dCas9-VP64-Blasticidin SAM CRISPRa Helper Construct 1 Plasmid DNA，10UG，SAMVP64BST-10UG，Sigma

General description

This product is a lentiviral plasmid that utilizes the EF1 alpha promoter to drive expression of dCas9-VP64 and blasticidin resistance cassette linked by a 2A peptide (EF1a-dCas9-VP64-2A-Blasticidin) allowing for easy selection following successful transfection or transduction. Use Sigma′s lentiviral dCas9-VP64 plasmid for generation of lentiviral particles and efficient production of stable cell lines expressing dCas9-VP64 for CRISPR based transcipritonal activation and genome wide gain of function screening. Sigma′s lenti-dCas9-VP64 plasmid is one part of a three part CRISPR system with individual dCas9-VP64, MS2-p65-HSF1 and gRNA expression vectors.

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Application

Functional Genomics/Target Validation

• Unbiased forward genetic screening
• Strong transcriptional activation in multiple cell lines
• Manufacture of dCas9-VP64 expressing Lentiviral Particles

Biochem/physiol Actions

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be rendered inactive (dCas9) with mutations to the two protein domains, RuvC and HnH (D10A and H840A respectively), which are responsible for nuclease activity. The nuclease deficient protein can then be fused with the transcriptional activator VP64 and used in conjunction with a guide RNA modified with MS2 RNA aptamers that function to recruit the additional transcriptional coactivators p65 and HSF1. The assembled SAM complex is then used as a cargo delivery system to target gene promoters, enabling site-specific transcriptional activation of the gene of interest.

Features and Benefits

• Highly specific and highly active
• Sequence verified high purity plasmid DNA
• Activates genes through transcriptional activation rather than cDNA based overexpression

Legal Information

CRISPR Use License Agreement

Lentiviral, WPRE and Evrogen Fluorophore Licenses