ES cells described herein, which are capable of maintaining their pluripotency after one or more electroporation in vitro and are capable of transmitting targeted genetic modifications to subsequent generations. In addition, in order to overcome the limitations associated with the inability of plasmids to accommodate large fragments of genomic DNA, and to overcome the low efficiency of introducing targeted genetic modification at an endogenous locus in rat ES cells, one or more targeted genetic modifications are performed in bacteria, for example, E. coli, by means of the use of bacterial homologous recombination (BHR) and large targeting vector (LTVEC). The LTVEC described herein contains, for example, a large fragment of endogenous rat genomic sequence with one or more modifications, or contains exogenous nucleic acid {eg, homologous or orthologous to human nucleic acid) flanked by rat arms of homology complementary to particular genomic regions.

4.2. Humanization of rat immunoglobulin loci

[00892] Humanization of an endogenous rat immunoglobulin heavy chain locus is performed by removing one or more nucleotide sequences of a rat immunoglobulin heavy chain (e.g., one or more endogenous V _H gene segments, one or more human D gene segments, and one or more human J _{H gene segments} ); and introducing a targeting vector into the modified immunoglobulin locus, e.g., a large targeting vector (LTVEC), comprising: (i) one or more non-rearranged human variable region nucleotide sequences (e.g., one or more human V _H gene segments, one or more human gene segments D and one or more human gene segments J _H ) or one or more rearranged human variable region nucleotide sequences (eg, one or more rearranged human VDJ gene segments); (ii) a selection cassette (for example, a neomycin resistance gene flanked by 1xP regions); and (iii) 5 'and 3' rat shoulders homology

[00893] Briefly, one or more endogenous rat immunoglobulin heavy chain variable region gene segments (ie, one or more V _H gene segments, one or more human D gene segments, and one or more human J _H gene segments) in rat IIR a clone is removed or inactivated by targeting the endogenous rat immunoglobulin heavy chain locus with a selection cassette flanked by rat homology arms. More specifically, a targeting vector is constructed containing a selection cassette (e.g., a neomycin resistance gene flanked by 1xP regions) flanked by 5 'and 3' rat homology arms that are complementary to target rat genomic sequences (e.g., upstream and downstream rat genomic DNA sequences, one or more V _H gene segments, one or more human D gene segments and one or more human J _H gene segments).

[00894] Thereafter, bacterial cells containing a large fragment of rat genomic DNA containing the rat locus of an immunoglobulin heavy chain are selected and introduced with a plasmid (eg, pABG) encoding a recombinase operably linked to a transiently inducible promoter. The targeting vector with the above construct is then introduced into recombination-competent bacterial cells. Following electroporation, bacterial cells are treated with an inducer (eg, arabinoside) to initiate homologous recombination between the targeting vector and the target rat genomic sequence in the IIR clone. The transformed cells are plated at high density and subjected to drug sensitivity screening to find drug resistant colonies. Drug resistant colonies are harvested and screened for targeted modification.

[00895] To facilitate the detection of targeted genetic modification, a high-throughput quantitative assay is used, namely allele modification assay (MOA), which allows large-scale screening of modified allele (s) on the parent chromosome after genetic modification. MOA analysis can be performed using a variety of analytical methods, including, but not limited to, quantitative PCR, such as real-time PCR (qPCR). For example, real-time PCR involves the use of a first primer set that recognizes a target locus and a second primer set that recognizes a non-target control locus. In addition, the primer group may contain a fluorescent probe that recognizes the amplified sequence. Alternatively, quantitative analysis can be performed using a variety of analytical methods, including, but not limited to, fluorescence in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization with immobilized probe (s), Invader Probes ®, MMP assays®, TaqMan® Molecular Beacon and Eclipse ™ probes. (See, for example, US2005 / 0144655, which is incorporated herein by reference in its entirety).

[00896] Then the bacterial cells containing the modified rat IIR clone, i. E. An IIR clone containing a rat genomic DNA sequence in which one or more endogenous heavy chain variable region gene segments (V _H , D and / or J _H gene segments) have been removed or inactivated is electroporated together with a large targeting vector (LTVEC) containing (i) one or more non-rearranged human variable region nucleotide sequences (e.g., one or more non-rearranged human V _H gene segments, one or more human D gene segments, and one or more human J _H gene segments) or one or more rearranged nucleotide sequences a human variable region (eg, one or more rearranged human VDJ gene segments).

[00897] The initiation of homologous recombination in bacterial cells and selection of positive clones is carried out as described above. Non-rearranged or rearranged human immunoglobulin heavy chain variable region nucleotide sequences when inserted into an endogenous immunoglobulin heavy chain locus become operably linked to an endogenous rat immunoglobulin heavy chain constant region nucleotide sequence. Alternatively, an endogenous rat heavy chain constant region locus can be inactivated, for example, by removing one or more rat heavy chain constant region (CH) gene segments from an endogenous heavy chain constant region locus and replacing a human heavy chain constant region with a nucleotide sequence.

[00898] Similarly, humanization of an endogenous κ or λ light chain locus of a rat immunoglobulin is performed by removing one or more endogenous rat κ and / or λ light chain immunoglobulin variable region nucleotide sequences (e.g., one or more endogenous rat V _κ gene segments and one or more endogenous rat gene segments J _κ ); and targeting the modified immunoglobulin light chain locus with a targeting vector, e.g., a large targeting vector (LTVEC), comprising: (i) one or more non-rearranged human immunoglobulin light chain variable region nucleotide sequences (e.g., one or more human V _κ and one or more human J _κ gene segments) or one or more rearranged human variable region nucleotide sequences (eg, one or more rearranged human V _κ -J _κ gene segments); (ii) a selection cassette (for example, a neomycin resistance gene flanked by 1xP regions); and (iii) 5 'and 3' rat shoulders homology.

[00899] Non-rearranged or rearranged human immunoglobulin light chain variable region nucleotide sequences when inserted into an endogenous immunoglobulin light chain locus become operably linked to an endogenous rat immunoglobulin light chain constant region nucleotide sequence.

[00900] The LTVEC thus obtained in bacterial cells contains, for example, a nucleotide insert that contains a humanized rat immunoglobulin heavy chain or light chain locus in which one or more endogenous rat variable heavy or light chain gene segments have been replaced one or more gene segments of a human variable region of the heavy or light chain; and rat shoulders homology {eg, for example, in the range of 5 kbp. up to 150 kb), complementary to specific target genomic sequences. Then the LTVEC containing the above genetic modification is linearized and electroporated into rat ES cells. Electroporated rat ES cells are plated at high density to select drug resistant ES cells containing the targeting vector. The selection process for drug susceptibility results in the removal of most of the seeded cells (~ 99%), leaving individual colonies, each of which is a clone derived from a single cell. Among the remaining cells, the majority of cells (~ 80-100%) contain a targeting vector integrated into a random region of the genome. Therefore, colonies are picked individually and genotyped to identify rat ES cells containing the targeting vector in the correct region of the genome (eg, using the allele modification analysis (MOA) described above).

[00901] To increase the efficiency of targeted genetic modification, rat ES cells are electroporated with expression vectors (or mRNA) that express ZFN 1 and 2 (or TALEN 1 and 2) together with LTVEC. The homology arms of the targeting vector are outside the target region of the ZFN, hence the targeting vector is not cleaved by the ZFN. The double-strand break created by ZFN stimulates homology-directed repair (HGR), which otherwise provides a very small percentage of repairs normally occurring in mammalian cells (compared to non-homologous end-joining; NGSC).

[00902] Alternatively, a type II CRISPR-associated nuclease (eg, Cas9), a guide RNA (including CRISPR-RNA (cr-RNA) and trans-activating CRISPR RNA (tracrRNA)) described herein can be introduced into bacterial cells together with LTVECs to increase the efficiency of homologous recombination at the target genomic locus. Electroporated cells are plated at high density and subjected to drug sensitivity screening to find drug resistant colonies. Drug resistant colonies are harvested and screened for targeted modification using the allele modification assay (MOA) described herein. After these procedures, you can achieve an improvement in targeting efficiency. For example, the magnitude of the improvement can be small (eg, 10% to 15% improvement) or large (eg, 10% to 80% improvement).

[00903] The selected rat ES cells containing the targeted genetic modification are then introduced into a rat host embryo, for example, a pre-morula or blastocyst rat embryo, and implanted into the uterus of a surrogate mother to obtain a founding rat (F0 rat) ... Thereafter, the founding rat is crossed with the wild-type rat to produce F1 offspring heterozygous for the genetic modification. By mating heterozygous F1 rats, progeny homozygous for genetic modification can be obtained.

4.3 (a). Replacement of rat IL2rg with human IL2 gamma receptor

[00904] Table 29 summarizes the genomic structure of the rat interleukin-2 gamma receptor locus, and the positions shown were taken from construct 5.0 of the rat genome standard sequence (ENSMBL). Il2rg is located on the X chromosome on the (-) chain.

