ZFIN ID: ZDB-PUB-150820-5
Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System
Yin, L., Jao, L.E., Chen, W.
Date: 2015
Source: Methods in molecular biology (Clifton, N.J.) 1332: 205-17 (Chapter)
Registered Authors: Chen, Wenbiao, Jao, Li-En
Keywords: CRISPR, Cas9, Genome editing, Mutagenesis, Zebrafish
MeSH Terms: Animals; Animals, Genetically Modified; CRISPR-Cas Systems*; Gene Targeting*; Mutagenesis, Site-Directed (all 10) expand
PubMed: 26285757 Full text @ Methods Mol. Biol.
ABSTRACT
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.
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