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.
Plasmid dCas9-VPR and Edit-R 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.
U2OS, HEK293T, MCF 10A and K562 stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transduced with sgRNA lentiviral particles targeting POU5F1 or TTN at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 4 days prior to analysis with 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.