Edit-R CRISPRa crRNA

Predesigned synthetic guide RNA for over-expression of human and mouse genes using CRISPR activation. Just search for your gene! Available as pooled or individual reagents.

Also Available As: Edit-R CRISPRa Lentiviral sgRNA

Genome-wide human and mouse synthetic crRNA reagents are specially designed for CRISPRa. In conjunction with dCas9-VPR expression, you can harness the power of the CRISPR-Cas9 system for activation of your favorite gene.

Enter a common gene identifier and click SEARCH to find products for your gene.

 
Edit-R Human CRISPRa crRNA pool
P-HUMAN-XX-0005 5 nmol - $280.00select
Edit-R Mouse CRISPRa crRNA pool
P-MOUSE-XX-0005 5 nmol - $280.00select
Edit-R Mouse CRISPRa Set of 4 crRNA
PQ-MOUSE-XX-0002 2 nmol - $349.00select
Edit-R Human CRISPRa Set of 4 crRNA
PQ-HUMAN-XX-0002 2 nmol - $349.00select
Edit-R Mouse CRISPRa crRNA
CA-MOUSE-XX-0002 2 nmol - $95.00select
Edit-R Human CRISPRa crRNA
CA-HUMAN-XX-0002 2 nmol - $95.00select

Efficient endogenous gene activation with an easy-to-use synthetic reagent

The CRISPR activation (CRISPRa) system is a unique adaptation of the classical CRISPR-Cas9 gene editing system which utilizes a catalytically deactivated or dead S. pyogenes Cas9 (dCas9) that is fused to one or more transcriptional activators. When paired with a well-designed guide RNA that targets a gene near a promoter region, or trancriptional start site (TSS), it promotes transcriptional activation.

Review our Applications page on CRISPRa to get an overview of the technology!

Highlights of Edit-R CRISPRa crRNA reagents

  • Available as individual reagents or a pool of four crRNAs to provide highly effective transcriptional activation (See Supporting Data tab)
  • Designs from a published algorithm by Horlbeck, et. al.[1] that demonstrates strong levels of gene activation from optimized designs (See References tab)
  • Chemically modified crRNAs provide additional stability against nuclease degradation and improve overall performance
  • For genes with alternative characterized start sites, distinct guide RNA designs are available (labeled P2)

Required components for an Edit-R CRISPRa gene activation experiment using synthetic crRNA:

  • A lentiviral expression plasmid or lentiviral particles for dCas9-VPR[2]; a mammalian codon-optimized S. pyogenes deactivated Cas9 fused to VPR activation domains (also compatible with SunTag technology[3])
  • A chemically synthesized trans-activating CRISPR RNA (tracrRNA) and
  • A predesigned CRISPRa crRNA for the gene of interest

How much crRNA & tracrRNA do I need?

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

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-crrna-individuals-pools-activation

Pooling of synthetic crRNAs can enhance transcriptional activation

crispra-crrna-individuals-pools-activation

Individual Edit-R CRISPRa crRNAs achieve robust target gene activation on their own, but when pooled together in a single reagent, enhanced activation levels can be achieved. 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 or. The pre-designed crRNAs were used either individually or pooled (to a total concentration of 25 nM). Cells were harvested 72 hours post-transfection and the relative gene expression was measured using RT-qPCR. The relative fold transcriptional activation for each gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC).


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

Efficient transcriptional gene activation with synthetic crRNA:tracrRNA in multiple 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 fold transcriptional activation for 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 with low or no basal expression are easier to activate to a robust level, while genes already expressed at high level are more difficult to further over-express. 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 23 genes with different basal level of expression.. Cells were harvested 72 hours post-transfection and the relative gene expression was measured using qRT-PCR. The CRISPRa-mediated fold transcriptional activation is shown in the upper graph where the genes are ordered from low to high level of basal transcript expression in samples treated with NTC control and is shown in the lower graph as basal target gene expression (compared to GAPDH control).


fluorescent-antibody-indicates-crispr-activation-crrna

Immunofluorescence analysis indicates effective CRISPRa gene activation using synthetic crRNAs

fluorescent-antibody-indicates-crispr-activation-crrna

U2OS-dCas9-VPR stable cells were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent (0.2 µL/well) with synthetic crRNA:tracrRNA pool targeting TFAP2C, EGFR, IL1R2 or POU5F1 (25 nM total concentration) or NTC control. 72 hours post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 followed by incubation with target specific primary antibodies and Dylight 550 conjugated secondary antibodies. Nuclei were stained with Hoescht 33342 Merged images are shown for cells treated with the target specific CRISPRa crRNA pools or NTC controls.


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 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.


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 calculated 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.


Edit-R-CRISPRa-workflow-diagram

CRISPRa workflow using Edit-R reagents

Edit-R-CRISPRa-workflow-diagram


References

  1. Horlbeck MA, Gilbert LA, et. al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. 2016 Sep 23;5. pii: e19760. doi: 10.7554/eLife.19760. PubMed 27661255
  2. Chavez A, Scheiman J et. al., Highly efficient Cas9-mediated transcriptional programming Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312. 10.1038/nmeth.3312 PubMed 25730490
  3. Tanenbaum ME, Gilbert LA, et. al., A protein tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159(3):635-646. doi:10.1016/j.cell.2014.09.039.