Establish and ensure successful conditions for CRISPR activation experiments.
CRISPR activation controls for gain-of-function experiments
CRISPR activation (CRISPRa) is based on the CRISPR-Cas9 system which requires two components to be delivered and/or expressed in the cell for efficient transcriptional activation: a guide RNA and a nuclease-deactivated Cas9 (dCas9) fused to transcriptional activators, such as dCas9-VPR. Insufficient amounts of either component will result in inefficient target gene expression.
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.
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
- Pooled or individual crRNA controls for assessment of optimal experimental conditions for gene activation
- Non-targeting controls to evaluate baseline cellular responses to CRISPRa components in the absence of gene target-specific crRNA
- Control sgRNAs targeting well-characterized genes to determine the effectiveness of your experimental conditions for maximum activation
- Non-targeting controls to evaluate baseline cellular responses to CRISPRa components in the absence of target-specific sgRNA
- 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.
- 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 synthetic crRNA:tracrRNA in 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 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 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 sgRNA lentiviral 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.