CRISPRi lentiviral sgRNA positive controls will selectively repress a well characterized human gene (either PPIB1 or SEL1L). Positive controls are recommended for optimization and ongoing monitoring of delivery conditions and dCas9-SALL1-SDS3 expression prior to experiments with target gene-specific guide RNAs.
- Select PPIB1 or SEL1L as your positive control gene
- Available as glycerol stocks and high-titer purified lentiviral particles (50 µL at ≥ 1 x 108 TU/mL)
- Ideal for optimization of experimental conditions and confirming successful gene repression
In addition to positive controls, it is recommended to use a CRISPRi lentiviral sgRNA non-targeting control to determine baseline levels of gene expression.
CRISPRi lentiviral sgRNA is well suited for long term repression
U2OS and A549 cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 50,000 cells/well in 24 well plates and transduced with CRISPRi sgRNA lentiviral particles targeting PPIB, SEL1L, BRCA1 or CANX at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 5 days, split into two populations, and allowed to recover for an additional day. At 7 days post-transduction one population was harvested for RT-qPCR analysis. The replicate populations of cells were cultured for 7 additional days before harvest. Total RNA was isolated from harvested plates and relative gene expression was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.