The CRISPR activation (CRISPRa) system is a variation of the canonical CRISPR-Cas9 used for creation of double-strand breaks in genomic DNA. It utilizes a nuclease-deactivated S. pyogenes Cas9 (dCas9), often called "dead Cas9", that is fused to one or more transcriptional activators. When paired with a well-designed guide RNA that targets a gene near a promoter region, the gene's native transcription start site is activated. Lentiviral dCas9-VPR Reagents are available with three different promoters (hCMV, mCMV, or hEF1a) and can be supplied as either purified high-titer lentiviral particles or purified plasmid DNA.
Review our Applications page on CRISPRa to get an overview of the technology!
- Choose from one of three different promoters for optimum expression in your cells of interest
- Provided as concentrated, purified lentiviral particles for immediate transduction, with a minimum ≥ 1 × 107 TU/mL functional titer, by qPCR titering
- Also available as certified endotoxin-free plasmid DNA for direct transfection into a packaging cell line and production of your own lentiviral particles
- Selection using blasticidin resistance marker (BlastR) for entire cell populations or for clonal expansion of individual cells
Not all RNA pol II promoters are equally active in different cellular environments
The activity of any given promoter controlling the transcription of dCas9-VPR nuclease can differ greatly from one biological context to another, resulting in variable dCas9-VPR expression levels and thus varying levels of gene activation. Choosing an optimal promoter for your cell line will be important for robust gene overexpression.SMARTchoice promoter options for expressing dCas9-VPR nuclease
|hCMV||human cytomegalovirus immediate early promoter|
|mCMV||mouse cytomegalovirus immediate early promoter|
|hEF1α||human elongation factor 1 alpha promoter|
CRISPRa gene activation workflow
CRISPR activation workflow with lentiviral dCas9-VPR and synthetic crRNA:tracrRNA (left side) or Lentiviral expressed sgRNA (right side).
Plasmid co-transfection workflow for CRISPRa
Plasmid dCas9-VPR and CRISPRa sgRNA plasmid can be co-transfected or co-electroporated into cells to achieve transcriptional activation. This method avoids lentiviral incorporation into the genome, but still provides the ability to enrich for transfected cells with antibiotics selection.
CRISPRa gene activation in U2OS cells is observed at 24 hours and increases at 48-72 hours
U2OS cells stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA targeting EGFR, IL1R2, POU5F1 or TFAP2C. The four pre-designed crRNAs for CRISPRa were used either individually or pooled (to a total concentration of 25 nM). Cells were harvested at 24, 48, and 72 hours post-transfection and the relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
Fold activation by CRISPRa varies by gene and depends on the endogenous gene expression level
U2OS cells stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA pools (25 nM) targeting genes with low to high basal transcript expression levels. Cells were harvested 72 hours post-transfection and the relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control. The fold activation is shown for the genes ranked from low to high basal transcript expression level in samples treated with NTC control and is shown in the lower graph as basal gene expression relative to GAPDH expression in the same samples.
|Shipping Condition||Dry Ice, Ambient|
|Storage Conditions||-80 C, -20 C|
|Stability at Recommended Storage Conditions||At least 12 months|