Description
General description
This product is a kit containing two lentiviral plasmids: one plasmid that utilizes the EF1 alpha promoter to drive expression of MS2-P65-HSF1 and a hygromycin resistance cassette linked by a 2A peptide (EF1a-MS2-P65-HSF1-2A-Hygromycin) and one plasmid that utilizes the EF1 alpha promoter to drive expression of dCas9-VP64 and blasticidin resistance cassette also linked by a 2A peptide (EF1a-dCas9-VP64-2A-Blasticidin). The vector composition allows for easy selection following successful transfection or transduction. Use Sigma′s lentiviral MS2-P65-HSF1 or dCas9-VP64 plasmids for generation of lentiviral particles and efficient production of stable cell lines expressing MS2-P65-HSF1 and/or dCas9-VP64. The two plasmids are part of a 3 part CRISPR system with individual dCas9-VP64, MS2-p65-HSF1 and gRNA expression vectors used for CRISPR based transcipritonal activation and genome wide gain of function screening.
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Application
Functional Genomics/Target Validation
Unbiased forward genetic screening
Strong transcriptional activation in multiple cell lines
Manufacture of dCas9-VP64 and MS2-p65-HSF1 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
Learn more about SAM CRISPR Activators at SigmaAldrich.com/CRISPRa
Other Notes
This kit contains 2 components:
2 Helper Constructs supplied at a concentration of 500 ng/μL in TE buffer (10μg of plasmid DNA)
20μL vial of dCas9-VP64-Blasticidin SAM CRISPRa Helper Construct 1 Plasmid DNA
20μL vial of MS2-P65-HSF1-Hygromycin SAM CRISPRa Helper Construct 2 Plasmid DNA
Legal Information
CRISPR Use License Agreement
Lentiviral, WPRE and Evrogen Fluorophore Licenses
