CRISPRi synthetic sgRNA non-targeting controls are designed and recommended for use as negative controls for transcriptional repression experiments using CRISPRi sgRNA. These non-targeting controls will engage the dCas9-SALL1-SDS3 complex, but will not target any PAM-adjacent sites in the human genome. Any observed alteration in gene expression levels or viability in cells treated with these controls can be used as a baseline response of the cells to the dCas9-SALL1-SDS3 complex for comparison to those treated with target-specific CRISPRi synthetic sgRNA.
In addition to non-targeting controls, it is recommended to use a CRISPRi synthetic positive control to optimize experimental conditions for efficient transcriptional repression.
Individual CRISPRmod CRISPRi sgRNAs achieve robust target gene repression independently, but when pooled together in a single reagent, display enhanced repression levels. U2OS cells stably expressing integrated dCas9-SALL1-SDS3 were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with CRISPRi synthetic sgRNA targeting BRCA1, PSMD7, SEL1L, or ST3GAL4. CRISPRi sgRNAs were used either individually or pooled (to a total concentration of 25 nM). Cells were harvested 72 hours post-transfection, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression for each gene was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to non-targeting control (NTC).