Methods and Compositions for the Targeted Modification of a Genome

• US 20150159174A1
• Filed: 10/15/2014
• Published: 06/11/2015
• Est. Priority Date: 12/11/2013
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Frendewey, David, Auerbach, Wojtek, Lai, Ka-Man Venus, Mujica, Alexander O., Lee, Jeffrey D., Droguett, Gustavo, Valenzuela, David M., Yancopoulos, George D., Trzaska, Sean, Hunt, Charleen, Gagliardi, Anthony, Kuno, Junko
• Granted Patent: US 9,546,384 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0362 Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
• CPC Class Codes: A01K 2267/0387 Animal model for diseases of the immune system
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C12N 15/1024 In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2015/8527 for producing animal models, e.g. for tests or diseases
• CPC Class Codes: C12N 2800/40 Systems of functionally co-operating vectors
• CPC Class Codes: C12N 2810/00 Vectors comprising a targeting moiety
• CPC Class Codes: C12N 2810/10 Vectors comprising a non-peptidic targeting moiety
• CPC Class Codes: C12N 2810/40 Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
• CPC Class Codes: C12N 2999/007 Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Compositions and methods are provided for modifying a genomic locus of interest in a eukaryotic cell, a mammalian cell, a human cell or a non-human mammalian cell using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Further methods combine the use of the LTVEC with a CRISPR/Cas system. Compositions and methods for generating a genetically modified non-human animal comprising one or more targeted genetic modifications in their germline are also provided.

Claims

1. A method for modifying a genome at a genomic locus of interest, comprising contacting the genome with a Cas protein, a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest, and a tracrRNA,

wherein the genome is contacted in the presence of a large targeting vector (LTVEC) that is at least 10 kb and comprises an insert nucleic acid flanked with:

(a) a 5′

homology arm that is homologous to a 5′

target sequence at the genomic locus of interest; and

(b) a 3′

homology arm that is homologous to a 3′

target sequence at the genomic locus of interest,

wherein following contacting with the Cas protein, CRISPR RNA, and tracrRNA in the presence of the LTVEC, the genome comprises a targeted genetic modification at the genomic locus of interest, wherein the modified genomic locus of interest comprises the insert nucleic acid.

2. The method of claim 1, wherein the genome is in a eukaryotic cell.

3. The method of claim 2, wherein the contacting comprises introducing the Cas protein, the CRISPR RNA, the tracrRNA, and the LTVEC into the eukaryotic cell.

4. The method of claim 3, wherein the CRISPR RNA and the tracrRNA are introduced as a single nucleic acid molecule comprising the CRISPR RNA and the tracrRNA.

5. The method of claim 3, wherein the CRISPR RNA and the tracrRNA are introduced separately.

6. The method of claim 3, wherein:

(a) the Cas protein is introduced into the eukaryotic cell in the form of a protein, a messenger RNA (mRNA) encoding the Cas protein, or a DNA encoding the Cas protein;

(b) the CRISPR RNA is introduced into the eukaryotic cell in the form of an RNA or a DNA encoding the CRISPR RNA; and

(c) the tracrRNA is introduced into the eukaryotic cell in the form of an RNA or a DNA encoding the tracrRNA.

7. The method of claim 6, wherein the Cas protein, the CRISPR RNA, and the tracrRNA are introduced into the eukaryotic cell as a protein-RNA complex.

8. The method of claim 6, 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 first nucleic acid encoding the Cas protein;

(b) the DNA encoding the CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding the CRISPR RNA; and

(c) the DNA encoding the tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a third nucleic acid encoding the tracrRNA;

wherein the first, second, and third promoters are active in the eukaryotic cell, and

wherein the first, second, and third expression constructs are on a single nucleic acid molecule or on multiple nucleic acid molecules.

10. The method of claim 6, 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 first nucleic acid encoding the Cas protein; and

(b) the DNA encoding the CRISPR RNA and the DNA encoding the tracrRNA are in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a gRNA comprising the CRISPR RNA and the tracrRNA;

wherein the first and second promoters are active in the eukaryotic cell, and

wherein the first and the second expression constructs are on a single nucleic acid molecule or on separate nucleic acid molecules.

12. The method of claim 1, wherein the targeted genetic modification comprises simultaneous deletion of an endogenous nucleic acid sequence at the genomic locus of interest and insertion of the insert nucleic acid at the genomic locus of interest.

