Edit-R CRISPRa Positive crRNA Controls

Validated synthetic crRNA pool or individual for experimental optimization of transcription activation experiments


Choose your human- or mouse-specific positive control gene to verify optimal experimental conditions and ensure ongoing dCas9-VPR expression.

The Edit-R CRISPRa positive controls will selectively activate a human or mouse Titin (TTN) or POU class 5 homeobox 1 (POU5F1). Positive controls are recommended for optimization and ongoing monitoring of delivery conditions and dCas9-VPR expression prior to experiments with target gene-specific guide RNAs.

The level of activation achieved will depend on your cell type and basal level of expression. If either TTN or POU5F1 is already expressed in your cells, there will be more modest activation than with a gene that is not expressed. Normalization of the activation level to a non-targeting control will provide a baseline for determining optimal dCas9-VPR expression, transfection efficiency, and timepoint for your assay.

Please note that Edit-R tracrRNA is required for use with Edit-R crRNA reagents.

crRNA nmol tracrRNA nmol 96-well plate 100 µL volume 24-well plate 500 µL volume 12-well plate 1000 µL volume 6-well plate 2500 µL volume
2 2 800 160 80 32
5 5 2000 400 200 80
10 10 4000 800 400 160
20 20 8000 1600 800 300
  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C
crispra-synthetic-crRNA-dCas9-VPR-stable-ttn-pou5f1

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

crispra-synthetic-crRNA-dCas9-VPR-stable-ttn-pou5f1

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 expression of each gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.


crispra-activation-basal-expression-relationship

Fold activation by CRISPRa varies by gene and depends on the endogenous gene expression level

crispra-activation-basal-expression-relationship

Genes that have low or no basal expression are easier to activate to a robust level, and genes already being expressed are more difficult to over-express further. 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 pools (25 nM) targeting genes with low to high basal transcript expression levels. Cells were harvested 72 hours post-transfection and the relative gene expression was measured using qRT-PCR. 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. The fold activation is shown for the genes ranked from low to high basal transcript expression level in samples treated with NTC control and is shown in the lower graph as basal gene expression relative to GAPDH expression in the same samples.


crispra-timecourse-crrna-tracrrna

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

crispra-timecourse-crrna-tracrrna

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 calculated using qRT-PCR. 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.


dose-curve-synthetic-crrna-indiv-pools-crispra

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

dose-curve-synthetic-crrna-indiv-pools-crispra

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 measured 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.