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

Methods Mol Biol. 2015;1332:205-17. doi: 10.1007/978-1-4939-2917-7_16.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

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

Substances

  • RNA, Guide
  • RNA, Messenger