[00905]

[00906] The lower diagram of Figure 25 shows the direction vector for creating 3.2 kbp. deletions in Il2rg. The targeting vector contains a reporter gene (uZPB) operably linked to an endogenous promoter and a self-destructive cassette flanked by loxP regions (unstained arrows). The self-destructive cassette contains the Crei gene operably linked to the murine Prm1 promoter and a selection cassette containing the neomycin resistance gene operably linked to the human ubiquitin promoter.

[00907] The Crei gene contains two exons encoding Cre recombinase, which are separated by an intron (Crei) to prevent its expression in a prokaryotic cell. See, for example, US patent 8697851 and US published patent application 2013-0312129, which describe in detail the self-destructive cassette, and which are fully incorporated herein by reference. The Cre expression cassette and the self-destructing cassette can be removed using the murine Prmi promoter, in particular in F0 male germ cells of rats. The targeting vector was electroporated into the rat ES cells obtained in Example 1, and cells were seeded at 15 cm with 2x density of neomycin resistant PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured, selected and maintained as described in Example 1.

[00908] A plasmid targeting vector was constructed to replace the full-length coding region of the rat interleukin-2 gamma receptor with the full-length human interleukin-2 gamma receptor coding region as shown in Figure 31. The targeting vector was electroporated into the rat ES cells obtained in Example 1 and the cells were seeded 15 cm with 2x density of neomycin-resistant PEM in 2i + 10 μM ROCKi. Specifically, 4 × 10 ⁶ cells were electroporated with 2 μg of the full-length Il2rg humanization vector at a voltage of 400 V, a capacitance of 100 μF, and a resistance of 0. Selection was performed using 75 μg / ml G418. Transformed rat ES cells were cultured, selected and maintained as described in Example 1.

[00909] As shown in Table 44, 168 colonies were screened and 6 targeted clones were obtained. The targeting efficiency was 3.57%. One clone was injected into blastocysts as described in Example 1, and one clone producing chimeras was obtained.

[00910] Clones were injected into blastocysts as described herein in Example 1. Received clones producing chimeric F0 rats. Blastocysts were transferred into pseudopregnant female recipients using standard methods and chimeric F0 rats were obtained. Received F0 rats that transmit targeting modification through the germ line.

4.3 (b) (i). Replacement of rat ectodomain IL2rg with human ectodomain IL2rg

[00911] Full-length humanization of the IL 2 gamma receptor has the advantage that rats having such a modified locus will produce human Il2rg; and this will allow the detection of human Il2rg in rats using antibodies specific to human Il2rg.

[00912] Ectogumanization (ie, replacement of the rat Il2rg ectodomain with the human Il2rg ectodomain) will result in an Il2rg polypeptide that will bind the human Il2rg ligands, but since the cytoplasmic domain remains rat, the ectogumanized form of Il2rg signaling will also interact with the rat Il2rg signaling domain. Figure 33 shows the sequence alignment of the human IL-2rg protein (SEQ ID No. 20; NP_000197.1); rat IL-2rg protein (SEQ ID No. 21; NP_543165.1); and a chimeric IL-2rg protein (SEQ ID NO: 22) containing the human IL-2rg ectodomain fused to the rat IL-2rg protein residue. The junction of human and rat IL-2rg is indicated by a vertical line.

[00913] Table 30 summarizes the genomic structure of the rat interleukin-2 gamma receptor locus, and the positions shown were taken from construct 5.0 of the rat genome standard sequence (ENSMBL). Il2rg is located on the X chromosome on the (-) chain. Additionally, the position of the Il2rg ectodomain is indicated.

[00914]

[00915] A plasmid targeting vector was constructed to replace the full-length coding region of the rat interleukin-2 gamma receptor ectodomain with a human ectodomain as shown in Figure 32. The targeting vector was electroporated into rat ES cells obtained in Example 1 and the cells were seeded at 15 cm at 2x density neomycin-resistant PEM in 2i + 10 μM ROCKi. Transformed rat ES cells were cultured, selected and maintained as described in Example 1.

[00916] As shown in Table 44, 192 colonies were screened and 13 targeted clones were obtained. The targeting efficiency was 6.77%.

[00917] Clones were injected into blastocysts as described herein in Example 1, and two clones producing chimeric rats were obtained. Received clones producing rats F0. Received F0 rats that transmit targeting modification through the germ line.

4.3 (b) (ii). Replacement of the rat IL2rg ectodomain with the IL2rg human ectodomain using a plasmid in combination with CRISPR / Cas9

[00918] Table 31 compares the results of experiments in which the humanized Il2rg ectodomain vector shown in Figure 32 was used alone to target the rat Il2rg locus or used in combination with CRISPR / Cas9 nuclease to target the rat Il2rg locus. In each experiment, electroporated cells were plated at high density and subjected to drug selection to find drug resistant colonies. Drug resistant colonies were harvested and screened for targeted modification by allele modification assay (MOA) as described herein. In particular, 4 × 10 ⁶ cells were electroporated with 2 μg of a vector with a humanized Il2rg ectodomain at a voltage of 400 V, a capacitance of 100 μF, and a resistance of 0. In the last experiment, 6 μg of the Cas9 expression plasmid and 3 μg of Il2rg nRNA2 or 3 μg of Il2rg nRNA4 were also electroporated. Selection was performed using 75 μg / ml G418. nRNA2 Il2rg has the sequence GAAGCTCTTTCTATACAATCTGG (SEQ ID NO. 91) and targets a 190 bp region. 3 'from exon 1 of rat Il2rg. nRNA4 Il2rg has the sequence CCCCCGAAAGGAGGAGCCCTAGG (SEQ ID NO. 92) and targets an 80 bp region. 5 'from the rat Il2rg stop codon (TGA) (see Figure 49).

[00919]

4.4 (a). Enhanced targeting by CRISPR / Cas9 endonucleases with large gene divisions of non-human animals with simultaneous replacement with human genes

[00920] Newly developed drugs against human diseases, such as fully human antibodies, are often highly specific for their targets in human cells and tissues and do not recognize homologous targets in rodents. This high level of selectivity makes it impossible to study the efficacy and mechanism of action of drugs in rodents before first use in humans.

[00921] A very effective solution to this problem is the creation of a genetically modified mouse or rat, in which the human gene encoding the target of the drug replaces the homologue of the rodent. One way to create such a modified allele in a rodent is to remove the rodent gene in the embryonic stem (ES) cell and insert the human gene exactly at the distant locus in a second step of modification. ES cells are then injected into a rodent embryo and implanted into the uterus of a rodent surrogate mother, which subsequently produces genetically modified babies that carry the humanized allele.

[00922] A more efficient way to create a humanized gene modification is the use of a large targeting vector (LTVEC), which directs the simultaneous removal of the rodent gene and replacement of it with a human counterpart. With the help of VELOCIGENE® genetic engineering methods, such a one-step humanization can be carried out with relatively high efficiency if the deletion of the rodent gene and the insertion of the human gene do not exceed about 20 thousand base pairs (kb). Larger one-step humanizations, resulting in the removal and replacement of more than 100 kbp, are possible with LTVEC and genetic engineering techniques such as VELOCIGENE® genetic engineering techniques, but due to reduced targeting efficiency often occurring with very large modifications , a successful result often requires screening of hundreds to thousands of ES cell clones to find the one carrying the required genetic modification.

[00923] To improve the efficiency of large humanization, we have developed methods that combine LTVEC targeting of genes with directed RNAs of short palindromic repeats, regularly arranged in groups, Cas9 endonucleases (CRISPR / Cas9). CRISPR / Cas9 nucleases are ribonucleoprotein enzymes composed of bacterial DNA-en to Cas9 nuclease linked to CRISPR RNA, which directs Cas9 to cleave a specific DNA sequence by Watson-Crick pairing between the guide RNA and one strand of target DNA. Due to the simplicity of the targeting mechanism, it is not difficult to design CRISPR / Cas9 endonucleases that create a double-stranded break near any genomic locus. Double-stranded breaks induce cellular genomic repair by non-homologous end-linkage (NHSC), which is error-prone and often leads to deletions and insertions at the double-stranded break site. An alternative mechanism for repairing double-strand breaks is homology-guided repair (HGR), in which an endogenous or exogenous piece of DNA, which has sequence identity or similarity to the damaged region, organically repairs the damaged ends through the action of the cellular apparatus of homologous recombination. UHR can result in ideal repair that retains the original sequence at the site of injury, or it can be used to drive an engineered modification, such as deletion, insertion, or replacement of a sequence at a double-strand break site. CRISPR / Cas9 can dramatically increase the rate of envisioned GHR acts by manipulating precise double-stranded cleavages at sites targeted for genetic modification.

