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).

Pooling of CRISPRa synthetic crRNAs enhances transcriptional activation

Individual CRISPRa crRNAs achieve robust target gene activation on their own, but when pooled together in a single reagent, enhanced activation levels can be achieved. U2OS cells stably expressing integrated CRISPRa dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRa synthetic crRNA:tracrRNA targeting EGFR or POU5F1. Four CRISPRa 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 measured using RT-qPCR. The relative fold transcriptional activation for each gene was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC).