Generation of targeted mutations in zebrafish using the CRISPR/Cas system

Linlin Yin, Li En Jao, Wenbiao Chen

Research output: Contribution to journalArticle

16 Scopus citations

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 vitrosynthesized 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 fl exible strategy of gene inactivation provides an efficient way to interrogate gene functions and genetic interactions in zebrafish.

Original languageEnglish (US)
Pages (from-to)205-217
Number of pages13
JournalMethods in Molecular Biology
Volume1332
DOIs
StatePublished - 2015

Keywords

  • Cas9
  • CRISPR
  • Genome editing
  • Mutagenesis
  • Zebrafish

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

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