CRISPRmod CRISPRa synthetic crRNA arrayed libraries enable one-gene-per-well investigation using high-content assays to answer in-depth biological questions. Further your drug discovery, pathway analysis, or disease progression studies with CRISPR activation screening.
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Highlights of CRISPRa crRNA libraries:
- Available as four individual crRNAs per gene or a pool of four crRNAs to provide highly effective transcriptional activation (see Supporting Data tab)
- crRNA pools provide robust activation while reducing costs, storage, and handling of libraries compared to working with four individual crRNAs
- Algorithm-generated designs (published by Horlbeck, et. al.1) demonstrate robust transcript activation (See References tab)
- Chemically modified CRISPRa crRNAs and tracrRNA provide additional stability against nuclease degradation and improve overall performance
- Conveniently arrayed in 96- or 384-well plates and offered as gene family collections. Echo-qualified 384-well plates are available upon request
Required components for an CRISPRa gene activation experiment using synthetic crRNA:
- A lentiviral expression plasmid or lentiviral particles for dCas9-VPR2; a mammalian codon-optimized S. pyogenes deactivated Cas9 fused to VPR activation domains
- CRISPRa crRNA are also compatible with SunTag technology3
- trans-activating CRISPR RNA (tracrRNA)
|Human CRISPRa crRNA Libraries||# genes (approximate)||Catalog # (Pool/Set of 4)|
|Cell Cycle Regulation||169||GP/GC-103205-xx|
|DNA Damage Response||240||GP/GC-106005-xx|
|G Protein-coupled Receptors||390||GP/GC-103605-xx|
*Druggable genome is made up of: Proteases, Protein Kinases, Phosphatases, Transcription Factors, Ubiquitin Enzymes, GPCRs, Ion Channels and Drug Targets.
Efficient transcriptional gene activation with synthetic crRNA in multiple 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 fold transcriptional activation for each gene was calculated with the Cq method using GAPDH as the reference gene and normalized to a non-targeting control.
CRISPRa gene activation in U2OS cells is observed at 24 hours and peaks at 48-72 hours
U2OS cells stably expressing integrated dCas9-VPR 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 guides 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 reference gene and normalized to a non-targeting control.
CRISPRa crRNAs and pools are highly effective at 25 nM working concentration
A CRISPRa dose curve 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 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 reference gene and normalized to a non-targeting control.
- 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.
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 2573049.
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. PubMed 25307933