# US 8,771,985 B2 · Genome editing of a Rosa locus using zinc-finger nucleases > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,985 B2 - Document ID: US8771985B2 - Status: Granted - Kind: B2 - Application number: 13/066,802 - Filed: 2011-04-25 - Published: 2012-01-19 - Granted: 2014-07-08 - Earliest claimed: 2010-04-26 - Assignees: Sangamo BioSciences, Inc.; Sigma-Aldvich Co. LLC - Applicants: Xiaoxia Cui; Gregory Davis; Philip D. Gregory; Michael C. Holmes; Edward J. Weinstein - Inventors: Xiaoxia Cui; Gregory Davis; Philip D. Gregory; Michael C. Holmes; Edward J. Weinstein - Web page: https://patentgrep.com/patent/US8771985B2 ## CPC - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - A01K 67/0275: Genetically modified vertebrates, e.g. transgenic - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12N 15/102: {Mutagenizing nucleic acids} - C12N 15/907: {in mammalian cells} - A01K 2207/05: Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose - A01K 2217/07: Animals genetically altered by homologous recombination - A01K 2227/105: Murine - A01K 2267/03: Animal model, e.g. for test or diseases - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C07K 2319/81: containing a Zn-finger domain for DNA binding - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract Disclosed herein are methods and compositions for genome editing of a Rosa locus, using fusion proteins comprising a zinc-finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins. ## Claims ### Claim 1 An isolated fusion protein comprising a nuclease domain and an engineered zinc finger DNA-binding domain, wherein the engineered zinc finger DNA-binding domain binds to a target site in a rat Rosa gene and the engineered zinc finger DNA-binding domain comprises four, five or six zinc finger DNA recognition regions designated and ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus and further wherein the engineered zinc finger DNA-binding domain comprises the zinc finger DNA recognition regions selected from the group consisting of: (i) F1: DRSDLSR (SEQ ID NO:38); F2: RSDDLTR (SEQ ID NO:39); F3: TSGHLSR (SEQ ID NO:40); F4: RSDNLSV (SEQ ID NO:41); and F5: RSANLTR (SEQ ID NO:42); (ii) F1: QSDHLTK (SEQ ID NO:43); F2: NSSNLSR (SEQ ID NO:44); F3: RSDHLTK (SEQ ID NO:45); F4: NSDHLSR (SEQ ID NO:46); and F5: RSDHLSR (SEQ ID NO:47); (iii) F1: RSDHLSE (SEQ ID NO:48); F2: RSAALAR (SEQ ID NO:49); F3: RSDHLST (SEQ ID NO:50); F4: QNAHRIT (SEQ ID NO:51); and F5: RSAVLSE (SEQ ID NO:52); (iv) F1: QSGDLTR (SEQ ID NO:17); F2: TSGSLTR (SEQ ID NO: 18); F3: RSANLTR (SEQ ID NO:42); F4: RSDHLTK (SEQ ID NO:45); and F5: NSDHLSR (SEQ ID NO:46); (v) F1: RSANLTR(SEQ ID NO:42); F2: QSGDLTR (SEQ ID NO: 17); F3: QSGDLTR (SEQ ID NO: 17); F4: RSANLAR (SEQ ID NO:53); and F5: RSDNLRE (SEQ ID NO:54); (vi) F1: RSDHLST (SEQ ID NO:50); F2: DNRDRIK (SEQ ID NO:55); F3: RSDTLSE (SEQ ID NO:56); F4: QSSHLAR (SEQ ID NO:57); and F5: QNAHRKT (SEQ ID NO:22); (vii) F1: QSGDLTR (SEQ ID NO:17); F2: QSGDLTR (SEQ ID NO: 17); F3: RSDNLTR (SEQ ID NO:58); F4: RSDNLSE (SEQ ID NO:21); and F5: QNAHRKT (SEQ ID NO:22); (viii) F1: DRSDLSR (SEQ ID NO:38); F2: RSDHLST (SEQ ID NO:50); F3: DNRDRIK (SEQ ID NO:55); F4: RSDTLSE (SEQ ID NO:56); and F5: QSSHLAR (SEQ ID NO:57); (ix) F1: QSGDLTR (SEQ ID NO:17); F2: RSDNLTR (SEQ ID NO:58); F3: RSDNLSE (SEQ ID NO:21); F4: QNAHRKT (SEQ ID NO:22); F5: RSDHLSE (SEQ ID NO:48); and F6: TSSTRKT (SEQ ID NO:59); (x) F1: TSGNLTR(SEQ ID NO:60); F2: QSGNLAR (SEQ ID No:61); F3: RSDALSV (SEQ ID NO:62); F4: DSSHRTR (SEQ ID NO:63); and F5: RSDVLSE (SEQ ID NO:64) (xi) F1: RSDNLSE (SEQ ID NO:21); F2: QNAHRKT (SEQ ID NO:22); F3: RSDHLSE (SEQ ID NO:48); F4: TSSTRKT (SEQ ID NO:59); and F5: TSGHLSR (SEQ ID NO:40); (xii) F1: TSGNLTR (SEQ ID NO:60); F2: QSGNLAR (SEQ ID NO:61); F3: RSDALSV (SEQ ID NO:62); and F4: DSSHRTR (SEQ ID NO:63); (xiii) F1: QRSNLVR (SEQ ID NO:65); F2: RSDHLTQ (SEQ ID NO:66); F3: QSGHLQR (SEQ ID NO:67); and F4: DRSHLAR (SEQ ID NO:68); (xiv) F1: RSDVLSE (SEQ ID NO:64); F2: QRNHRTT (SEQ ID NO:69); F3: TKRSLIE (SEQ ID NO:70); F4: TSSNLSR (SEQ ID NO:71); F5: RSDDLSK (SEQ ID NO:25); and F6: DNRDRIK (SEQ ID NO:55); (xv) F1: RSDHLSA (SEQ ID NO:72); F2: QSGHLSR (SEQ ID NO:24); F3: RSDHLSR (SEQ ID NO:47); F4: QNDNRIK (SEQ ID NO:73); and F5: QSGNLAR (SEQ ID NO:61); (xvi) F1: NNRDLIN (SEQ ID NO:74); F2: TSSNLSR (SEQ ID NO:71); F3: RSDVLSE (SEQ ID NO:64); F4: QRNHRTT (SEQ ID NO:69); F5: TKRSLIE (SEQ ID NO:70); and F6: TSSNLSR (SEQ ID NO:71). ### Claim 2 (depends on 1) The fusion protein of claim 1, wherein the nuclease domain comprises a Type IIS restriction endonuclease cleavage domain or cleavage half-domain. ### Claim 3 (depends on 1) A polynucleotide encoding the isolated fusion protein of claim 1. ### Claim 4 (depends on 1) An isolated cell comprising the fusion protein of claim 1. ### Claim 5 (depends on 3) An isolated cell comprising the polynucleotide of claim 3. ### Claim 6 (depends on 4) The isolated cell of claim 4, wherein the cell is a rat embryonic stem cell. ### Claim 7 (depends on 1) A composition comprising the isolated fusion protein of claim 1 and a pharmaceutically acceptable excipient. ### Claim 8 (depends on 3) A composition comprising the polynucleotide of claim 3 and an acceptable excipient. ### Claim 9 (depends on 1) A method for cleaving one or more Rosa genes in an isolated rat cell, the method comprising: introducing, into the isolated rat cell, one or more fusion proteins of claim 1 into the cell, such that the one or more Rosa genes are cleaved. ### Claim 10 (depends on 9) The method of claim 9, wherein the one or more fusion proteins are introduced by transformation of isolated rat cell with polynucleotides encoding the fusion proteins. ### Claim 11 (depends on 9) A method of introducing an exogenous polynucleotide sequence into the genome of an isolated rat cell, the method comprising cleaving one or more Rosa genes by the method of claim 9; and contacting the isolated rat cell with an exogenous polynucleotide sequence; wherein cleavage of the one or more Rosa genes stimulates integration of the exogenous polynucleotide sequences into the genome by homologous recombination. ### Claim 12 (depends on 11) The method of claim 11, wherein the exogenous polynucleotide sequence is integrated into the genome via nucleic acid replication processes. ### Claim 13 (depends on 11) The method of claim 11, wherein the exogenous polynucleotide is integrated into the genome via non-homology dependent targeted integration. ### Claim 14 (depends on 9) A method of modifying a Rosa gene sequence in the genome of an isolated rat cell, the method comprising: cleaving one or more Rosa genes by the method of claim 9, wherein (i) a first zinc finger nuclease cleaves at a first cleavage site and a second zinc finger nuclease cleaves at a second cleavage site; (ii) the Rosa gene sequence is located between the first cleavage site and the second cleavage site; (iii) cleavage of the first and second cleavage sites results in a modification of the gene sequence by non-homologous end-joining or homology directed repair. ### Claim 15 (depends on 14) The method of claim 14, wherein the modification comprises a deletion. ### Claim 16 (depends on 14) The method of claim 14, wherein the modification comprises insertion of an exogenous sequence. ### Claim 17 (depends on 1) A kit comprising the isolated fusion protein of claim 1. ### Claim 18 (depends on 3) A kit comprising the polynucleotide of claim 3. ### Claim 19 (depends on 17) The kit of claim 17, further comprising additional components selected from the group consisting of one or more exogenous sequences, instructions for use, and combinations thereof. ### Claim 20 (depends on 18) The kit of claim 18, further comprising additional components selected from the group consisting of one or more exogenous sequences, instructions for use, and combinations thereof.