Generation of Targeted Mutations in Zebrafish Using the CRISPR/Cas System.
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.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
Linlin
L
Yin
Li-En
LE
Jao
Wenbiao
W
Chen
D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D018390:Gene Targeting; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*
D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger