Dickinson DJ, Ward JD, Reiner DJ, Goldstein B
Nat Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641. PubMed Article
Our Cas9-sgRNA plasmids contain both the Cas9 gene and an sgRNA gene. The inclusion of both components on a single plasmid facilitates archival and distribution, and makes it simpler to prepare injection mixes. The Cas9 coding sequence is codon-optimized and includes synthetic introns for efficient expression in C. elegans , and is driven by the eft-3 promoter for germline expression. The sgRNA is driven by the R07E5.16 U6 RNA promoter. Compared to the C. elegans CRISPR plasmids from John Calarco's lab, which are also available on Addgene, the biggest differences are 1) Inclusion of both components on the same plasmid, and 2) Use of a different U6 promoter to drive sgRNA expression. We have not compared the two systems side by side but would expect them to be similar in terms of efficiency.
The Supplementary Information in the publication contains a protocol for using the plasmids. A Cas9-triggered homologous recombination website has been created for sharing protocols and information.
Please note: eft-3 has officially been changed to eef-1A.1 Please see the eef-1A.1 WormBase entry for details.
Plasmids from Article
|47549||pDD162 (Peft-3::Cas9 + Empty sgRNA)||Cas9 + sgRNA plasmid that can be modified to cleave any Cas9 target site in the C. elegans genome.||
|47550||pDD122 (Peft-3::Cas9 + ttTi5605 sgRNA)||Cas9 + sgRNA plasmid that is targeted to a genomic site near the ttTi5605 Mos1 insertion allele||
|47551||pDD104 (Peft-3::Cre)||Cre recombinase expression plasmid for removal of floxed selectable markers in the C. elegans germline||