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Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.
Jao LE, Wente SR, Chen W
Proc Natl Acad Sci U S A. 2013 Aug 5.
PubMed Article

We have synthesized a Cas9 coding sequence with nuclear localization signals (nls) at both its amino and carboxyl termini and codons optimized for zebrafish expression (nls-zCas9-nls). Injection of in vitro-transcribed nls-zCas9-nls mRNA with a gene-specific chimeric guide RNA consistently resulted in high mutagenic efficiency in both somatic and germline cells. Injected fish often exhibit loss-of-function phenotype, indicating prevalent biallelic inactivation. The injected fish produces predominantly mutation-carrying progeny. In addition, multiple loci can be efficiently targeted simultaneously in the same fish.

Chen lab plasmid cloning figure

Resources:

A protocol for synthesizing gRNAs: Icon gRNA plasmid construction protocol (66.8 KB)

Plasmids from Article

ID Plasmid  
46757pT3TS-nCas9n
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46759pT7-gRNA
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46760pT7EGFPgRNA
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46761pT7tyrgRNA
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47929pCS2-nCas9n
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47930pT7goldRNA
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47931pT7mitfagRNA
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47932pT7ddx19gRNA
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