Boxem Lab CRISPR Plasmids Available from Addgene
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.
These plasmids are described in:
CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans . Waaijers S, Portegijs V, Kerver J, Lemmens BB, w Tijsterman M, van den Heuvel S, Boxem M. Genetics. 2013 Aug 26. PubMed PMID 23979586.
Individual plasmids can be ordered via the links below:
|47941||pMB60: Can be used to generate synthetic guide RNA 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 eft-3 (eef-1A.1) promoter.|
|47945||pMB63: Expresses C. elegans optimized Cas9 from the eft-3 (eef-1A.1) promoter.|
|47946||pMB66: Expresses C. elegans optimized Cas9::mEGFP from the hap-16.48 promoter.|
|47947||pMB67: Expresses C. elegans optimized Cas9 from the hap-16.48 promoter.|
Please note: eft-3 has officially been changed to eef-1A.1 Please see the eef-1A.1 WormBase entry for details.