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 language | English (US) |
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Pages (from-to) | 205-217 |
Number of pages | 13 |
Journal | Methods in Molecular Biology |
Volume | 1332 |
DOIs | |
State | Published - 2015 |
Keywords
- Cas9
- CRISPR
- Genome editing
- Mutagenesis
- Zebrafish
ASJC Scopus subject areas
- Molecular Biology
- Genetics
- Medicine(all)