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 ...

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons. Causative mutations in the global RNA-processing proteins TDP-43 and FUS among others, as well as their aggregation in ALS patients, have identified defects in RNA metabolism as an important feature in this disease. Lethal congenital contracture syndrome 1 and lethal arthrogryposis with anterior horn cell disease are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1. In this study, we carried out the first screening of GLE1 in ALS patients (173 familial and 760 sporadic) and identified 2 deleterious mutations (1 splice site and 1 nonsense mutation) and 1 missense mutation. Functional analysis of the deleterious mutants revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells, both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA. These results suggest a haploinsufficiency mechanism and point to a causative role for GLE1 mutations in ALS patients. This further supports the involvement of global defects in RNA metabolism in ALS.

Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F 1 offspring of the infected fish. From F 1 fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes. By inbreeding fish carrying proviral integrations in 25 different genes, we were able to demonstrate that in Ϸ50% of the gene ''hits,'' the mRNA transcript levels were reduced by >70%, with the highest probability for mutation occurring if the integration was in an exon or first intron. Based on these data, the mutagenic frequency for the retrovirus is nearly one in five integrations. In addition, a strong mutagenic effect is seen when murine leukemia virus integrates specifically in the first intron of genes but not in other introns. Three of 19 gene inactivation events had embryonic defects. Using the strategy we outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F1 fish. This is a 20-to 30-fold increase in efficiency when compared with the current resequencing approach [targeting induced local lesions in genomes (TILLING)] used in zebrafish for identifying mutations in genes. Combining this increase in efficiency with cryopreservation of sperm samples from the F 1 fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene.genetics ͉ retrovirus

Several strategies have been developed to generate targeted gene disruptions in zebrafish.Here we developed a simple targeted gene inactivation strategy in zebrafish using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. By injecting two simple in vitro-synthesized components [Cas9 mRNA and single guide (sgRNA)] into one-cell-stage embryos, mutations of the target gene could be efficiently generated. We used a codon-optimized version of Cas9 to improve its translation efficiency in zebrafish. In addition, we designed a cloning-free strategy to facilitate the synthesis of sgRNA. The system allows biallelic inactivation of multiple genes simultaneously by co-injecting a mix of sgRNAs with a single Cas9 construct. This flexible strategy of gene inactivation provides an efficient way to interrogate gene functions and genetic interactions in zebrafish.

At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT—enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs)—phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions.

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