CRISPRmod CRISPRa Synthetic sgRNA Positive Controls

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CRISPRmod CRISPRa Synthetic sgRNA Positive 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 CRISPRa positive controls will selectively activate a well characterized human or mouse gene. 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. 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.

Highlights

Choose from human or mouse Titin (TTN) or POU class 5 homeobox 1 (POU5F1).
Use to determine optimal experimental conditions for efficient gene activation.

How much CRISPRa sgRNA do I need?

This table provides the approximate number of experiments that can be carried out for lipid transfection methods at the recommended sgRNA working concentration (25 nM) in various plate/well formats using either a pool or individual sgRNA. Calculations do not account for pipetting errors.

sgRNA 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	800	160	80	32
5	2000	400	200	80
10	4000	800	400	160

Individual CRISPRa crRNAs achieve robust target gene activation on their own, or when pooled together in a single reagent

tnt-pou5f1-mouse

NIH-3T3 cells stably expressing integrated CRISPRa dCas9-VPR (driven by mCMV promoter) were plated at 10,000 cells/well and transfected using DharmaFECT 1 Transfection Reagent (0.2 uL/well) with CRISPRa synthetic single guide RNA (sgRNA) targeting ttn or pou5f1 genes. Four CRISPRa sgRNAs were used either individually or pooled (to an individual or pooled concentration of 25 nM). Cells were harvested at 72 hours post-transfection and gene expression was assessed using RT-qPCR. Relative fold transcriptional activation for each gene was calculated with the Cq method using beta-actin as the housekeeping gene and normalized to an experiment using non-targeting control sgRNA.

Gene activation using CRISPRa synthetic single guide RNA in dCas9-VPR stable human and mouse cell lines

stable-human-mouse-controls

Human U2OS or mouse NIH-3T3 cells stably expressing integrated CRISPRa dCas9-VPR (driven by hEF1a promoter in human and mCMV promoter in mouse cells) were plated at 10,000 cells/well and transfected using DharmaFECT 1 Transfection Reagent (0.2 uL/well) with 25 nM individual or pooled CRISPRa synthetic single guide RNA (sgRNA) targeting either human (TTN or POU5F1) mouse (pou5f1 or ttn) gene targets. Cells were harvested at 72 hours post-transfection and gene expression was assessed using RT-qPCR. Relative fold transcriptional activation for each gene was calculated with the Cq method using beta-actin as the housekeeping gene and normalized to an experiment using non-targeting control sgRNA.

sgRNA dose response curve demonstrates effective gene activation at 25 mM sgRNA concentration

pou5f1-il1r2-titration

U2OS-dCas9-VPR stable cells were plated at 10,000 cells/well and transfected using 0.2 uL/well DharmaFECT 1 Transfection Reagent with CRISPRa synthetic single guide RNA (sgRNA) targeting POU5F1 or IL1R2 at six concentrations (0, 1, 5, 10, 25 and 100 nM). At 72 hours post-transfection, cell lysates were used to generate total cDNA, from which target gene expression was assessed using RT-qPCR. Relative expression of each gene was calculated with the Cq method using beta-actin mRNA as the housekeeping gene and normalized to a non-targeting control. Data points were fit to a one phase exponential decay equation.

Robust CRISPRa gene activation peaks between 24 to 72 hours

U2OS-dCas9-VPR stable cells were plated at 5,000 cells/well and transfected using 0.2 uL / well DharmaFECT 1 Transfection Reagent with 25 nM CRISPRa synthetic single guide RNA (sgRNA) targeting POU5F1, IL1R2, or non-targeting control (NTC). Transfected cells were passaged every 2-3 days to avoid overconfluency. Cells were harvested at 72 hours post-transfection and gene expression was assessed using RT-qPCR. Relative fold transcriptional activation for each gene was calculated with the Cq method using beta-actin as the housekeeping gene and normalized to an experiment using non-targeting control sgRNA.