Transcriptional activation of gene targets in SAM system expressing cell lines

U2OS and A375 cells stably expressing dCas9-VP64 and MS2-p65-HSF1 (dCas9-SAM) were plated at 10,000 cells/well and transfected using DharmaFECT 4 (U2OS) or DharmaFECT 1 (A375) Transfection Reagent with synthetic crRNA:tracrRNA (25nM). Four predesigned crRNAs targeting each gene were pooled (to a total concentration of 25nM). 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 ΔΔCq method using PPIB as the housekeeping gene and normalized to a non-targeting control.

Transcriptional activation of gene targets in U2OS-dCas9-SAM cell lines: individual and pooled crRNA

U2OS cells stably expressing dCas9-VP64 and MS2-p65-HSF1 (dCas9-SAM) were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA (25nM). Four predesigned crRNAs targeting each gene were transfected individually or pooled (to a total concentration of 25nM). 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 Δ Δ Cq method using PPIB as the housekeeping gene and normalized to a non-targeting control.

Transcriptional activation of gene targets in A375-dCas9-SAM cell lines: individual and pooled crRNA

A375 cells stably expressing dCas9-VP64 and MS2-p65-HSF1 (dCas9-SAM) were plated at 10,000 cells/well and transfected using DharmaFECT 1 Transfection Reagent with synthetic crRNA:tracrRNA (25nM). Four predesigned crRNAs targeting each gene were transfected individually or pooled (to a total concentration of 25nM). 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 Δ ΔCq method using PPIB as the housekeeping gene and normalized to a non-targeting control.

CRISPRa SAM workflow

Horizon's CRISPRmod MS2 tracrRNA (also called SAM tracrRNA) contains an MS2 aptamer on stem loop 2 that recruits MCP-p65-HSF1. Combined with a catalytically inactivated Cas9-VP64 and a gene specific CRISPRa crRNA, this utilization of the synergistic activation mediator (SAM) system achieves robust transcriptional activation.