Genome editing in rats using zinc-finger nucleases

• US 20100218264A1
• Filed: 12/03/2009
• Published: 08/26/2010
• Est. Priority Date: 12/04/2008
• 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: Geurts, Aron M., Urnov, Fyodor, Cui, Xiaoxia
• Granted Patent: US 9,206,404 B2
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/054 inducing loss of function
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/10 Mammal
• 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 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• 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: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• 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 15/8509 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 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• 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 one or more loci in a rat, 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. A method for modifying one or more endogenous cellular genes in a rat cell, the method comprising:

introducing, into the rat cell, one or more polynucleotides encoding one or more zinc finger nucleases (ZFNs) that bind to a target site in the one or more genes under conditions such that the ZFN(s) is (are) expressed and the one or more endogenous cellular genes are cleaved and modified.

2. The method of claim 1, wherein the modification comprises introducing an exogenous sequence into the genome of a rat cell by homologous recombination stimulated by cleavage of the one or more endogenous cellular genes.

3. The method of claim 2, wherein the exogenous sequence is integrated physically into the genome.

4. The method of claim 2, wherein the exogenous sequence is integrated into the genome by copying of the exogenous sequence into the host cell genome via nucleic acid replication processes.

5. The method of claim 4, wherein the nucleic acid replication process comprises homology-directed repair of the double strand break.

6. The method of claim 4, wherein the nucleic acid replication process comprises non-homology dependent targeted integration.

7. The method of claim 1, wherein the modification results from non-homologous end joining following cleavage.

8. The method of claim 7, wherein first and second zinc finger nucleases cleave the genome at two sites and further wherein the non-homologous end joining results in a deletion between the first and second cleavage sites.

9. The method of claim 1, wherein the zinc finger nucleases comprise a cleavage domain or cleavage half-domain of a Type IIS restriction endonuclease.

10. A method for germline disruption of one or more target genes in rat, the method comprising modifying one or more gene sequences in the genome of one or more cells of a rat embryo the method comprising:

modifying one or more of the target genes in one or more cells of a rat embryo according to the method of claim 1; and

allowing the rat embryo to develop, wherein that the modified gene sequences are present in at least a portion of gametes of the sexually mature rat.

11. The method of claim 10, wherein the modification comprises integration of an exogenous sequence into the genome of a rat cell by homologous recombination stimulated by cleavage of the one or more endogenous cellular genes.

12. The method of claim 10, wherein the modification results from non-homologous end joining following cleavage.

13. A method of creating one or more heritable mutant alleles in rat loci of interest, the method comprising

modifying one or more loci in the genome of one or more cells of a rat embryo by the method of claim 1;

raising the rat embryo to sexual maturity; and

allowing the sexually mature rat to produce offspring;

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

14. The method of claim 13, wherein the modification comprises integration of an exogenous sequence into the genome of a rat cell by homologous recombination stimulated by cleavage of the one or more endogenous cellular genes.

15. The method of claim 13, wherein the modification results from non-homologous end joining following cleavage.

16. A rat comprising one or more modified alleles, produced by the method of claim 1.

17. A rat comprising one or more heritable mutant alleles produced by the method of claim 10.

18. A rat comprising one or more heritable mutant alleles produced by the method of claim 13.