Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System
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.
Key wordsCRISPR Cas9 Genome editing Mutagenesis Zebrafish
- 34.Varshney GK, Pei W, LaFave MC et al (in press) High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome ResGoogle Scholar