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Volume 1332 of the series Methods in Molecular Biology pp 205-217

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

  • Linlin YinAffiliated withDepartment of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine
  • , Li-En JaoAffiliated withDepartment of Cell Biology and Human Anatomy, School of Medicine, University of California Davis
  • , Wenbiao ChenAffiliated withDepartment of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine Email author 

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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.

Key words

CRISPR Cas9 Genome editing Mutagenesis Zebrafish