[00924] To perform an accurate one-step removal of all or part of the rodent gene and simultaneous replacement of all or part of its human homologue, we introduced three nucleic acid molecules into ES cells of rodents by electroporation: (1) LTVEC; (2) a plasmid or mRNA encoding a Cas9 endonuclease; and (3) a plasmid encoding a CRISPR single guide RNA (sRNA), or sRNA itself. An LTVEC containing all or part of a human gene that encodes a gene product (protein or RNA) flanked by rodent DNA homology arms is engineered to drive the act of GH, which removes the rodent gene and inserts the human gene. The humanizing LTVEC also carries a drug susceptibility selection cassette that drives the expression of an enzyme (eg, neomycin phosphotransferase) that confers antibiotic resistance (eg, G418). ES cells that ingested LTVEC and incorporated it into their genome were able to grow and form colonies in a Petri dish in growth medium containing the antibiotic. Since we introduced 500 to 1000 times more CRISPR / Cas9-encoding nucleic acid molecules than LTVEC molecules, most LTVEC-containing drug-resistant colonies also contained, at least temporarily, CRISPR / Cas9 components. We collected drug-resistant colonies and screened them by allele loss (Valenzuela, D. ei al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21: 652-660; Frendewey, D. et al. (2010) The loss-of-allele assay for ES cell screening and mouse genotyping, Methods Enzymol. 476: 295-307; incorporated by reference in its entirety) to identify clones that contained correctly targeted humanized allele.

[00925] In one particular experiment, LTVECs were designed to generate 68 kbp. removal of the murine Lrp5 gene (related to low density lipoprotein receptor protein 5) and simultaneous replacement of 91 kbp. fragment of the homologous gene of human LRP 5 (Figure 34). LTVEC contained 91 kbp. a fragment of the human LRP 5 gene, flanked by homology arms containing 7 kb. and 33 kbp. genomic DNA derived from portions of the murine Lrp5 locus that flank 68 kbp. the sequence of the murine Lrp5 gene to be deleted. In separate experiments, we combined a humanized Lrp5 LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of eight ssRNAs (gA, gB, gB2, gC, gD, gE2, gE, gF) designed to create double-stranded breaks in the region the murine Lrp5 gene to be deleted. ssRNAs were designed to avoid recognition of any sequence in the inserted portion of the human LRP5 gene.

[00926] The results of humanizing the Lrp5 gene using CRISPR / Cas9 are shown in Table 32. When only LTVECs were added to ES cells, we found that 1.0% of the drug resistant clones tested carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, the combination of LTVEC with Cas9 endonucleases directed by seven of the eight studied sRNAs (sRNA-5'A, sRNA-5'B, sRNA-5'B2, sRNA-C, sRNA-D, sRNA-3'E2, and sRNA -3'F; sequences are given in Table 33), resulted in correctly targeted monoallelic heterozygous mutations with efficiencies ranging from 2.1 to 7.3%, which corresponds to a 2-9-fold increase in one-step targeting of humanized genes compared with the use of one LTVEC. In the case of Ca89-driven cleavage by sRNA-5'B2, in addition to monoallelic targeting, we detected biallelic homozygous humanization with a frequency of 1%. Humanized ES cells homozygous for Lrp5 can be transformed by genetic engineering VELOCIMOUSE® (Poueymirou, W.T. et al. (2007) F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyzes, Nature Biotech. 25:91 -99, fully incorporated herein by reference) directly into fully ES derived mice cells ready for phenotype and drug efficacy studies.

[00927]

[00928]

[00929] The enhanced targeting of large Lrp5 humanization with CRISPR / Cas9 endonucleases is significant compared to equivalent experiments conducted with zinc finger nucleases (ZFN). We obtained four ZFNs designed to create double-stranded breaks in the region of the mouse Lrp5 gene targeted for deletion (Figure 34). ZFN targeted the sequence near the 5 'end of the deletion (a), one targeted the sequence in the middle of the deletion (b), and two targeted sequences near the 3' end of the deletion (c, d). In separate experiments, we combined humanizing Lrp5 LTVEC with a plasmid encoding one of four ZFNs (a-d) designed to create double-stranded breaks in the region of the murine Lrp5 gene that were intended to be deleted. We determined that all ZFNs were active and capable of inducing Lrp5 mutations in IHSCs (data not shown), but in combination with LTVEC, no enhanced HHR-mediated gene targeting was comparable to LTVEC alone.

[00930] The increase in targeting efficiency during humanization of large Lrp5 using CRISPR / Cas9 endonucleases is also significant compared to a series of experiments on humanization using ZFN. In these experiments, a series of ZFN humanizations was performed in which the size of the target mouse gene deletion and the human gene insert was increased (Table 34; Figure 35). Figure 35A illustrates the percent targeting efficiency of LTVECs targeting genes with increasing deletion size. LTVEC was used alone (gray squares) or in combination with ZFN (black squares). Figure 35B illustrates the percent targeting efficiency of LTVEC with increasing size human gene inserts. Again LTVECs were used alone (gray triangles) or in combination with ZFN (black triangles). As shown in Table 34 and Figure 35, the ability of ZFN-mediated DNA cleavage to increase the targeting efficiency of LTVEC disappeared when the size of the target mouse gene deletion exceeded 24.7 kb. and when the size of the human gene insert exceeded 22.2 kbp. (Table 34; Figure 35A). In contrast, CRISPR / Cas9 was able to increase the efficiency of LTVEC targeting of the Lrp5 gene, which involved deletion of the 68.3 kb mouse gene. and insertion of a human gene from 91.0 kbp. (Table 32; Figure 34). This indicates that CRISPR / Cas9 endonucleases are capable of increasing the efficiency of LTVEC targeting in situations where other nucleases (for example, zinc finger nucleases) cannot.

[00931]

[00932] Comparable experiments were performed to humanize other murine genes. In one experiment, LTVECs were designed to generate 45 kbp. deletion of the murine Trpa1 gene (representative of 1 subfamily of cationic channels with transient receptor potential) and simultaneous replacement of 55 kbp. a fragment of the homologous human TRPA1 gene (Figure 36). LTVEC contained 55 kbp. a fragment of the human TRPA1 gene, flanked by homology arms containing 41 kb. and 58 kb. genomic DNA derived from portions of the mouse Trpa1 locus that flanks 45 kbp. the sequence of the mouse Trpa1 gene to be deleted. In separate experiments, we combined a humanized Trpa1 LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of eight ssRNAs (gA, gB, gB2, gC, gD, gE2, gE, gF) designed to create double-stranded breaks in the region the murine Trpa1 gene to be deleted. sRNAs were designed to avoid recognition of any sequence in the inserted portion of the human TRPA1 gene.

[00933] The results of CRISPR / Cas9 humanization of the Trpa1 gene are shown in Table 35. When only LTVECs were added to ES cells, we found that 1.0% of the drug resistant clones tested carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, combining LTVEC with the Cas9 endonuclease directed by seven of the eight ssRNAs tested (A, A2, B, C, D, and F; the sequences are shown in Table 43) resulted in correctly targeted monoallelic heterozygous mutations or biallelic compound heterozygous or homozygous mutations with an efficiency ranging from 1.0 to 3.1%. In the case of Cas9-driven cleavage by nRNA A and nRNA F, we identified compound heterozygous mutations with a frequency of 1.0%.

[00934]

[00935] In another experiment, LTVECs were designed to generate 55 kbp. deletion of the mouse gene Folk1 (glutamate carboxypeptidase 2) and simultaneous substitution of 61 kbp. a fragment of the homologous human gene F0LH1 (Figure 37). LTVEC contained 61 kbp. a fragment of the human gene F0LH1, flanked by homology arms containing 22 kb. and 46 kbp. genomic DNA derived from portions of the mouse Folh1 locus that flanks 55 kbp. the sequence of the mouse Folk1 gene to be deleted. In separate experiments, we combined a humanized Folh1 LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of six ssRNAs (gA, gA2, gC, gD, gE, and gE2) designed to create double-stranded breaks in the mouse Folh1 gene region. intended to be deleted. ssRNAs were designed to avoid recognition of any sequence in the inserted portion of the human F0LH1 gene.

[00936] The results of CRISPR / Cas9 humanization of the Folh1 gene are shown in Table 36. When only LTVECs were added to ES cells, we found that none of the 96 drug resistant clones tested carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, the combination of LTVEC with the Cas9 endonuclease directed by three of the six ssRNAs tested (A, D, and E2; sequences are shown in Table 43) resulted in correctly targeted monoallelic heterozygous mutations with efficiencies ranging from 1.0 to 3.1 %.

[00937]

[00938] In another experiment, LTVECs were designed to generate 76 kbp. deletion of the murine gene complement component 5 (C5 or Hc) and simultaneous replacement of 97 kbp. a fragment of the homologous human gene C 5 (Figure 38). LTVEC contained 97 kbp. a fragment of the human C5 gene, flanked by homology arms containing 34.1 kbp. and 31.2 kb. genomic DNA derived from portions of the mouse C5 (Hc) locus that flanks 76 kbp. the sequence of the mouse C5 (Hc) gene to be deleted. In separate experiments, we combined a humanized C5 (Hc) LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of six ssRNAs (gA, gB, gC, gD, gE, and gE2) designed to create double-stranded breaks in the gene region murine C5 (Hc) to be removed. sRNAs were designed to avoid recognition of any sequence in the inserted portion of the human C5 gene.

[00939] The results of CRISPR / Cas9 humanization of the C5 (Hc) gene are shown in Table 37. When only LTVECs were added to ES cells, we found that 1.0% of the drug resistant clones studied carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, the combination of LTVEC with the Cas9 endonuclease directed by all six ssRNAs studied (A, B, C, D, E, and E2; the sequences are shown in Table 43) resulted in correctly targeted monoallelic heterozygous mutations or biallelic compound heterozygous or homozygous mutations with an efficiency ranging from 4.2 to 16.7%. In the case of Cas9-driven cleavage by nRNA A and E, we identified compound heterozygous mutations with a frequency of 5.2% and 4.2%, respectively.

