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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


A protocol for synthesizing gRNAs: gRNA plasmid construction protocol 66.8 KB

Plasmids from Article

ID Plasmid Purpose
46759pT7-gRNAgRNA cloning vector for in vitro transcription of target gRNA
47929pCS2-nCas9nexpression of an optimized Cas9 for genome-editing in zebrafish
47930pT7goldRNAIn vitro transcription of golden gRNA
47931pT7mitfagRNAin vitro transcription of mitfa gRNA
47932pT7ddx19gRNAin vitro transcription of ddx19 gRNA

Antibodies from Article