VEGF Signaling pp 205-217 | Cite as

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

Part of the Methods in Molecular Biology book series (MIMB, volume 1332)

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 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Molecular Physiology and BiophysicsVanderbilt University School of MedicineNashvilleUSA
  2. 2.Department of Cell Biology and Human Anatomy, School of MedicineUniversity of California DavisDavisUSA
  3. 3.Department of Molecular Physiology and BiophysicsVanderbilt University School of MedicineNashvilleUSA

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