[00940]

[00941] In another experiment, LTVECs were designed to generate 38 kbp. deletion of the murine gene Adamts5 (disintegrin and metalloproteinase with thrombospondin motifs 5) and simultaneous substitution of 43 kbp. a fragment of the homologous human gene ADAMTS5 (Figure 39). LTVEC contained 43 kb. a fragment of the human ADAMTS5 gene, flanked by homology arms containing 22 kb. and 46 kbp. genomic DNA derived from portions of the murine Adamts5 locus that flanks 38 kbp. the sequence of the murine Adamts5 gene to be deleted. In separate experiments, we combined a humanized Adamts5 LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of eight ssRNAs (gA, gA2, gB, gC, gD, gE, gE2, and gF) designed to create double-stranded breaks in the region gene for murine Adamts5, intended for deletion. ssRNAs were designed to avoid recognition of any sequence in the inserted portion of the human ADAMTS5 gene.

[00942] The results of CRISPR / Cas9 humanization of the Adamts5 gene are shown in Table 38. When only LTVECs were added to ES cells, we found that none of the 96 drug resistant clones tested carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, the combination of LTVEC with the Cas9 endonuclease directed by two of the eight ssRNAs tested (B and F; sequences are shown in Table 43) resulted in correctly targeted monoallelic heterozygous mutations or biallelic compound heterozygous mutations with a 1.0% efficiency. In the case of Cas9-driven cleavage by E2 nRNA, we identified compound heterozygous mutations with a frequency of 1.0%.

[00944] In another experiment, LTVECs were designed to generate 102 kbp. deletion of the murine gene Erbb4 (receptor tyrosine protein kinase erbB-4) and simultaneous substitution of 127 kbp. a fragment of the homologous human gene ERBB4 (Figure 40). LTVEC contained 127 kbp. a fragment of the human ERBB4 gene, flanked by homology arms containing 48 kb. and 26 so on. genomic DNA derived from portions of the mouse Erbb4 locus that flanks 102 kbp. the sequence of the mouse Erbb4 gene to be deleted. In separate experiments, we combined a humanized Erbb4 LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of eight ssRNAs (gA, gB, gB2, gC, gD, gE, gE2, and gF) designed to create two strand breaks in region of the mouse Erbb4 gene intended for deletion. sRNAs were designed to avoid recognition of any sequence in the inserted portion of the human ERBB4 gene.

[00945] The results of CRISPR / Cas9 humanization of the Erbb4 gene are shown in Table 39. When only LTVECs were added to ES cells, we found that none of the 96 drug resistant clones tested carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, the combination of LTVEC with the Cas9 endonuclease directed by one of the eight ssRNAs tested (D; sequence is shown in Table 43) resulted in correctly targeted monoallelic heterozygous mutations or biallelic compound heterozygous mutations with an efficiency of 1.0%. In the case of Cas9-driven cleavage by nRNA D, we identified compound heterozygous mutations with a frequency of 1%.

[00947] In another experiment, LTVECs were designed to generate 110 kbp. deletion of the murine gene Ror1 (transmembrane receptor of tyrosine protein kinase ROR1) and simultaneous substitution of 134 kbp. a fragment of the homologous human ROR1 gene (Figure 41). LTVEC contained 134 kbp. a fragment of the human ROR1 gene, flanked by homology arms containing 41.8 kbp. and 96.4 kbp. genomic DNA derived from portions of the mouse Ror1 locus that flanks 110 kbp. the sequence of the murine Ror1 gene to be deleted. In separate experiments, we combined a humanized Ror1 LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of six ssRNAs (gA, gB, gC, gD, gE, and gF) designed to create double-stranded breaks in the mouse Ror1 gene region. intended to be deleted. sRNAs were designed to avoid recognition of any sequence in the inserted portion of the human ROR1 gene.

[00948] The results of CRISPR / Cas9 humanization of the Ror1 gene are shown in Table 40. When only LTVECs were added to ES cells, we found that none of the 96 drug resistant clones tested carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, the combination of LTVEC with the Cas9 endonuclease directed by two of the six ssRNAs tested (D and F; sequences are shown in Table 43) resulted in correctly targeted monoallelic heterozygous mutations or biallelic mutations with a 1.0% efficiency. In the case of Cas9-driven cleavage by F nRNA, we also identified compound heterozygous mutations with a frequency of 1%.

[00950] In another experiment, + LTVEC was designed to generate 79 kbp. deletion of the murine gene Dpp4 (dipeptidyl peptidase 4) and simultaneous substitution of 82 kbp. a fragment of the homologous human DPP4 gene (Figure 42). LTVEC contained 82 kbp. a fragment of the human DPP4 gene, flanked by the 5 'and 3' arms of homology, containing 46 kb each. genomic DNA derived from portions of the murine Dpp4 locus that flanks 79 kbp. the sequence of the murine Dpp4 gene to be deleted. In separate experiments, we combined a humanized Dpp4 LTVEC with a plasmid encoding Cas9 and a second plasmid encoding one of eight ssRNAs (gA, gB, gB2, gC, gD, gE, gE2, and gF) designed to create double-stranded breaks in the region the murine Dpp4 gene to be deleted. ssRNAs were designed to avoid recognition of any sequence in the inserted portion of the human DPP4 gene.

[00951] The results of CRISPR / Cas9 humanization of the Dpp4 gene are shown in Table 41. When only LTVECs were added to ES cells, we found that 2.1% of the drug resistant clones tested carried the correctly targeted monoallelic heterozygous humanized allele. In contrast, combining LTVEC with the Cas9 endonuclease directed by any of the eight ssRNAs studied (A, B, B2, C, D, E, E2, and F; the sequences are shown in Table 43) resulted in correctly targeted monoallelic heterozygous mutations with efficacy in the range from 2.1 to 7.3%.

[00953] Summarized results on humanization of various murine genes using CRISPR / Cas9 are shown in Table 42. In the first row, the targeted gene locus is indicated. The second row shows the size of the deletion (Del.) Of the endogenous murine locus and the size of the insert (Bst.) Of the corresponding human locus. The remaining columns indicate the number of colonies (out of 96) for each condition characterized by directed monoallelic heterozygous mutations, biallelic compound heterozygous mutations, or biallelic homozygous mutations, “no nRNA” corresponds to one LTVEC, while the rest of the rows correspond to LTVEC plus the corresponding nRNA (indicated by the relative position in the deleted locus).

Example 5. Generalized data on targeted modification of rat genomic loci

[00956] Table 44. Pooled data on targeting rats with various types of vectors and nuclease agents discussed in Examples 3 and 4.

[00958] Table 45 summarizes the targeting of rat ES cells by plasmids or LTVECs in combination with CRISPR / Cas9. Two nRNAs were examined separately for each target locus: Rag2, ApoE and Il2rg. The CRISPR / Cas9 cleavage efficiency was> 20% at all three loci. An increase in targeting efficiency and an increase in biallelic targeting were observed when CRISPR / Cas9 was used in combination with targeting plasmids and LTVEC.

[00960] Table 46 summarizes germline transmission data for targeted modification of rat genomic loci. Germline transmission has been confirmed for ApoE-targeted rats and Ilrg-targeted rats. Rat ES cells were XY (male) and heterozygous targeted. Therefore, when targeted ES cells contribute to the germ line, approximately 50% of the spermatozoa derived from ES cells will carry the mutant allele and produce heterozygous F1 offspring.

Example 6. Creation, maintenance and targeting of human induced pluripotent stem cells

6.1. Obtaining human iPS cells

[00962] This example describes the production of human iPS cells from non-pluripotent human cells. PiggyBac vectors (System Biosciences) (PB-600A_CAGGS Bst XI (0.64 μg / μL) and PB-200 (0.99 μg / μL) containing genes that encode four reprogramming factors (h0ct4, hSox2, hKLF-4, hMYC), operably linked to the CM7 promoter, were introduced into neonatal human foreskin fibroblasts using RED and BLUE Genein ™ transfection reagents (GlobalStem) The transfected cells were incubated on NuFFI feeder cells in E7 medium to insert vectors and express reprogramming factors DMEM. / F-12, NaHCO ₃ , L-ascorbic acid, insulin, transferrin, selenium and FGF-2.

[00963] Puromycin sensitivity selection was started 10 days after transfection using 2 μg / ml puromycin in E7 medium. On day 21, colonies were selected and cultured in mTeSR ™ medium containing DMEM / F-12, NaHCO ₃ , L-ascorbic acid, insulin, transferrin, selenium, FGF-2, TGF-β1, glutathione, L-glutamine, certain lipids , thiamine, trace elements B and C, β-mercaptoethanol, bovine serum albumin, pipicolic acid, lithium chloride and GABA. On days 29 to 57, cells were expanded and passaged in mTeSR ™ medium until -50% confluence in 6-well plates. On days 65 to 73, propagation and passage were continued using mTeSR ™ medium and gentle cell separation reagent (Stem Cell Technologies). On day 76, the medium was changed to medium with low osmolarity VG2i for additional propagation, passage and maintenance of cells containing naive or naive-like chiPSCs.

