Jao LE, Wente SR, Chen W
Proc Natl Acad Sci U S A. 2013 Aug 5. PubMed Article
We have synthesized a Cas9 coding sequence with nuclear localization signals (nls) at both its amino and carboxyl termini and codons optimized for zebrafish expression (nls-zCas9-nls). Injection of in vitro-transcribed nls-zCas9-nls mRNA with a gene-specific chimeric guide RNA consistently resulted in high mutagenic efficiency in both somatic and germline cells. Injected fish often exhibit loss-of-function phenotype, indicating prevalent biallelic inactivation. The injected fish produces predominantly mutation-carrying progeny. In addition, multiple loci can be efficiently targeted simultaneously in the same fish.
A protocol for synthesizing gRNAs: gRNA plasmid construction protocol (66.8 KB)