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Predesigned synthetic and lentiviral reagents for gene activation
CRISPR activation (CRISPRa) is a powerful method for overexpressing genes for functional analysis. In conjunction with nuclease-deactivated Cas9 (dCas9) fused to activator domains, these guide RNAs offer an efficient method for targeted transcriptional activation.
Offering coverage of the human and mouse genomes, these synthetic RNAs are modified for nuclease resistance and available as pools of four or individual crRNA reagents.
Lentiviral particles or glycerol stocks of single guide RNA are predesigned for genome-wide coverage of human and mouse genes in an optimized vector backbone.
All Edit-R CRISPRa guide RNAs are predesigned with the algorithm described by Horlbeck et. al., who performed a systematic study of protospacer-adjacent motif (PAM) sites that are adjacent to transcriptional start sites (TSS) to identify features that contribute to CRISPRa activity.
In addition to expression of an S. pyogenes catalytically
deactivated or dead Cas9 (dCas9) fused to activation domains, such as dCas9-VPR or SunTag-VP64, the CRISPR-Cas9 system requires a specific RNA moiety which complexes with the dCas9-VPR and targets a region adjacent to a gene’s transcriptional start site (TSS). These guide RNAs take two forms:
Offering coverage of the human and mouse genomes, these synthetic RNAs are modified for nuclease resistance and available as pooled or individual crRNA reagents.
Edit-R trans-activating CRISPR RNA (tracrRNA) is a synthetic, HPLC-purified, long RNA required for use with Edit-R crRNA to form the complex that programs dCas-VPR. It is modified for nuclease resistance.
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
Upload your own gene list to customize and order plates of predesigned crRNA for CRISPRa studies across tens or thousands of genes
Lentiviral particles or glycerol stocks of vector-based single guide RNA are predesigned for genome-wide coverage of human and mouse genes
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
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 in U2OS cells stably expressing integrated dCas9-VPR using different Edit-R guide RNA types 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 ΔCq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
Transfection reagent-mediated delivery or electroporation can result in efficient target gene activation when using Edit-R plasmids expressing dCas9-VPR and a single guide RNA (sgRNA) targeting the transcriptional start site of your gene of interest. Synthetic crRNA:tracrRNA is not recommended for co-transfection with plasmid for CRISPRa.