Jao, L.E., Wente, S.R., and Chen, W. (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proceedings of the National Academy of Sciences of the United States of America. 110(34):13904-13909.
A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic
mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation
of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type
II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system
has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish
with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75–99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes
were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient
germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting
in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and
scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.