CRISPRa Controls

Positive and non-targeting controls to ensure successful gene activation

CRISPRmod CRISPRa requires two components be delivered and/or expressed in the cell for efficient transcriptional activation: a CRISPRa-designed guide RNA and a nuclease-deactivated Cas9 (dCas9) fused to the transcriptional activator system (VPR). Insufficient amounts of either component will result in inefficient activation of the target gene, so proper controls should be used to ensure successful gene activation. Visit our CRISPRa applications page to learn more.

Controls should be used for:

  • Optimizing CRISPRa crRNA:tracrRNA transfection conditions
  • Determining the optimal promoter for driving lentiviral dCas9-VPR expression
  • Identifying optimal co-transfection conditions with dCas9-VPR plasmid with either CRISPRa crRNA:tracrRNA or sgRNA plasmid
  • Ensuring experimental consistency and controlling for any possible background effects

 

CRISPR activation controls for gain-of-function experiments

To establish optimal experimental conditions and ensure ongoing successful activation, it is recommended to use a species-specific CRISPRa positive control for a characterized gene target. The CRISPRa positive controls target human or mouse Titin (TTN) or POU class 5 homeobox 1 (POU5F1) genes.

 

Detection of activation

mRNA and protein levels may change to different degrees and with different timing in CRISPRa experiments; this should be taken into consideration when detecting expression levels.

qPCR: Detect changes in mRNA compared to baseline expression levels to ascertain fold increase in transcript expression.

Western blotting: Determine changes in protein level compared to untreated and non-targeting controls

Non-targeting (negative) controls are designed to target no annotated genomic region; therefore, the baseline expression of any given gene should not be altered, and any non-specific effects can be observed.

CRISPRa Controls

CRISPRa synthetic crRNA positive controls
  • Pooled or individual crRNA controls for assessment of optimal experimental conditions for gene activation
CRISPRa synthetic crRNA non-targeting controls
  • Non-targeting controls to evaluate baseline cellular responses to CRISPRa components in the absence of gene target-specific crRNA
CRISPRa lentiviral sgRNA positive controls
  • Control sgRNAs targeting well-characterized genes to determine the effectiveness of your experimental conditions for maximum activation
CRISPRa lentiviral sgRNA non-targeting controls
  • Non-targeting controls to evaluate baseline cellular responses to CRISPRa components in the absence of target-specific sgRNA

 

Additional CRISPRa related products

CRISPRa dCas9-VPR
  • Lentiviral particles, purified plasmid or mRNA that express nuclease-deactivated Cas9 fused to transcriptional activators. When complexed with a guide RNA will trigger an endogenous gene’s expression.
tracrRNA
  • Trans-activating CRISPR RNA (tracrRNA) is a synthetic, HPLC-purified, long RNA required for use with crRNA to form the complex that programs dCas-VPR. It is modified for nuclease resistance.
Efficient transcriptional gene activation with CRISPRa synthetic crRNA:tracrRNA in dCas9-VPR stable cells
Efficient transcriptional gene activation with synthetic crRNA:tracrRNA in dCas9-VPR stable cells

Efficient transcriptional gene activation with CRISPRa 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 CRISPRa synthetic crRNA:tracrRNA (25 nM) targeting POU5F1 and TTN. K562 cells were electroporated with CRISPRa 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 ΔΔCq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.

 

Efficient transcriptional gene activation with CRISPRa lentiviral sgRNA in dCas9-VPR stable cells
Efficient transcriptional gene activation with lentiviral sgRNA in dCas9-VPR stable cells

U2OS, HEK293T, MCF 10A and K562 stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transduced with CRISPRa lentiviral sgRNA particles targeting POU5F1 or TTN at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 4 days prior to analysis with RT-qPCR. The relative expression of each gene was calculated with the ΔΔCq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.