Co-transfect or electroporate cells with CRISPRi synthetic sgRNA and CRISPRi dCas9-SALL1-SDS3 mRNA. Then enrich cell populations using fluorescence or puromycin resistance options. This system is best suited for rapid, transient gene repression studies.

Transduce cells with dCas9-SALL1-SDS3 lentiviral particles to generate CRISPRi-ready, stable dCas9-SALL1-SDS3 expressing cells. Then introduce CRISPRi synthetic sgRNA specific to your target gene(s). This system is ideal for screening, extended time point assays or when working with difficult-to-transfect cell types.

CRISPRmod CRISPRi synthetic sgRNAs achieve maximal repression at 48-72 hours post-transfection in both dCas9-KRAB and Cas9-SALL1-SDS3-expressing cells. U2OS cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with pools of pre-designed synthetic sgRNA (25nM) targeting CBX1, HBP1, or SEL1L. Cells were harvested at 24, 48, 72, 96, 120, and 144 hours post-transfection, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression of each target gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC).

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 synthetic sgRNA targeting BRCA1, PSMD7, SEL1L, or ST3GAL4. The pre-designed 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).

CRISPRi transcriptional repression varies by gene but does not appear to be dependent on endogenous expression levels. Data has been compiled from multiple transfections performed in U2OS cells stably expressing integrated dCas9-SALL1-SDS3 with the following matching conditions. Cells were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic sgRNA pools (25 nM) targeting 21 genes with different basal levels of expression. Cells were harvested 72 hours post-transfection, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression of each target gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC). The CRISPRi-mediated target gene repression is shown in the left graph where the genes are ordered from low to high level of basal transcript expression. In the right graph target gene expression is plotted against fragments per kilobase of transcript per million (FPKM), a representation of basal gene expression based on RNA-Seq data from the parental U2OS cell line. In U2OS cells PPIB is expressed at a basal level roughly one hundredfold greater than that of SOX2, but both genes can be repressed to roughly 20-25 percent of their basal expression with CRISPRmod CRISPRi reagents.