Figure 7. CRISPRi Strongly Represses Gene Expression of Both Protein-Coding and Non-Coding Genes, Resulting in Reproducible Phenotypes.
(A–C) Cells expressing a negative control sgRNA or an sgRNA targeting SEL1L or B4GALNT1 were incubated with cholera toxin and fractionated to quantify cholera toxin present in the cytosolic and membrane fractions by Western blot. B4GALNT1 repression blocks toxin uptake whereas SEL1L repression prevents toxin retro-translocation from the membrane fraction to the cytosol. (D) Validation of CTx-DTA screen phenotypes with single sgRNA re-test experiments. Data is represented as the mean ± standard deviation of replicates (N=3). (E) CRISPRi knockdown of 13 hit genes (28 sgRNAs; same sgRNAs as 7D) identified in the CTx-DTA screen was quantified by qPCR. The gray shaded region denotes sgRNAs showing at least 90% knockdown for each gene. (F) CRISPRi knockdown of 6 lncRNA genes was quantified by qPCR. 2–3 sgRNAs computationally predicted to target each gene were cloned and transduced into K562 cells expressing dCas9-KRAB. (G) K562 cells expressing dCas9-KRAB were transduced with either a non-targeting sgRNA or an sgRNA targeting the XIST locus (sgXIST-1). The cells were then stained with DAPI and an RNA FISH probe for the XIST transcript. 200 non-apoptotic interphase cells in each condition were scored for XIST RNA coating. XIST is undetectable in cells transduced with sgXIST-1. Scale bar represents 5μm.