Fig. 4: A CRISPR-activation screen identifies RAC3 in prion-induced ferroptosis.
A Schematic of a whole-genome CRISPR-activation depletion screen. HT-1080 conditionally expressing PrPC (PRNPtet) were transduced with a synergistic activation mediator pooled human library and dCas9-VP64 lentiviruses. PrPC expression was induced for 3 days with doxycycline or vehicle, followed by treatment with 500 nM IKE, resulting in a viability loss of 5–10%. Normalized guide frequencies amplified from viable cells in sensitized PrPC cells were scored against (-)doxycycline control cells to identify enriched or depleted guides. B Fishtail plot of prion-facilitated ferroptosis by differential CRISPRa guide representation. Effect size is determined by the average PRNPtet (+)doxycycline / (−)doxycycline ratio. Depleted guides (Effect size <1) are positive regulators of PrPC-facilitated ferroptosis. C Inverse relationship of RAC3 and PRNP in PrPC OE or RAC3 OE, respectively, by qPCR and Western blot. Relative mRNA expression is shown as mean ± SD of n = 3 technical replicates. D Gene effect characterized by principal component analysis. CRISPR gene effect (individual gene knockout effect on viability and growth, scaled for whole-genome libraries) in 1095 cell lines is displayed in two dimensions. The distance between two genes in the scatter plot can be interpreted to have similar or inverse effects on cellular processes across individual cell lines, relative to all other cell lines. Significance was determined by two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.
