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.