This kit contains 2 components :
2 Helper Constructs with a minimum p24 titer of 1x10⁶ VP/mL 
•  8 x 25μl vials of dCas9-VP64-Blasticidin SAM CRISPRa Helper Construct 1 Lentiviral Transduction Particles
•  8 x 25μl vials of MS2-P65-HSF1-Hygromycin SAM CRISPRa Helper Construct 2 Lentiviral Transduction Particles

•  Highly specific and highly active
•  Sequence verified high purity, high titer lentiviral particles
•  Activates genes through transcriptional activation rather than cDNA based overexpression

This product is a kit containing two sets of ready to use lentiviral particles enabling immediate transduction for strong expression in a wide range of both dividing and non-dividing cell lines. MS2-P65-HSF1 lentiviral particles efficiently and stably integrate MS2-P65-HSF1 and hygromycin resistance cassette linked by a 2A peptide and driven by the EF1 alpha promoter (EF1a-dCas9-VP64-2A-Blasticidin). dCas9-VP64 lentiviral particles efficiently and stably integrate dCas9-VP64 and blasticidin resistance cassette linked by a 2A peptide and driven by the EF1 alpha promoter (EF1a-dCas9-VP64-2A-Blasticidin). They are ideal for avoiding the tedious lentivirus production process and are part of a three part CRISPR system with individual dCas9-VP64. MS2-p65-HSF1 and gRNA expression vectors.

Unit Definition

VP/mL is the concentration unit of measure for viral titer estimated by p24 assay.

Functional Genomics/Target Validation
• Unbiased forward genetic screening
• Strong transcriptional activation in multiple cell lines
• Creation of cell lines stably expressing dCas9-VP64 and MS2-p65-HSF1.

Principle

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.