6.2. LTVEC targeting in human iPS cells

[00964] This example describes the use of LTVEC targeting in human iPS cells. As shown in Figure 51, we introduced the following nucleic acid molecules by electroporation into human iPS cells propagated in VG2i medium: (1) LTVEC (0.67 μg); (2) plasmid encoding endonuclease Cas9 (5 μg); and (3) a plasmid encoding a single CRISPR guide RNA (nRNA) (10 μg). In one group of samples, Cas9 and nRNA were excluded. Specifically, 3 × 10 ⁶ cells were electroporated at a voltage of 700 V, a capacitance of 25 μF, and a resistance of 400 ohms. LTVEC contained 16.7 kbp. nucleic acid containing the murine genes Adam6a and Adam6b, flanked by arms of homology containing 34 kb. and 105 kbp. genomic DNA from genomic regions that flank 4.1 kb. sequence of the human ADAM6 locus to be deleted. The LTVEC also carries a drug susceptibility screening cassette that drives the expression of an enzyme that confers antibiotic resistance (hygromycin). The human ADAM6 nRNA used had the following sequence: GTATAGCCCTGTTACACATT (SEQ ID No. 94).

[00965] Cells that took up LTVEC and inserted it into their genomes were able to grow and form colonies in a GELTREX ™ coated culture dish in growth medium containing an antibiotic. Since we introduced 500 to 1000 times more CRISPR / Ca89-encoding nucleic acid molecules than LTVEC molecules, most LTVEC-containing drug-resistant colonies also contained, at least temporarily, CRISPR / Cas9 components. We collected drug-resistant colonies and screened them by allele loss (Valenzuela et at. (2003) Nat. Biotech. 21: 652-660; Frendewey et at. (2010) Methods Enzymol. 476: 295-307; in full volume incorporated herein by reference) to identify clones that contained a correctly targeted allele.

[00966] The results of LTVEC targeting using the CRISPR / Cas9 locus ADAM6 are shown in Table 47.

[00967] When LTVEC alone was added to human iPS cells, the targeting efficiency was observed to be 3.1%. In contrast, combining LTVEC with Cas9 directed by ADAM6 nRNA resulted in a targeting efficiency of 7.3%.

6.3. Effect of Low Osmolarity Environment on Human IPS Cell Morphology

[00968] This example describes the effect of salt concentration, ionic strength and / or osmolarity on the pluripotency state of human iPS cells in culture. Human iPS cells were cultured on MATRIGEL ™ or GELTREX ™ substrate in the medium described in Table 48 or in mTeSR ™ -hLIF medium.

[00969] When the main medium used was DMEM, this medium was called 2i medium. When the main medium used was VG-DMEM, this low osmolarity medium was called VG2i medium. The osmolarity of the VG2i medium (233 mOsm / kg) is lower than the osmolarity of the traditional 2i medium (261 mOsm / kg).

[00970] As shown in Figure 52, human iPS cells cultured on MATRIGEL ™ in medium 2i for 8 days (Figure 52A) or 12 days (Figure 52B) showed morphology characteristic of iPS cells in the primed state, in particular growth in the epithelial monolayer and the appearance of apico-basal polarity.

[00971] The mTeSR-hLIF medium and the VG2i medium were further evaluated for their effect on the morphology and pluripotency state of human iPS cells. In this study, human iPS cells were cultured on MATRIGEL ™ or NuFF feeder cells in mTeSR ™ -hLIF medium (Figures 53A and 53C) or VG2i medium (Figures 53B and 53D) for 6 days. When cultured in mTeSR ™ -hLIF medium on MATRIGEL ™ or NuFF feeder cells, human iPS cells showed morphology characteristic of the primed state of pluripotency, in particular growth in the epithelial monolayer and the appearance of apico-basal polarity. Several cells cultured in mTeSR ™ -hLIF medium began to exhibit morphology characteristic of three-dimensional aggregation. In contrast, when cultured in VG2i medium on MATRIGEL ™ or NuFF feeder cells, human iPS cells showed morphology characteristic of the naive state of pluripotency, in particular, growth in round, hemispherical colonies and lack of apico-basal polarity.

[00972]

6.4. Effect of low osmolarity media on the expression of pluripotency markers in human iPS cells

[00973] This example describes the effect of salt concentration, ionic strength and / or osmolarity on the expression of pluripotency markers in human iPS cells that have been reprogrammed from a primed state to a naive state. After 24 days of culture in VG2i medium on MATRIGEL ™ substrate, reprogrammed naive human iPS cells were stained for expression of alkaline phosphatase or NANOG. The reprogrammed cells were observed to express at high levels both alkaline phosphatase (Figure 54A) and NANOG (Figures 54B and 54C), which indicate a naive state of pluripotency.

6.5. Effect of Low Osmolarity Medium on Enzymatic Separation and Subculturing of Human IPS Cells

[00974] In this example, human iPS cells that have been reprogrammed naively with VG2i low osmolarity medium were enzymatically separated using trypsin to obtain a single cell suspension (Figure 55A). The cell suspension was passaged on new GELTREX ™ coated plates for subculture in VG2i medium. It was observed that after 1 day (Figure 55B) and 4 days (Figure 55C), the subcultured cells continued to exhibit morphology characteristic of cells in the naive state of pluripotency. In particular, the cells grew as round, hemispherical colonies and did not show apico-basal polarity. It turned out that enzymatic separation could be carried out in the absence of an ROCK inhibitor, which is usually required to prevent activation of proapoptotic pathways. This suggests that pro-apoptotic pathways are not so strongly activated during enzymatic separation and subculturing of naive human iPS cells under the conditions defined herein.

[00975] All publications and patent applications mentioned in the description correspond to the level of specialists in the field of technology to which this invention belongs. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and separately incorporated by reference. Unless otherwise indicated by context, any embodiment, aspect, step, or feature of the invention may be used in combination with each other. Range hints include any integers within the specified range, any subrange within the specified range. Indication of multiple ranges includes constituent parts of such ranges.

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LIST OF SEQUENCES

<110> Frendaway, David

Auerbach, Wojtek

Lai, Ka-Man Venus

Peasant, Alexander O.

Lee, Jeffrey D.

Drogett, Gustavo

Trzaska, Sean

Hunt, Charlene

Gagliardi, Anthony

Kunou, Junko

Valenzuela, David M.

Yankopoulos, George D.

<120> Methods and compositions for targeted genome modification

<130> 057766/453460

<150> US 61 / 914,768

<151> 2013-12-11

<150> US 62 / 017,416

<151> 2014-06-26

<150> US 62 / 029.261

<151> 2014-07-25

<150> US 62 / 052,906

<151> 2014-09-19

<150> US 62 / 059,527

<151> 2014-10-03

<150> US 62 / 064,384

<151> 2014-10-15

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<223> guide RNA (nRNA)

<400> 2

guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu 60

ggcaccgagu cggugcuuuu 80

<210> 3

<211> 42

<212> RNA

<213> Artificial sequence

<220>

<223> guide RNA (nRNA)

<400> 3

guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cg 42

<210> 4

<211> 30

<212> RNA

<213> Artificial sequence

<220>

<223> crRNA

<400> 4

guuuuagagc uagaaauagc aaguuaaaau 30

<210> 5

<211> 33

<212> RNA

<213> Artificial sequence

<220>

<223> crRNA

<400> 5

guuuuagagc uagaaauagc aaguuaaaau aag 33

<210> 6

<211> 26

<212> RNA

<213> Artificial sequence

<220>

<223> crRNA

<400> 6

gaguccgagc agaagaagaa guuuua 26

<210> 7

<211> 12

<212> RNA

<213> Artificial sequence

<220>

<223> tracrRNA

<400> 7

aaggcuaguc cg 12

<210> 8

<211> 50

<212> RNA

<213> Artificial sequence

<220>

<223> tracrRNA

<400> 8

aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 50

<210> 9

<211> 203

<212> Protein

<213> Mus Musculus

<400> 9

Met Lys Val Leu Ala Ala Gly Ile Val Pro Leu Leu Leu Leu Val Leu

1 5 10 15

His Trp Lys His Gly Ala Gly Ser Pro Leu Pro Ile Thr Pro Val Asn

20 25 30

Ala Thr Cys Ala Ile Arg His Pro Cys His Gly Asn Leu Met Asn Gln

35 40 45

Ile Lys Asn Gln Leu Ala Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe

50 55 60

Ile Ser Tyr Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn Asn Val Glu

65 70 75 80

Lys Leu Cys Ala Pro Asn Met Thr Asp Phe Pro Ser Phe His Gly Asn

85 90 95

Gly Thr Glu Lys Thr Lys Leu Val Glu Leu Tyr Arg Met Val Ala Tyr

100 105 110

Leu Ser Ala Ser Leu Thr Asn Ile Thr Arg Asp Gln Lys Val Leu Asn

115 120 125

Pro Thr Ala Val Ser Leu Gln Val Lys Leu Asn Ala Thr Ile Asp Val

130 135 140

Met Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Asn Lys Tyr

145 150 155 160

Arg Val Gly His Val Asp Val Pro Pro Val Pro Asp His Ser Asp Lys

165 170 175

Glu Ala Phe Gln Arg Lys Lys Leu Gly Cys Gln Leu Leu Gly Thr Tyr

180 185 190

Lys Gln Val Ile Ser Val Val Val Gln Ala Phe

195,200

<210> 10

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Binding site ZFN1a

<400> 10

caggccctga accgc 15

<210> 11

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Cutting area ZFN1

<400> 11

ttctgg 6

<210> 12

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Binding site ZFN1b

<400> 12

gattacctgc gctggg 16

<210> 13

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Binding area ZF21a

<400> 13

ttcaccctcc gcacc 15

<210> 14

<211> 7

<212> DNA

<213> Artificial sequence

<220>

<223> Cutting area ZFN2

<400> 14

tgctgag 7

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Binding area ZF21b

<400> 15

tatccagatc caggggtt 18

<210> 16

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> conserved domain of the homing nuclease family

