CRISPRmod CRISPRi requires two components be delivered and/or expressed in the cell for efficient transcriptional repression: a CRISPRi-designed guide RNA and a nuclease-deactivated Cas9 (dCas9) fused to transcriptional repressors (SALL1 and SDS3). Insufficient amounts of either component will result in inefficient knockdown of the target gene, so proper controls should be used to ensure successful gene repression. Visit our CRISPRi applications page to learn more.
Controls should be used for:
- Optimizing CRISPRi sgRNA transfection conditions
- Determining the optimal promoter for driving lentiviral dCas9-SALL1-SDS3 expression
- Identifying optimal co-transfection conditions with dCas9-SALL1-SDS3 mRNA
- Ensuring experimental consistency and controlling for any possible background effects
CRISPRi controls for loss-of-function experiments
It is recommended to use a CRISPRi positive control for a characterized gene target to establish optimal experimental conditions and ensure ongoing successful gene repression. Our CRISPRi positive controls have the option to target human PPIB1 or SEL1L genes.
Non-targeting (negative) controls are guide RNAs with no complementary target in the annotated human genome, so the baseline expression of any given gene should not be altered. mRNA and protein levels may change over time in CRISPRi experiments; this should be taken into consideration when detecting expression levels. Use qPCR and Western blotting to determine changes in mRNA and protein levels compared to untreated and non-targeting controls to observe gene knockdown.
CRISPRi control reagents
- Pooled or individual sgRNA controls for assessment of optimal experimental conditions for gene repression
- Non-targeting controls to evaluate baseline cellular responses to CRISPRi components in the absence of gene target-specific sgRNA
- Control sgRNAs targeting well-characterized genes to determine the effectiveness of your experimental conditions for maximum repression
- Non-targeting controls to evaluate baseline cellular responses to CRISPRi components in the absence of target-specific sgRNA
Robust gene knockdown with CRISPRi synthetic sgRNA positive controls
|K562 and Jurkat cells were nucleofected with dCas9-KRAB or dCas9-SALL1-SDS3 mRNA (2 µg), and pooled CRISPRi synthetic sgRNAs (5 µM) targeting PPIB and SEL1L via a Lonza 96-well Shuttle system. WTC-11 hiPS cells were nucleofected with dCas9-KRAB or dCas9-SALL1-SDS3 mRNA (1 µg) and pooled CRISPRi synthetic sgRNA (3 µM) targeting PPPIB or SEL1L via a Lonza 96-well Shuttle system. Cells were harvested 72 hours post-nucleofection. Total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative gene expression for each target gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC).|