CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans.

Waaijers S, Portegijs V, Kerver J, Lemmens BB, Tijsterman M, van den Heuvel S, Boxem M
Genetics. 2013 Aug 26. PubMed Article

We adapted the S. pyogenes CRISPR/Cas9 system for use in C. elegans . We provide four different vectors for expression of Cas9, and two vectors for the delivery of the synthetic guide RNA (sgRNA). Each of the Cas9 vectors contains the Cas9 coding sequence optimized for C. elegans expression, flanked by two nuclear localization signals. pMB62 and pMB63 drive expression from the eft-3 promoter, while pMB66 and pMB67 use the hsp-16.48 heat shock promoter. All vectors include the germline expression compatible tbb-2 3’UTR. For both the eft-3 and hsp-16.48 vectors we generated variants with and without a C-terminal EGFP fusion.

For the sgRNA, vector pMB60 allows in vitro production of RNA from the T7 promoter, while pMB70 is designed for in vivo transcrition controlled by the regulatory sequences of an RNA polymerase III transcribed U6 snRNA. In both vectors, the target site sequence can be added by inserting an oligonucleotide linker into BsaI digested vector.

Please note: eft-3 has officially been changed to eef-1A.1 Please see the eef-1A.1 WormBase entry for details.

Figure 1: Maps of the four Cas9 expression plasmids (pMB62, pMB63, pMB66, and pMB67) and the two sgRNA vectors (pMB60 and pMB70).

Plasmids from Article

ID	Plasmid	Purpose
47941	pMB60	Can be used to generate synthetic guide in vitro from T7 promotor
47943	pMB70	Can be used to express a CRISPR guide RNA in C. elegans from U6 regulatory sequences
47944	pMB62	Expresses C. elegans optimized Cas9::mEGFP from the eef-1A.1 (eft-3) promoter
47945	pMB63	Expresses C. elegans optimized Cas9 from the eef-1A.1 (eft-3) promoter
47946	pMB66	Expresses C. elegans optimized Cas9::mEGFP from the hsp-16.48 promoter
47947	pMB67	Expresses C. elegans optimized Cas9 from the hsp-16.48 promoter