Fig. 5: A genome wide CRISPR-based activation screen identifies molecular modifiers of sensitivity to ATR inhibition in PAX3-FOXO1-expressing ARMS cells.
From: Therapeutic targeting of ATR in alveolar rhabdomyosarcoma
a Schematic representation of the genome wide CRISPRa screen experimental design. b Enrichment score for the GSEA hallmark pathways based on sgRNA enrichment. c Waterfall plot showing the positive robust rank aggregation (RRA) score of sgRNAs in Rh4 cells incubated in the presence of AZD6738 for 9 days compared to DMSO treated cells as analyzed using MAGeCK. FOSB (d, P = 0.014; 5.45 × 10−6; 1.17 × 10−6), FOSL1 (e, P = 9.09 × 10−11; 5.16 × 10−6; 2.19 × 10−7) and FOSL2 (f P = 9.87 × 10−10; 1.49 × 10−7; 1.15 × 10−4) mRNA expression measured using RT-qPCR in Rh30 cells expressing dCas9, lentiMPH and sgRNAs targeting FOSB, FOSL1 or FOSL2 (n = 3). Western immunoblot of FOSB (g) and FOSL1 (h) in Rh30 cells stably expressing dCas9, lentiMPH and sgRNAs targeting FOSB and FOSL1, respectively. Relative cell viability of Rh30 cells stably expressing dCas9, lentiMPH and sgRNAs targeting FOSB (i), FOSL1 (j) and FOSL2 (k) in the presence of varying concentrations of AZD6738. l Western immunoblot of RPA32 phosphorylation at T21 in Rh4 cells expressing sgRNAs targeting FOS family members FOSB, FOSL1 or FOSL2. m Quantification of RPA32 phosphorylation at T21compared to the corresponding non-targeting control, (P = 2.66 × 10−6; 1.78 × 10−4, respectively). All statistical analyses correspond to two-sided student’s t-test; data presented as mean value ± error bars representing standard deviation.
