CRISPRa lentiviral sgRNA non-targeting controls are recommended as negative controls for experiments using CRISPRa lentiviral sgRNAs in human and mouse cells. They are designed to have a minimum of three mismatches or gaps to all potential PAM-adjacent targets in human, mouse and rat genomes. When using these controls, changes in cellular viability or gene expression likely reflect nonspecific cellular responses that can be used as a baseline for comparison to cells treated with target-specific reagents.
- CRISPRa guide RNA non-targeting control sequences are cloned into the same optimized lentiviral vector as CRISPRa gene targeting sgRNAs
- Proprietary alignment tools used to verify at least three mismatches or gaps to any potential target in human, mouse or rat genomes
- Available as glycerol stock and high-titer purified lentiviral particles
CRISPRa workflow diagram with stable dCas9-VPR expression
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.
Plasmid co-transfection workflow for CRISPRa
Plasmid dCas9-VPR and 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.
Transductions with lentiviral sgRNA particles in different dCas9-VPR cell lines
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.