<400> 16

Leu Ala Gly Leu Ile Asp Ala Asp Gly

fifteen

<210> 17

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Binding site ZFN1a

<400> 17

caggccctga accgc 15

<210> 18

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> Cutting area ZFN1

<400> 18

ttctgg 6

<210> 19

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Binding site ZFN1b

<400> 19

gattacctgc gctggg 16

<210> 20

<211> 369

<212> Protein

<213> Homo sapiens

<400> 20

Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu

1 5 10 15

Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly

20 25 30

Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp

35 40 45

Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val

50 55 60

Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro

65 70 75 80

Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn

85 90 95

Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr

100 105 110

Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe

115 120 125

Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln

130 135 140

Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu

145 150 155 160

Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn

165 170 175

Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp

180 185 190

Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe

195 200 205

Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg

210 215 220

Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp

225 230 235 240

Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe

245 250 255

Leu Phe Ala Leu Glu Ala Val Val Ile Ser Val Gly Ser Met Gly Leu

260 265 270

Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro

275 280 285

Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His

290 295 300

Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser

305 310 315 320

Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro

325 330 335

Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn

340 345 350

Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu

355 360 365

Thr

<210> 21

<211> 368

<212> Protein

<213> Rattus norvegicus

<400> 21

Met Leu Lys Pro Leu Leu Pro Ser Arg Ser Phe Leu Leu Leu Gln Leu

1 5 10 15

Leu Leu Leu Arg Val Gly Trp Ser Ser Lys Val Leu Met Ser Ser Gly

20 25 30

Asn Glu Asp Thr Lys Ser Asp Leu Leu Leu Thr Ser Met Asp Leu Lys

35 40 45

His Leu Ser Val Pro Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val

50 55 60

Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro

65 70 75 80

Gln Pro Thr Asn Leu Thr Met His Tyr Arg Tyr Lys Gly Ser Asp Asn

85 90 95

Asn Thr Phe Gln Glu Cys Ser His Tyr Leu Phe Ser Lys Glu Ile Thr

100 105 110

Ser Gly Cys Gln Ile Gln Lys Glu Asp Ile Gln Leu Tyr Gln Thr Phe

115 120 125

Val Val Gln Leu Gln Asp Pro Gln Lys Pro Gln Arg Arg Ala Glu Gln

130 135 140

Lys Leu Asn Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu

145 150 155 160

Thr Leu Tyr Asn Leu Ser Glu Ser Gln Val Glu Leu Arg Trp Lys Ser

165 170 175

Arg Tyr Ile Glu Arg Cys Leu Gln Tyr Leu Val Gln Tyr Arg Ser Asn

180 185 190

Arg Asp Arg Ser Trp Thr Glu Gln Ile Val Asp His Glu Pro Arg Phe

195 200 205

Ser Leu Pro Ser Val Asp Glu Gln Lys Leu Tyr Thr Phe Arg Val Arg

210 215 220

Ser Arg Phe Asn Pro Ile Cys Gly Ser Thr Gln Gln Trp Ser Lys Trp

225 230 235 240

Ser Gln Pro Ile His Trp Gly Ser His Thr Ala Glu Glu Asn Pro Ser

245 250 255

Leu Phe Ala Leu Glu Ala Val Leu Ile Pro Val Gly Thr Met Gly Leu

260 265 270

Ile Ile Thr Leu Ile Phe Val Tyr Cys Trp Leu Glu Arg Met Pro Arg

275 280 285

Ile Pro Ala Ile Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly

290 295 300

Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Thr Glu Ser Leu

305 310 315 320

Gln Pro Asp Tyr Ser Glu Arg Phe Cys His Val Ser Glu Ile Pro Pro

325 330 335

Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Gly Ser Pro Cys Ser Leu

340 345 350

His Ser Pro Tyr Trp Pro Pro Pro Cys Tyr Ser Leu Lys Pro Glu Ala

355 360 365

<210> 22

<211> 368

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric IL-2 gamma receptor containing receptor protein

IL-2 gamma containing receptor ectodomain

IL-2 human gamma

<400> 22

Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu

1 5 10 15

Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly

20 25 30

Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp

35 40 45

Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val

50 55 60

Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro

65 70 75 80

Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn

85 90 95

Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr

100 105 110

Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe

115 120 125

Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln

130 135 140

Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu

145 150 155 160

Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn

165 170 175

Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp

180 185 190

Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe

195 200 205

Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg

210 215 220

Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp

225 230 235 240

Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe

245 250 255

Leu Phe Ala Leu Glu Ala Val Leu Ile Pro Val Gly Thr Met Gly Leu

260 265 270

Ile Ile Thr Leu Ile Phe Val Tyr Cys Trp Leu Glu Arg Met Pro Arg

275 280 285

Ile Pro Ala Ile Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly

290 295 300

Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Thr Glu Ser Leu

305 310 315 320

Gln Pro Asp Tyr Ser Glu Arg Phe Cys His Val Ser Glu Ile Pro Pro

325 330 335

Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Gly Ser Pro Cys Ser Leu

340 345 350

His Ser Pro Tyr Trp Pro Pro Pro Cys Tyr Ser Leu Lys Pro Glu Ala

355 360 365

<210> 23

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> target genomic sequence that is linked

with guide RNA (nRNA)

<220>

<221> note

<222> (2) ... (21)

<223> n = A, T, C or G

<220>

<221> note

<222> (0) ... (0)

