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
We thank Shawn Burgess for communicating unpublished data. This work was supported by R01DK088686 (to W.C.) from the NIH.
- 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