Format

Send to

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

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

Author information

1
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.

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.

PMID:
26285757
DOI:
10.1007/978-1-4939-2917-7_16
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center