<223> n can be 1-20 nucleotides

<400> 23

gnnnnnnnnn nnnnnnnnnn ngg 23

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 24

gggaacccac agcatactcc 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 25

gaatcatgca cggctacccc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 26

tgctcctatg gggaggcgcg 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 27

cttggataac attgataccc 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 28

ggggcagagc ccttatatca 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 29

tcgctcacat taatccctag 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 30

gtactgggga atcggtggtc 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 31

cacgcactcc aaatttatcc 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 32

ctaagtgtgt atcagtacat 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 33

tgccctgcac aataagcgca 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 34

actcattgaa acgttatggc 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 35

agtaagggtg gattaaattc 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 36

gccatctaga ttcatgtaac 20

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 37

gactagaaat gttctgcacc 20

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 38

tgaaccaatt gtgtagcctt 20

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 39

aatagtggta aagcaccatg 20

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 40

gtgtgctaag gatcgaagtc 20

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 41

caccgagatg cttgggtatt 20

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 42

tgtaaccgcc ctgaatgacc 20

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 43

aaaagggcat cataaatccc 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 44

tcaaaaatag tcatacacct 20

<210> 45

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 45

ggtctctagt acattgtaga 20

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 46

atcacaaacc agttaaccgg 20

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 47

tttcagacga gccgacccgg 20

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 48

ctgtcaacag tgccgcgttt 20

<210> 49

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 49

tgtgtgtcat agcgatgtcg 20

<210> 50

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 50

aacaggtacc ctatcctcac 20

<210> 51

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 51

tcgtggttgc atgcgcactg 20

<210> 52

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 52

ggcccggacc tagtctctct 20

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 53

agtctgtaaa gttagcagtc 20

<210> 54

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 54

ggtggtggtg ctgacggaca 20

<210> 55

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 55

tatgagatca acactcgcta 20

<210> 56

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 56

ccaaggactt ccccacgtta 20

<210> 57

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 57

tgcttccctt atgcaagatt 20

<210> 58

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 58

ttaggtaccc tatttgaata 20

<210> 59

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 59

tgcagtgggt gacaggtcca 20

<210> 60

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 60

agggttatac tgacgttgtg 20

<210> 61

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 61

tgtctttcaa ggagggctac 20

<210> 62

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 62

tgatgtgcag tcagacaaag 20

<210> 63

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 63

tgcactatgg ttgactatga 20

<210> 64

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 64

ggaatattct aataggaagt 20

<210> 65

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 65

aagtgctgta ccattctagc 20

<210> 66

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 66

taatcaatag acaacctcgt 20

<210> 67

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 67

tcattcctaa tggtattata 20

<210> 68

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 68

agggtacata gatggcatcg 20

<210> 69

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 69

ctctttaaca attaccactt 20

<210> 70

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 70

tgtgggcctt tgctgatcac 20

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 71

aatctatgat cctatggcct 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 72

tgccaatagc agtgacttga 20

<210> 73

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 73

gggaagaatg ggctattgtc 20

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 74

ggttgtttgt gctgatgacg 20

<210> 75

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 75

ccgtcctagg ccttctacgt 20

<210> 76

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 76

actagtagac ctgaggggtt 20

<210> 77

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 77

gctccagtgt ttaggccttg 20

<210> 78

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 78

ggcaagctga aaacgcatgc 20

<210> 79

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 79

gtagatcgct ttccactacc 20

<210> 80

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 80

gaactccact gctcgtgagc 20

<210> 81

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 81

ataggtgggc actattgaag 20

<210> 82

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 82

atgggaaggt ttataccagc 20

<210> 83

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 83

cggtgtaaaa acaacgggaa 20

<210> 84

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 84

gacccgcagu cccagcgucg 20

<210> 85

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 85

actgagatca atgaccccga 20

<210> 86

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 86

gggtcgcccg gaacctctac 20

<210> 87

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 87

gcaggccctg aaccgcttct tgg 23

<210> 88

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 88

cctgcgctgg gtgcagacgc ttt 23

<210> 89

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 89

ccagctactt gctcgtacaa 20

<210> 90

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 90

cccctcagat tcacgtgcgt 20

<210> 91

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 91

gaagctcttt ctatacaatc tgg 23

<210> 92

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> guide sequence

<400> 92

cccccgaaag gaggagccct agg 23

<210> 93

<211> 56

<212> DNA

<213> Rattus norvegicus

<400> 93

ttctgagcct cagccgacca acctcactat gcactatagg tatgagaagg gggagg 56

<210> 94

<211> 20

<212> DNA

<213> aArtificial sequence

<220>

<223> guide sequence

<400> 94

gtatagccct gttacacatt 20

<---

Claims (109)

