Pooling of synthetic crRNAs can enhance transcriptional activation

Individual Edit-R 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 dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA 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 measured using RT-qPCR. The relative fold transcriptional activation for each gene was calculated with the ∆∆Cq method using GAPDH as the reference gene and normalized to a non-targeting control (NTC).

Edit-R CRISPRa crRNAs and pools are highly effective at 25 nM working concentration

A dose curve of Edit-R CRISPRa crRNA:tracrRNA targeting two different genes demonstrates that a working concentration of 25 nM achieves robust target gene 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 targeting EGFR or POU5F1. The pre-designed crRNAs were used either individually or pooled at four concentrations (1, 5, 25, 100 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 reference gene and normalized to a non-targeting control.

Efficient transcriptional gene activation with synthetic crRNA in multiple dCas9-VPR stable cells

Efficient transcriptional gene activation with synthetic crRNA:tracrRNA in dCas9-VPR stable cells. HEK293T, U2OS, MCF 10A, NIH/3T3 stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT Transfection Reagents with synthetic crRNA:tracrRNA (25 nM) targeting POU5F1 and TTN. K562 cells were electroporated with synthetic crRNA:tracrRNA (400 nM) targeting POU5F1 and TTN. 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 reference gene and normalized to a non-targeting control.

CRISPRa gene activation in U2OS cells is observed at 24 hours and peaks at 48-72 hours

Edit-R CRISPRa synthetic crRNAs and pools achieve maximal activation at 48-72 hours post-transfection in dCas9-VPR-expressing cells. 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 targeting EGFR, IL1R2, POU5F1 or TFAP2C. The four pre-designed crRNAs for CRISPRa were used either individually or pooled (to a total concentration of 25 nM). Cells were harvested at 24, 48, and 72 hours post-transfection and the relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using GAPDH as the reference gene and normalized to a non-targeting control.

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