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 20;110(34):13904-9. doi: 10.1073/pnas.1308335110.
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PubMed
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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.
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A protocol for synthesizing gRNAs: gRNA plasmid construction protocol 66.8 KB
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 46757 | pT3TS-nCas9n | expression of an optimized Cas9 for genome editing in zebrafish |
| 46759 | pT7-gRNA | gRNA cloning vector for in vitro transcription of target gRNA |
| 46760 | pT7EGFPgRNA | |
| 46761 | pT7tyrgRNA | |
| 47929 | pCS2-nCas9n | expression of an optimized Cas9 for genome editing in zebrafish |
| 47930 | pT7goldRNA | In vitro transcription of golden gRNA |
| 47931 | pT7mitfagRNA | in vitro transcription of mitfa gRNA |
| 47932 | pT7ddx19gRNA | in vitro transcription of ddx19 gRNA |