Genome editing of a Rosa locus using nucleases

• US 20110265198A1
• Filed: 04/25/2011
• Published: 10/27/2011
• Est. Priority Date: 04/26/2010
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Holmes, Michael C., Gregory, Philip D.
• Granted Patent: US 9,567,573 B2
• Time in Patent Office: Days
• US Class Current: 800/21
• 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 DNA binding domain 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. A fusion protein comprising a nuclease and one or more engineered TALE repeat domains.

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

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

4. The fusion protein of claim 3, wherein the cleavage domain or cleavage half-domain is naturally occurring or engineered.

5. A polynucleotide encoding a fusion protein according to claim 1.

6. A cell comprising a fusion protein according to claim 1.

7. A cell comprising a polynucleotide according to claim 5.

8. The cell of claim 6, wherein the cell is an embryo cell.

9. A composition comprising a fusion protein according to claim 1 and a pharmaceutically acceptable excipient.

10. A composition comprising a polynucleotide according to claim 5 and a pharmaceutically acceptable excipient.

11. A method for cleaving one or more Rosa genes in a cell, the method comprising:

introducing, into the cell, one or more fusion proteins according to claim 1 into the cell, such that the one or more Rosa gene are cleaved.

12. The method of claim 11, wherein the fusion proteins are introduced as polynucleotides encoding the fusion proteins.

13. A method of introducing an exogenous polynucleotide sequence into the genome of a cell, the method comprising:

cleaving one or more Rosa genes according to claim 11; and

contacting the cell with an exogenous polynucleotide sequence;

wherein cleavage of the one or more genes stimulates integration of the exogenous polynucleotide sequence into the genome by homologous recombination.

14. The method of claim 13, wherein the exogenous polynucleotide sequence is physically into the genome.

15. The method of claim 13, wherein the exogenous polynucleotide sequence is integrated into the genome via nucleic acid replication processes.

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

17. A method of modifying a Rosa gene sequence in the genome of cell, the method comprising:

cleaving one or more Rosa genes according to claim 11, wherein cleavage results in a modification of the gene sequence by non-homologous end joining or homology directed repair.

18. The method of claim 17, wherein the modification comprises a deletion.

19. The method of claim 17, wherein the modification comprises insertion of an exogenous sequence.

20. A method of generating a transgenic animal, the method comprising;

modifying a Rosa gene sequence in an embryo cell according claim 17; and

allowing the embryo to develop into an animal.

21. The method of claim 20, wherein the modification comprises one or more random mutations at defined positions.

22. The method of claim 20, wherein the modification comprises insertion of a humanized gene.

23. The method of claim 22, wherein the humanized gene is associated with drug metabolism.

24. The method of claim 20, wherein the animal is a sexually mature animal and the modified gene sequences are present in at least a portion of gametes of the sexually mature animal.

25. A method of creating one or more heritable mutant alleles in at least one Rosa locus of interest, the method comprising:

generating a transgenic animal according to claim 20, wherein the embryo is raised to sexual maturity; and

allowing the sexually mature animal to produce offspring;

wherein at least some of the offspring comprise the mutant alleles.

26. A kit comprising a fusion protein according to claim 1.

27. A kit comprising a polynucleotide according to claim 5.

28. The kit of claim 26, further comprising additional components selected from the group consisting of one or more exogenous sequences, instructions for use, and combinations thereof.

29. The kit of claim 27, further comprising additional components selected from the group consisting of one or more exogenous sequences, instructions for use, and combinations thereof.