CRISPRa dCas9-VPR lentiviral particles express a human codon-optimized version of the nuclease-deactivated Cas9 gene, fused to three transcriptional activators (VP64, p65 and Rta), and two nuclear localization signals (NLS). 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.
Review our CRISPRa application page to get an overview of the technology.
- Generate your own stable "CRISPRa ready" dCas9-VPR-expressing cell line.
- Concentrated, purified lentiviral particles are ready for immediate transduction (minimum ≥1×107 TU/mL functional titer, by qPCR).
- Also available as certified endotoxin-free plasmid DNA for direct transfection into a packaging cell line and production of your own lentiviral particles.
- Blasticidin resistance gene offers easy cell population selection or for clonal expansion of individual cells.
- Choose from one of three different promoters for optimum expression in your cells of interest (see options below).
Requirements for a CRISPRa experiment using dCas9-VPR lentiviral particles
- CRISPRa dCas9-VPR lentiviral particles.
- CRISPRa lentiviral sgRNA for your target gene (see Workflow tab).
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 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
|hCMV||human cytomegalovirus immediate early promoter|
|mCMV||mouse cytomegalovirus immediate early promoter|
|hEF1α||human elongation factor 1 alpha promoter|
CRISPRa workflow diagram with stable dCas9-VPR expression
CRISPR activation workflow with lentiviral dCas9-VPR and synthetic crRNA:tracrRNA (left side) or Lentiviral expressed sgRNA (right side).
CRISPRa plasmid co-transfection workflow
CRISPRa workflow for plasmid co-transfection of lentiviral dCas9-VPR and expressed sgRNA.
CRISPRa gene activation fold depends on the endogenous gene expression level
Genes with low or no basal expression are easier to activate to a robust level, while genes already expressed at high level are more difficult to further over-express. U2OS-dCas9-VPR stable cells were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRa synthetic crRNA:tracrRNA pools (25 nM) targeting 23 genes with different basal level of expression. Cells were harvested 72 hours post-transfection and the relative gene expression was measured using RT-qPCR. The CRISPRa-mediated fold transcriptional activation is shown in the upper graph where the genes are ordered from low to high level of basal transcript expression in samples treated with NTC control and is shown in the lower graph as basal target gene expression (compared to GAPDH control).
CRISPRa gene activation is observed at 24 hours and peaks at 48-72 hours
U2OS-dCas9-VPR stable cells were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRa synthetic crRNA:tracrRNA targeting EGFR, IL1R2, POU5F1 or TFAP2C. Four CRISPRa crRNAs 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 measured 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.