13. The method of claim 12, wherein the deleted endogenous nucleic acid sequence is from about 30 kb to about 110 kb, and the insert nucleic acid is from about 40 kb to about 140 kb.

14. The method of claim 1, wherein the targeted genetic modification is a biallelic genetic modification.

15. The method of claim 14, wherein the biallelic genetic modification comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid at the genomic locus of interest in two homologous chromosomes.

16. The method of claim 14, wherein the modified genome is compound heterozygous or hemizygous at the genomic locus of interest.

18. The method of claim 16, wherein the targeted genetic modification at the genomic locus of interest in one chromosome comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid.

19. The method of claim 16, wherein the targeted genetic modification comprises:

(1) deletion of an endogenous nucleic acid sequence at the genomic locus of interest in first and second homologous chromosomes; and

(2) insertion of the insert nucleic acid into the genomic locus of interest in the first homologous chromosome and disruption of the genomic locus of interest in the second homologous chromosome.

20. The method of claim 1, wherein the LTVEC is at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, or at least 200 kb.

22. The method of claim 1, wherein the insert nucleic acid is at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, or at least 300 kb.

23. The method of claim 1, wherein the insert nucleic acid is about 40 kb to about 140 kb.

24. The method of claim 2, wherein the eukaryotic cell is a mammalian cell.

25. The method of claim 24, wherein the mammalian cell is a fibroblast.

26. The method of claim 2, wherein the eukaryotic cell is a non-human pluripotent cell or a human pluripotent cell.

28. The method of claim 26, wherein the non-human pluripotent cell is a rodent pluripotent cell.

29. The method of claim 28, wherein the rodent pluripotent cell is a mouse or rat embryonic stem (ES) cell.

31. The method of claim 26, wherein the human pluripotent cell is an embryonic stem (ES) cell, an adult stem cell, a developmentally restricted progenitor cell, or an induced pluripotent stem (iPS) cell.

34. The method of claim 1, wherein the Cas protein is Cas9 and/or the CRISPR target sequence is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence.

37. The method of claim 1, wherein the sum total of the 5′

and the 3′

homology arms of the LTVEC is 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 120 kb, or from about 120 kb to 150 kb.

38. The method of claim 2, wherein the targeted genetic modification comprises:

(a) replacement of an endogenous nucleic acid sequence with a homologous or an orthologous nucleic acid sequence;

(b) deletion of an endogenous nucleic acid sequence;

(c) deletion of an endogenous nucleic acid sequence,

wherein the deletion ranges 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, or from about 150 kb to about 200 kb, from about 200 kb to about 300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb;

(d) insertion of an exogenous nucleic acid sequence;

(e) insertion of an exogenous nucleic acid 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, from about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to about 300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400 kb;

(f) insertion of an exogenous nucleic acid sequence comprising a homologous or an orthologous nucleic acid sequence;

(g) insertion of a chimeric nucleic acid sequence comprising a human and a non-human nucleic acid sequence;

(h) insertion of a conditional allele flanked with site-specific recombinase target sequences;

(i) insertion of a selectable marker or a reporter gene operably linked to a promoter active in the eukaryotic cell;

or

(j) a combination thereof.

41. The method of claim 1, wherein:

(a) 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, 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 kb but less than about 500 kb, at least about 500 kb but less than about 1 Mb, at least about 1 Mb but less than about 1.5 Mb, at least about 1.5 Mb but less than about 2 Mb, at least about 2 Mb but less than about 2.5 Mb, or at least about 2.5 Mb but less than about 3 Mb;

or

(b) 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, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at least 190 kb, or at least 200 kb.

43. The method of claim 1, wherein the 5′

target sequence and the 3′

target sequence are separated by from about 30 kb to about 110 kb.

44. The method of claim 1, wherein the genomic locus of interest comprises the Interleukin-2 receptor gamma locus, the ApoE locus, the Rag1 locus, the Rag2 locus, both of the Rag1 and the Rag2 loci, the Adamts5 locus, the Trpa1 locus, the Folh1 locus, the Erbb4 locus, the Lrp5 locus, the C5 (Hc) locus, the Ror1 locus, or the Dpp4 locus.

