Figure 6.

Multiplex targeting of CCNU sensitizers identified through Genome-wide CRISPRi screens. (A) Schematic of genome-wide CRISPRi screens in GBM cells stably expressing dCas9-Zim3. Dual sgRNA libraries were transduced via lentivirus. Parallel screens were performed in the presence of vehicle or CCNU in LN18 as well as T98G cells. Phenotypes of CRISPRi suppression of target genes were quantified using targeted sequencing of integrated sgRNA barcodes. (B) Waterfall plot of all gene targets from genome-wide CRISPRi screens in LN18 (left) and T98G (right) ranking the log2 ratio of sgRNA barcodes in the CCNU vs. vehicle conditions at the endpoint of each screen. Phenotypes are the average across 3 replicate screens. Light gray = non-targeting sgRNA. Dark gray = gene targeting sgRNA. (C) Gene set enrichment analyses of CCNU screens, ranked by the topmost negative enrichment terms. (D) RT-qPCR of BRIP1 or MGMT mRNA normalized to RPLP0 housekeeping gene, in polyclonal LN18 cells following delivery of CRISPRoff mRNA and the indicated combination of sgRNAs. (E) as in (D) but for T98G. (F) Cell viability dose–response curves for CCNU sensitivity in LN18 (left) and T98G (G) polyclonal populations following delivery of CRISPRoff and the indicated combinations of sgRNAs. IC50 is calculated for each condition.