1. An in vitro method for modifying a genome at a genomic locus of interest in a human induced pluripotent stem cell obtained from non-pluripotent cells transformed to express a pluripotent state, comprising introducing into a human induced pluripotent stem cell: (a) Cas9 protein, messenger RNA (mRNA) coding for Cas9 protein, or DNA coding for Cas9 protein; (b) a guide RNA containing CRISPR RNA that hybridizes to the target CRISPR sequence at the genomic locus of interest, and tracrRNA, or DNA, encoding the guide RNA; (c) a large targeting vector (LTVEC) that is at least 10 kbp. and contains a nucleotide insert flanked by: (i) a 5'-arm homology that is homologous to the 5'-target sequence at the genomic locus of interest, wherein the 5'-arm of homology is 5 kb or more. up to 200 kbp, and (ii) a 3 'arm of homology that is homologous to a 3' target sequence at the genomic locus of interest, wherein the 3 'arm of homology is 5 kb or more. up to 200 kbp, whereby the guide RNA is designed to avoid recognition of any sequence in the nucleotide insert, while after introducing into a human induced pluripotent stem cell (a) the Cas9 protein, the mRNA coding for the Cas9 protein, or the DNA coding for the Cas9 protein, (b) the guide RNA or DNA encoding a guide RNA, and (c) LTVEC there is a modification of the genome of the human induced pluripotent stem cell so that it contains targeted genetic modification, including the removal of a region of interest genomic locus, where the deletion is from about 30 kb. up to about 200 kb, and / or the introduction of a nucleotide insert at the genomic locus of interest, where the insert is from about 30 kb. up to about 200 kbp, moreover, if the targeted genetic modification includes the introduction of a nucleotide insert into the genomic locus of interest, where the insert is from about 30 kbp. up to about 200 kb, then the LTVEC is at least 40 kb. 2. The method of claim 1, wherein the targeted genetic modification comprises deleting a region of the genomic locus of interest and the guide RNA is designed to create a double-stranded break within the region of the genomic locus of interest to be deleted. 3. The method of claim 1 or 2, wherein the target CRISPR sequence is within the 5'end region of the genomic locus of interest to be deleted. 4. The method of claim 3, wherein the target CRISPR sequence is 50-1000 bp from the endpoint of the deletion. 5. The method of claim 1 or 2, wherein the target CRISPR sequence is within the region of the 3 'end of the genomic locus of interest to be deleted. 6. The method of claim 5, wherein the target CRISPR sequence is 50-1000 bp from the end point of the deletion. 7. The method of claim 1 or 2, wherein the target CRISPR sequence is located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. 8. The method of claim 1 or 2, wherein the target CRISPR sequence is located within a coding region of a gene or within a regulatory region that affects gene expression. 9. The method of claim 1 or 2, wherein the guide RNA comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8. 10. The method of claim 9, wherein the guide RNA comprises SEQ ID NO: 3, 4, 5, or 7. 11. The method according to claim 1 or 2, wherein CRISPR RNA and tracrRNA are introduced together in a single nucleic acid molecule containing CRISPR RNA and tracrRNA. 12. The method of claim 11, wherein one nucleic acid molecule comprises CRISPR RNA and tracrRNA fused together to form a single guide RNA (sRNA). 13. The method according to claim 1 or 2, wherein CRISPR RNA and tracrRNA are added separately. 14. The method according to claim 1 or 2, wherein the Cas9 protein, CRISPR RNA and tracrRNA are introduced as a protein-RNA complex. 15. The method according to claim 1 or 2, where: (I) (a) the DNA encoding the Cas9 protein is in the form of a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding a Cas9 protein; (b) the DNA encoding CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding CRISPR RNA; and (c) the DNA encoding tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a third nucleic acid encoding tracrRNA; wherein the first, second and third promoters are active in human induced pluripotent stem cells, and where the first, second and third expression constructs are in the same nucleic acid molecule or in separate nucleic acid molecules; or (Ii) (a) the DNA encoding the Cas9 protein is in the form of a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding a Cas9 protein; and (b) DNA encoding CRISPR RNA and DNA encoding tracrRNA are in the form of a second expression construct containing a second promoter operably linked to a second nucleic acid encoding a guide RNA (nRNA) containing CRISPR RNA and tracrRNA; wherein the first and second promoters are active in human induced pluripotent stem cells, and where the first and second expression constructs are in the same nucleic acid molecule or in separate nucleic acid molecules. 16. The method of claim 1 or 2, wherein the targeted genetic modification comprises simultaneously removing an endogenous nucleotide sequence at the genomic locus of interest and introducing a nucleotide insert at the genomic locus of interest. 17. The method according to p. 16, where the deleted endogenous nucleotide sequence is from 30 kb. to about 110 kb, and the nucleotide insert is from about 40 kb. up to about 140 kbp 18. The method of claim 1 or 2, wherein the targeted genetic modification is a biallelic genetic modification. 19. The method according to claim 18, where (a) biallelic genetic modification includes the removal of an endogenous nucleotide sequence and the introduction of a nucleotide insert at the genomic locus of interest in two homologous chromosomes; or (b) the modified human induced pluripotent stem cell is compound heterozygous or hemizygous at the genomic locus of interest. 20. The method of claim 19, wherein targeted genetic modification at a genomic locus of interest on one chromosome comprises removing an endogenous nucleotide sequence and introducing a nucleotide insert. 21. The method of claim 20, wherein the targeted genetic modification comprises: (1) removing an endogenous nucleotide sequence at a genomic locus of interest on the first and second homologous chromosomes and (2) introducing a nucleotide insert into a genomic locus of interest on the first homologous chromosome and disrupting the genomic locus of interest on the second homologous chromosome. 22. The method according to claim 1 or 2, wherein the LTVEC is at least 40 kbp. or where the directed genetic modification comprises deleting a region of the genomic locus of interest, the deletion being at least 30 kb. and the LTVEC is at least 15 kbp. 23. The method of claim 1 or 2, wherein the targeted genetic modification comprises introducing a nucleotide insert at a genomic locus of interest, wherein the nucleotide insert is at least 40 kb; or where the targeted genetic modification comprises deleting a region of the genomic locus of interest, the deletion being at least 30 kb, and introducing a nucleotide insert at the genomic locus of interest, the nucleotide insertion being at least 10 kb .O. 24. The method of claim 1 or 2, wherein the nucleotide insert is from about 40 kbp. up to about 140 kbp 25. The method of claim 1 or 2, wherein the target CRISPR sequence is directly flanked by a protospacer adjacent motif (PAM) sequence. 26. The method according to claim 1 or 2, where (a) 5'- and 3'-arms of LTVEC homology in total are from 10 kbp. up to 150 kbp or from 30 kbp. up to 150 kb in length and / or (b) LTVEC is from 100 kbp. up to 300 kbp in length. 27. The method of claim 1 or 2, wherein the targeted genetic modification comprises: (a) replacement of an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) removal of the endogenous nucleotide sequence; (c) deletion of an endogenous nucleotide sequence, the deletion range being at least 30 kb. up to about 3 m.p .; (d) insertion of an exogenous nucleotide sequence; (e) insertion of an exogenous nucleotide sequence in the range of at least 30 kb. up to about 400 kbp; (f) insertion of an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (g) insertion of a chimeric nucleotide sequence containing a human and non-human nucleotide sequence; (h) insertion of a conditioning allele flanked by target sequences of site-specific recombinase; (i) insertion of a selectable marker or reporter gene operably linked to a promoter active in a human induced pluripotent stem cell; or (j) a combination of these. 28. The method of claim 1 or 2, wherein the targeted genetic modification comprises deleting a region of the genomic locus of interest, the deletion being at least 40 kb, or wherein the targeted genetic modification further comprises introducing a nucleotide insert into the genomic of interest a genomic locus, where the insert is at least 30 kb, and the deletion of a region of the genomic locus of interest, the deletion is at least 10 kb. 29. The method of claim 1 or 2, wherein the deletion region of the genomic locus of interest is from about 30 kbp. up to about 110 kbp. 30. The method of claim 1 or 2, wherein the targeted genetic modification comprises deleting a region of the genomic locus of interest, wherein the deletion is at least 30 kb, and introducing a nucleotide insert at the genomic locus of interest, wherein the insert is at least 30 kb. 31. The method of claim 1 or 2, wherein the genomic locus of interest is an endogenous locus of a human induced pluripotent stem cell. 32. The method of claim 1 or 2, wherein the genomic locus of interest comprises a heterologous or exogenous DNA segment that has been integrated into the genome of a human induced pluripotent stem cell. 33. The method of claim 1 or 2, wherein the genomic locus of interest is an immunoglobulin locus. 34. The method of claim 33, wherein the immunoglobulin locus encodes: (a) the amino acid sequence of the variable region of the heavy chain of a mammalian immunoglobulin; or (b) the amino acid sequence of the light chain variable region of a mammalian immunoglobulin. 35. The method of claim 34, wherein the immunoglobulin locus comprises: (a) a non-rearranged nucleotide sequence of the λ and / or κ light chain variable region of a mammal; or (b) a rearranged nucleotide sequence of the λ and / or κ light chain variable region of a mammal. 36. The method of claim 1 or 2, wherein the genomic locus of interest is a T cell receptor locus. 37. The method of claim 36, wherein the T cell receptor locus is an alpha T cell receptor locus. 38. The method of claim 1 or 2, wherein the genomic locus of interest comprises Il2rg locus, ApoE locus, Rag1 locus, Rag2 locus, both Rag1 and Rag2, Adamts5 locus, Trpa1 locus, Folh1 locus, Erbb4 locus, Lrp5 locus, Rag2 locus C5 (Hc), Ror1 locus or Dpp4 locus. 39. The method of claim 1 or 2, wherein the nucleotide insert comprises a genomic nucleotide sequence that encodes the amino acid sequence of an immunoglobulin heavy chain variable region. 40. The method of claim 39, wherein the nucleotide insert comprises: (a) one or more functional human V _H gene segments comprising V _H 1-2, V _H 1-3, V _H 1-8, V _H 1-18, V _H 1-24, V _H 1-45, V _H 1-46, V _H 1-58, V _H 1-69, V _H 2-5, V _H 2-26, V _H 2-70, V _H 3-7, V _H 3-9, V _H 3-11, V _H 3-13, V _H 3- 15, V _H 3-16, V _H 3-20, V _H 3-21, V _H 3-23, V _H 3-30, V _H 3-30-3, V _H 3-30-5, V _H 3-33, V _H 3-35, V _H 3-38, V _H 3-43, V _H 3-48, V _H 3-49, V _H 3-53, V _H 3-64, V _H 3- 66, V _H 3-72, V _H 3-73, V _H 3-74, V _H 4-4, V _H 4-28, V _H 4-30-1, V _H 4-30-2, V _H 4-30-4, V _H 4-31, V _H 4-34, V _H 4-39, V _H 4-59, V _H 4-61, V _H 5-51, V _H 6-1, V _H 7-4-1, V _H 7-81, or a combination thereof; (b) one or more functional human D gene segments including D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21, D3 -3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24 , D6-6, D6-13, D6-19, D6-25, D7-27, or a combination thereof; or (c) one or more functional J _H gene segments including J _H 1, J _H 2, J _H 3, J _H 4, J _H 5, J _H 6, or a combination thereof. 41. The method of claim 1 or 2, wherein the nucleotide insert comprises a genomic nucleotide sequence that encodes the amino acid sequence of an immunoglobulin light chain variable region. 42. The method of claim 41, wherein the nucleotide insert comprises: (a) one or more human Vκ gene segments, including Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8, Vκ1-9, Vκ2- 10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1- 35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40, or a combination thereof; (b) one or more human Vλ gene segments, including Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2- 18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, or a combination thereof; or (c) one or more human Jκ gene segments comprising Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, or a combination thereof. 43. The method of claim 1 or 2, wherein the nucleotide insert comprises a polynucleotide encoding at least a T cell receptor region. 44. The method of claim 43, wherein the T cell receptor is an alpha T cell receptor. 45. The method of claim 1 or 2, wherein the nucleotide insert comprises at least one disease allele. 46. The method of claim 45, wherein the nucleotide insert comprises at least one human disease allele from a human gene. 47. The method of claim 1 or 2, wherein the targeted genetic modification comprises replacing an endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence. 48. The method of claim 47, wherein the targeted genetic modification comprises removing an endogenous nucleotide sequence and adding the orthologous nucleotide sequence to the genomic locus of interest. 49. The method of claim 48, wherein the targeted genetic modification comprises removing a region of the genomic locus of interest, wherein the deletion is at least 30 kb, and introducing a nucleotide insert at the genomic locus of interest, wherein the insert is at least at least 30 kbp 50. The method of claim 49, wherein the remote area is 30 kbp. to about 110 kb, and the nucleotide insert is from about 40 kb. up to about 140 kbp 51. The method according to claim 1 or 2, wherein the LTVEC is from 100 kbp. up to 300 kb, 5'- and 3'-arms of homology are in total from 30 kb. up to 150 kb and directed genetic modification includes the deletion of a region of interest genomic locus, the deletion is from 30 kb. up to 110 kb, and the introduction of a nucleotide insert in the genomic locus of interest, the insert is from 40 kb. up to 140 kbp 52. The method according to claim 1 or 2, wherein the LTVEC is from 20 kbp. up to 250 kbp in length, with the 5'- and 3'-arms of homology in the total amount from 20 kbp. up to 200 kb in length, and where the directed genetic modification comprises deleting a region of the genomic locus of interest, the deletion being from 30 kb. up to 110 kb, and the introduction of a nucleotide insert in the genomic locus of interest, the insert is from 40 kb. up to 140 kb, and where the guide RNA is designed to create a double-stranded break in the region of interest of the genomic locus to be deleted. 53. The method of claim 1 or 2, wherein the non-pluripotent cells transformed to express the pluripotent state express reprogramming genes including Oct4, Sox2, Klf4 and Myc. 54. The method of claim 1 or 2, wherein the human induced pluripotent stem cell is maintained in a low osmolarity medium containing a basic medium and additives, the low osmolarity medium comprising: (a) a leukemia inhibiting factor (LIF) polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and (c) a MEK inhibitor, the low osmolarity medium having an osmolarity of 200-250 mOsm / kg. 55. The method of claim 1 or 2, wherein the CRISPR target sequence is immediately adjacent to the 5 'target sequence or 3' target sequence. 56. The method of claim 1 or 2, wherein the Cas9 protein comprises a nuclear localization signal. 57. The method of claim 1 or 2, wherein the 5 'and 3' arms of LTVEC homology total at least 10 kbp. 58. The method of claim 1 or 2, wherein the target CRISPR sequence is a native sequence that is endogenous to a human induced pluripotent stem cell. 59. The method according to claim 1 or 2, wherein the Cas9 protein contains a nuclear localization signal and wherein the target CRISPR sequence is located anywhere between the 5'-target sequence and the 3'-target sequence, or the target CRISPR sequence is immediately adjacent to the 5'-target sequence or a 3'-target sequence. 60. The method of claim 1 or 2, wherein the 5 'and 3' arms of LTVEC homology total at least 10 kb, wherein the target CRISPR sequence is a native sequence that is endogenous to to a human induced pluripotent stem cell, and wherein the CRISPR target sequence is located anywhere between the 5 'target sequence and the 3' target sequence, or the CRISPR target sequence is immediately adjacent to the 5 'target sequence or 3' target sequence. 61. The method of claim 1 or 2, wherein the 5 'and 3' arms of LTVEC homology total at least 10 kb, the genomic locus of interest being native to human induced pluripotent stem the cell and wherein the target CRISPR sequence is located anywhere between the 5'-target sequence and the 3'-target sequence, or the target CRISPR sequence is immediately adjacent to the 5'-target sequence or 3'-target sequence. 62. The method of claim 1 or 2, wherein the targeted genetic modification comprises introducing a nucleotide insert at a genomic locus of interest, or wherein the targeted genetic modification comprises deleting a region of the genomic locus of interest and introducing a nucleotide insert at the genomic locus of interest. 63. The method of claim 62, wherein the target CRISPR sequence is a native sequence that is endogenous to a human induced pluripotent stem cell, and wherein the 5 'and 3' arms of LTVEC homology in total are from 10 kb .O. up to 150 kbp

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