Genome editing of a Rosa locus using zinc-finger nucleases

• US 8,771,985 B2
• Filed: 04/25/2011
• Issued: 07/08/2014
• Est. Priority Date: 04/26/2010
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc., Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sangamo Biosciences, Inc., Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Cui, Xiaoxia, Davis, Gregory, Gregory, Philip D., Holmes, Michael C., Weinstein, Edward J.
• Primary Examiner(s): Steadman, David J
• Assistant Examiner(s): HOLLAND, PAUL J
• Application Number:
  US13/066,802
• Publication Number:
  US 20120017290A1
• Time in Patent Office: 1,170 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2207/05 Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
• CPC Class Codes: A01K 2217/07 Animals genetically altered by homologous recombination
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: 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
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

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

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).

2. The fusion protein of claim 1, wherein the nuclease domain comprises a Type IIS restriction endonuclease cleavage domain or cleavage half-domain.

3. A polynucleotide encoding the isolated fusion protein of claim 1.

4. An isolated cell comprising the fusion protein of claim 1.

5. An isolated cell comprising the polynucleotide of claim 3.

6. The isolated cell of claim 4, wherein the cell is a rat embryonic stem cell.

7. A composition comprising the isolated fusion protein of claim 1 and a pharmaceutically acceptable excipient.

8. A composition comprising the polynucleotide of claim 3 and an acceptable excipient.

9. 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.

10. 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.

11. 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.

12. The method of claim 11, wherein the exogenous polynucleotide sequence is integrated into the genome via nucleic acid replication processes.

13. The method of claim 11, wherein the exogenous polynucleotide is integrated into the genome via non-homology dependent targeted integration.

14. 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.

15. The method of claim 14, wherein the modification comprises a deletion.

16. The method of claim 14, wherein the modification comprises insertion of an exogenous sequence.

17. A kit comprising the isolated fusion protein of claim 1.

18. A kit comprising the polynucleotide of claim 3.

19. 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.

20. 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.