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.
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.