Method of deleting an IgM gene in an isolated rat cell
View Patent ↗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.
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.