CRISPRmod CRISPRa synthetic 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.

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.

CRISPRa synthetic crRNA

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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 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 CRISPRa application page to get an overview of the technology!

Highlights of 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. 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 CRISPRa gene activation using synthetic crRNA

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

How much CRISPRa 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

Efficient gene activation with CRISPRa synthetic crRNA in multiple dCas9-VPR stable cell lines

Efficient transcriptional gene activation

HEK293T, U2OS, MCF 10A, NIH/3T3 stably expressing integrated CRISPRa 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 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.

Pooling of CRISPRa synthetic crRNAs enhances transcriptional activation

Pooling of synthetic crRNAs can enhance transcriptional activation

Individual 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 CRISPRa dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRa synthetic crRNA:tracrRNA targeting EGFR or POU5F1. Four CRISPRa 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 gene activation fold depends on the endogenous gene expression level

Fold activation by CRISPRa varies by gene

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-dCas9-VPR stable cells were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRa 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 RT-qPCR. 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).

Immunofluorescence analysis demonstrates effective CRISPRa gene activation

Immunofluorescence analysis indicates effective CRISPRa gene activation using synthetic 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 CRISPRa 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 gene activation is observed at 24 hours and peaks at 48-72 hours

CRISPRa gene activation in U2OS cells is observed at 24 hours

U2OS-dCas9-VPR stable cells were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRa synthetic crRNA:tracrRNA targeting EGFR, IL1R2, POU5F1 or TFAP2C. Four CRISPRa crRNAs 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 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 dose curve demonstrates highly effective gene activation at 25 nM working concentration

CRISPRa crRNAs and pools are highly effective at 25 nM

U2OS-dCas9-VPR stable cells were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRa 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.