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.genome engineering | RNA-guided mutagenesis | pigmentation R ecent methodological innovations that exploit zinc finger nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs) have made it possible to introduce sitespecific genomic modifications in cell culture systems and in a wide range of species (1, 2). These designer nucleases target and cleave specific genomic sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed (3).CRISPR systems in eubacteria and archaea use small RNAs and CRISPR-associated proteins (Cas) proteins to target and cleave invading foreign DNAs (4-6). One of the simplest CRISPR systems is the type II CRISPR system from Streptococcus pyogenes: an endonuclease Cas9 and two small RNAs, CRISPR RNA (crRNA) and a transacting RNA (tracrRNA), are sufficient for RNA-guided cleavage of foreign DNAs (7). Recent studies show that a single guide RNA (gRNA) chimera that mimics the crRNA:tracrRNA complex can guide Cas9 to introduce sitespecific DNA double-stranded breaks in vitro (3) and in mammalian cell lines (8-11), bacteria (12, 13), yeast (14), zebrafish (15, 16), and mice (17) with ...

The genetic loci agouti and extension control the relative amounts of eumelanin (brown-black) and phaeomelanin (yellow-red) pigments in mammals: extension encodes the receptor for melanocyte-stimulating hormone (MSH) and agouti encodes a novel 131-amino-acid protein containing a signal sequence. Agouti, which is produced in the hair follicle, acts on follicular melanocytes to inhibit alpha-MSH-induced eumelanin production, resulting in the subterminal band of phaeomelanin often visible in mammalian fur. Here we use partially purified agouti protein to demonstrate that agouti is a high-affinity antagonist of the MSH receptor and blocks alpha-MSH stimulation of adenylyl cyclase, the effector through which alpha-MSH induces eumelanin synthesis. Agouti was also found to be an antagonist of the melanocortin-4 receptor, a related MSH-binding receptor. Consequently, the obesity caused by ectopic expression of agouti in the lethal yellow (Ay) mouse may be due to the inhibition of melanocortin receptor(s) outside the hair follicle.

To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.