Edit-R CRISPRa Lentiviral sgRNA Positive Controls


Validated Edit-R CRISPRa Lentiviral sgRNA Positive Controls for optimization of experimental conditions and quantification of gene activation efficiency.

Edit-R CRISPRa Lentiviral sgRNA Positive controls are designed to specifically activate the human and mouse Titin (TTN) or POU class 5 homeobox 1 (POU5F1) genes. It is recommended that positive controls always be used to optimize experimental conditions and included in every gene activation experiment to confirm successful delivery and dCas9-VPR activity.

The level of activation observed 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.

Highlights

  • Species-specific lentiviral sgRNAs targeting the TTN and POU5F1 genes
  • Available as glycerol stocks and high-titer purified lentiviral particles (50 µL at ≥ 1 x 108 TU/mL)
  • Ideal for optimization of experimental conditions and confirming successful gene activation
  
HazardousNo
Shipping ConditionDry Ice
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-80 C
Edit-R-CRISPRa-workflow-diagram

Edit-R CRISPRa workflow diagram with stable dCas9-VPR expression

Edit-R-CRISPRa-workflow-diagram

CRISPR activation workflow with lentiviral dCas9-VPR and synthetic crRNA:tracrRNA (left) or Lentiviral expressed sgRNA (right) as a purified lentiviral particle or plasmid prepared from glycerol stock.


CRISPRa-co-transfection-workflow-diagram

Plasmid co-transfection workflow for CRISPRa

CRISPRa-co-transfection-workflow-diagram

Plasmid dCas9-VPR and Edit-R CRISPRa sgRNA plasmid can be co-transfected or co-electroporated into cells to achieve transcriptional activation. This method avoids lentiviral incorporation into the genome, but still provides the ability to enrich for transfected cells with antibiotics selection.


CRISPRa_lentiviral_sgRNA_positive_controls

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

CRISPRa_lentiviral_sgRNA_positive_controls

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 ∆∆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.


edit-r-crispra-guide-rna-types-dcas9-vpr-cells

Comparable activation is achieved with different CRISPRa guide RNA types

edit-r-crispra-guide-rna-types-dcas9-vpr-cells

CRISPRa in U2OS cells stably expressing integrated dCas9-VPR was compared using different guide RNAs and delivery methods. CRISPRa using synthetic crRNA:tracrRNA: Cells were plated at 10,000 cells/well and were transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA (25 nM) targeting ASCL1, EGFP, POU5F1 and TTN genes. Cells were harvested 72 hours post-transfection and the relative gene expression was calculated using qRT-PCR. The relative expression of each gene was ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control. CRISPRa with lentiviral sgRNA transduction: Cells were plated at 10,000 cells/well and were transduced with CRISPRa sgRNA lentiviral particles targeting ASCL1, EGFR, 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 ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control. CRISPRa with lentiviral sgRNA plasmid transfection: cells were plated at 10,000 cells/well and transfected with CRISPRa sgRNA plasmids (100 ng) targeting ASCL1, EGFR, POU5F1 or TTN using DharmaFECT kb Transfection Reagent. Cells were harvested 72 hours post-transfection (without puromycin selection) and the relative gene expression was calculated using qRT-PCR. The relative expression of each gene was ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.