Method of deleting an IgM gene in an isolated rat cell

• US 9,206,404 B2
• Filed: 12/03/2009
• Issued: 12/08/2015
• Est. Priority Date: 12/04/2008
• Status: Active Grant

• Current Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.), Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.), Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Cui, Xiaoxia, Geurts, Aron M., Urnov, Fyodor
• Primary Examiner(s): WILSON, MICHAEL C
• Application Number:
  US12/592,852
• Publication Number:
  US 20100218264A1
• Time in Patent Office: 2,196 Days
• Field of Search: 435/325, 800/8, 800/21, 800/14, 800/18
• US Class Current: 1/1
• 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 making a deletion in an endogenous IgM gene, the method comprising:

introducing mRNA into an isolated rat cell, wherein the mRNA comprises

(i) mRNAs encoding a first zinc finger nuclease (ZFN) that binds to a first target site in an endogenous rat IgM gene wherein the first ZFN comprises a cleavage domain and a zinc finger protein, wherein the first zinc finger protein-comprises the following recognition helices in the following order;

DRSHLTR (SEQ ID NO;

41);

RSDALTQ (SEQ ID NO;

40);

DRSDLSR (SEQ ID NO;

28);

RSDALAR (SEQ ID NO;

39);

RSDSLSA (SEQ ID NO;

38); and

TSSNRKT (SEQ ID NO;

37), and

(ii) mRNA encoding a second ZFN that binds to a second target site in the endogenous rat IgM gene, wherein the second ZFN comprises a cleavage domain and a zinc finger protein comprising the following recognition helices in the following order;

NKVGLIE (SEQ ID NO;

46);

TSSDLSR (SEQ ID NO;

45);

RSDHLSR (SEQ ID NO;

44);

RSDNLSE (SEQ ID NO;

43); and

QNAHRKT(SEQ ID NO;

42),

such that the first and second ZFNs are expressed, dimerize and cleave the endogenous rat IgM gene and non-homologous end joining (NHEJ) occurs causing the cell to have a deletion in the endogenous rat IgM gene.

2. The method of claim 1, wherein the cleavage domain is a Type IIS restriction endonuclease cleavage domain.