Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System.

Yin L, Jao LE, Chen W
Methods Mol Biol. 2015 1332: 205-17

PMID: 26285757 · DOI:10.1007/978-1-4939-2917-7_16

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.

MeSH Terms (10)

Animals Animals, Genetically Modified CRISPR-Cas Systems Gene Targeting Mutagenesis, Site-Directed Mutation RNA, Guide RNA, Messenger RNA Editing Zebrafish

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