CRISPRa crRNA non-targeting controls are designed and recommended for use as negative controls for transcriptional activation experiments using CRISPRa crRNA individuals and pools. These non-targeting controls will hybridize with tracrRNA and engage the dCas9-VPR complex, but will not target any PAM-adjacent sites in the human or mouse genome. Any observed alteration in gene expression levels or viability in cells treated with these controls can be used as a baseline response of the cells to the dCas9-VPR complex for comparison to those treated with target-specific CRISPRa crRNAs and pools.
- Proprietary alignment tools used to verify at least three mismatches or gaps to any potential target in human or mouse genomes
- Choose an individual or pooled crRNA control to match your experimental crRNA reagent
Remember to order tracrRNA for use with your controls!
CRISPRa workflow diagram with stable dCas9-VPR expression
CRISPR activation workflow with lentiviral dCas9-VPR and synthetic crRNA:tracrRNA (left) or Lentiviral expressed sgRNA (right) as a purified lentiviral particle or plasmid prepared from glycerol stock.
Pooling of synthetic crRNAs can enhance transcriptional activation
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 (25 nM) targeting EGFR or POU5F1. The pre-designed crRNAs were used either individually or pooled (to a total concentration of 25 nM). 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.