The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F₁ generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.

The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea. L. crocea is a temperate-water migratory fish that belongs to the order Perciformes and the family Sciaenidae. In China, the annual yield from L. crocea aquaculture exceeds that of any other net-cage-farmed marine fish species. L. crocea also exhibits peculiar behavioral and physiological characteristics and is especially sensitive to various environmental stresses. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea. Further genomic analyses showed the significant expansion of several gene families, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, and provided a genetic basis for the peculiar physiological characteristics of L. crocea. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. These findings provide novel insights into the mechanisms of fish stress adaptation.

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. To understand the molecular basis for antiviral defense in this species, we used Illumia paired-end sequencing to characterize the spleen transcriptome of polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced large yellow croakers. The library produced 56,355,728 reads and assembled into 108,237 contigs. As a result, 15,192 unigenes were found from this transcriptome. Gene ontology analysis showed that 4,759 genes were involved in three major functional categories: biological process, cellular component, and molecular function. We further ascertained that numerous consensus sequences were homologous to known immune-relevant genes. Kyoto Encyclopedia of Genes and Genomes orthology mapping annotated 5,389 unigenes and identified numerous immune-relevant pathways. These immune-relevant genes and pathways revealed major antiviral immunity effectors, including but not limited to: pattern recognition receptors, adaptors and signal transducers, the interferons and interferon-stimulated genes, inflammatory cytokines and receptors, complement components, and B-cell and T-cell antigen activation molecules. Moreover, the partial genes of Toll-like receptor signaling pathway, RIG-I-like receptors signaling pathway, Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway, and T-cell receptor (TCR) signaling pathway were found to be changed after poly(I:C) induction by real-time polymerase chain reaction (PCR) analysis, suggesting that these signaling pathways may be regulated by poly(I:C), a viral mimic. Overall, the antivirus-related genes and signaling pathways that were identified in response to poly(I:C) challenge provide valuable leads for further investigation of the antiviral defense mechanism in the large yellow croaker.