9. (canceled)

11. (canceled)

17. (canceled)

21. (canceled)

27. (canceled)

30. (canceled)

32-33. -33. (canceled)

35-36. -36. (canceled)

39-40. -40. (canceled)

42. (canceled)

45-47. -47. (canceled)

48. A method for producing an F0 generation non-human animal that comprises a targeted genetic modification at a genomic locus of interest, the method comprising:

(a) contacting a genome in a non-human ES cell with a Cas protein, a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest, and a tracrRNA to form a modified non-human ES cell,

wherein the genome is contacted in the presence of a large targeting vector (LTVEC) that is at least 10 kb and comprises an insert nucleic acid flanked with a 5′

homology arm that is homologous to a 5′

target sequence at the genomic locus of interest and a 3′

homology arm that is homologous to a 3′

target sequence at the genomic locus of interest, and

wherein following contacting with the Cas protein, CRISPR RNA, and tracrRNA in the presence of the LTVEC, the genome comprises a targeted genetic modification at the genomic locus of interest, wherein the modified genomic locus of interest comprises the insert nucleic acid;

(b) identifying the modified non-human ES cell comprising the targeted genetic modification at the genomic locus of interest;

(c) introducing the modified non-human ES cell into a non-human host embryo; and

(d) gestating the non-human host embryo in a surrogate mother,

wherein the surrogate mother produces the F0 generation non-human animal comprising the targeted genetic modification at the genomic locus of interest.

49. The method of claim 48, wherein the CRISPR RNA and the tracrRNA are introduced into the non-human ES cell as a single nucleic acid molecule comprising the CRISPR RNA and the tracrRNA.

50. The method of claim 48, wherein the CRISPR RNA and the tracrRNA are introduced into the non-human ES cell separately.

51. The method of claim 48, wherein:

(a) the Cas protein is introduced into the non-human ES cell in the form of a protein, a messenger RNA (mRNA) encoding the Cas protein, or a DNA encoding the Cas protein;

(b) the CRISPR RNA is introduced into the non-human ES cell in the form of an RNA or a DNA encoding the CRISPR RNA; and

(c) the tracrRNA is introduced into the non-human ES cell in the form of an RNA or a DNA encoding the tracrRNA.

52. The method of claim 51, wherein the Cas protein, the CRISPR RNA, and the tracrRNA are introduced into the non-human ES cell as a protein-RNA complex.

53. The method of claim 51, 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 first nucleic acid encoding the Cas protein;

(b) the DNA encoding the CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding the CRISPR RNA; and

(c) the DNA encoding the tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a third nucleic acid encoding the tracrRNA,

wherein the first, second, and third promoters are active in the non-human ES cell, and

wherein the first, second, and third expression constructs are on a single nucleic acid molecule or on multiple nucleic acid molecules.

55. The method of claim 51, 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 first nucleic acid encoding the Cas protein; and

(b) the DNA encoding the CRISPR RNA and the DNA encoding the tracrRNA are in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a gRNA comprising the CRISPR RNA and the tracrRNA;

wherein the first and second promoters are active in the non-human ES cell, and

wherein the first and the second expression constructs are on a single nucleic acid molecule or on separate nucleic acid molecules.

57. The method of claim 48, wherein the targeted genetic modification comprises simultaneous deletion of an endogenous nucleic acid sequence at the genomic locus of interest and insertion of the insert nucleic acid at the genomic locus of interest.

58. The method of claim 48, wherein the targeted genetic modification is a biallelic genetic modification.

59. The method of claim 58, wherein the biallelic genetic modification comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid at the genomic locus of interest in two homologous chromosomes.

60. The method of claim 48, wherein the modified non-human ES cell is compound heterozygous or hemizygous at the genomic locus of interest.

62. The method of claim 60, wherein the targeted genetic modification at the genomic locus of interest in one chromosome comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid.

63. The method of claim 60, wherein the targeted genetic modification comprises:

(1) deletion of an endogenous nucleic acid sequence at the genomic locus of interest in first and second homologous chromosomes; and

(2) insertion of the insert nucleic acid into the genomic locus of interest in the first homologous chromosome and disruption of the genomic locus of interest in the second homologous chromosome.

64. The method of claim 48, wherein the Cas protein is Cas9.

54. (canceled)

56. (canceled)

61. (canceled)

65-123. -123. (canceled)