PUBLICATION
Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System
- Authors
- Yin, L., Jao, L.E., Chen, W.
- ID
- ZDB-PUB-150820-5
- Date
- 2015
- Source
- Methods in molecular biology (Clifton, N.J.) 1332: 205-17 (Chapter)
- Registered Authors
- Chen, Wenbiao, Jao, Li-En
- Keywords
- CRISPR, Cas9, Genome editing, Mutagenesis, Zebrafish
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- CRISPR-Cas Systems*
- Gene Targeting*
- Mutagenesis, Site-Directed
- Mutation*
- RNA Editing
- RNA, Guide, Kinetoplastida/genetics
- RNA, Messenger/genetics
- Zebrafish/genetics*
- PubMed
- 26285757 Full text @ Meth. Mol. Biol.
Citation
Yin, L., Jao, L.E., Chen, W. (2015) Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System. Methods in molecular biology (Clifton, N.J.). 1332:205-17.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping