Gersbach Lab CRISPR Plasmids Available from Addgene
These mammalian expression plasmids encode the nuclease-null dCas9 (D10A, H840A) with HA and FLAG epitope tags either alone or fused to the transcriptional activator VP64. When directed to its target in gene promoters by the sgRNA expressed in trans from pSPgRNA, the dCas9-VP64 fusion protein activates the expression of downstream genes. Multiple sgRNAs targeted to the same promoter act synergistically to activate gene expression. The dCas9 protein serves as a negative control of the function of the VP64 activator and can also be used to inhibited gene expression via steric hindrance.
These plasmids are described in:
RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat. Methods. 2013 July. PubMed PMID 23892895.
Individual plasmids can be ordered via the links below:
|47106||pcDNA-dCas9: Expresses inactivated S. pyogenes dCas9 (D10A, H840A) in mammalian cells.|
|47107||pcDNA-dCas9-VP64: Expresses inactivated S. pyogenes dCas9 (D10A, H840A) fused to VP64 transactivator domain in mammalian cells.|
|47108||pSPgRNA: Expresses a S. pyogenes Cas9/dCas9 guide RNA in mammalian cells.|
We also provide a collection of reagents for CRISPR/Cas9-based multiplex genome engineering in diverse cell types. This set of plasmids is described in:
Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. Nucleic Acids Res.. 2014. PubMed PMID 25122746.
|53186||phH1-gRNA: Expresses the S. pyogenes sgRNA from the H1 promoter.|
|53187||pmU6-gRNA: Expresses the S. pyogenes sgRNA from the mouse U6 promoter.|
|53188||phU6-gRNA: Expresses the S. pyogenes sgRNA from the human U6 promoter.|
|53189||ph7SK-gRNA: Expresses the S. pyogenes sgRNA from the 7SK promoter.|
|53190||pLV hUbC-Cas9-T2A-GFP: Co-expresses human optimized S. pyogenes Cas9 and GFP.|
|53191||pLV hUbC-dCas9-T2A-GFP: Co-expresses human optimized S. pyogenes dCas9 and GFP.|
|53192||pLV hUbC-dCas9 VP64-T2A-GFP: Co-expresses human optimized S. pyogenes dCas9-VP64 and GFP|