Panel a depicts a schematic diagram of the experimental workflow, showing the steps involved in the CRISPR knockout screen, including the treatment of cells with RSL3 and the subsequent analysis of guide RNA abundance. Panel b and Panel c show volcano plots representing the gene-level log2 fold changes and Robust Rank Aggregation scores for chronic and acute ferroptosis treatments, respectively. Genes with significantly depleted or enriched guide RNAs are highlighted, with the top 10 significant genes labeled. Panel d is a Venn diagram showing the overlap of genes with guide RNAs depleted after chronic RSL3 treatment and those enriched in the dead cell population after acute RSL3 treatment. Panel e is a schematic representation illustrating the regulation of coenzyme Q and cholesterol synthesis by the SCAP-SREBP pathway. Panel f and Panel g are bar graphs showing the relative gene expression levels of SREBP-target genes in cyto-STARD7 cells and PARL knockout cells, respectively, with error bars representing the 95 percent confidence interval and p-values indicated above the graphs.
Genome-wide CRISPR KO screen to identify pathways regulating CoQ distribution. (a) Ferroptosis-resistant HeLa cells, cyto-STARD7, stably expressing Cas9 were infected with the genome-wide VBC library (each gene targeted by five guide RNAs). The cells were then selected with G418 and subjected to two different types of treatments (n = 2 biological replicates). First, for chronic ferroptosis treatment, cells were cultured with DMSO or 100 nM RAS-selective lethal 3 (RSL3) for 2 wk, after which they were collected, and gDNA was isolated for PCR amplification of genome-integrated gRNAs followed by deep sequencing. Second, for acute treatment, cells were treated with 500 nM RSL3 for 48 h. After treatment, cells were trypsinized, stained with a live–dead stain, and sorted into gates indicating live and dead cells. gDNA was isolated from both the live and dead cell pellets, and gRNA abundance was analyzed by deep sequencing. (b and c) The x-axis shows gene-level LFCs; the y-axis shows Robust rank aggregation (RRA) scores for b chronic and c acute ferroptosis treatment. Genes with guide RNAs significantly depleted (b) or enriched (c) are highlighted with red circles. The top 10 significant genes are labeled in the Volcano plots. A gene was considered a positive hit if FDR <0.05. (d) Venn diagram showing 12 genes with guide RNAs depleted after chronic RSL3 treatment and 81 genes with guide RNAs enriched in the dead cell population after acute RSL3 treatment. Three genes overlap between the two screens: SCD, FSP1, and SCAP. (e) Schematic representation showing that both CoQ and cholesterol synthesis are regulated by the SCAP–SREBP pathway. Inhibition of SCAP prevents the recruitment of the SCAP–SREBP complex to the Golgi, inhibiting the transcription of SREBP target genes, including HMGCR, a rate-limiting enzyme in the mevalonate pathway, thereby blocking both CoQ and cholesterol synthesis. The scheme was created using BioRender. (f and g) Relative gene expression levels of SREBP-target genes, including SCD, SQLE, HMGCR, and INSIG1, were calculated using the ΔΔCt method with TaqMan probes. HPRT was used as the reference gene. Results were normalized to WT HeLa cells, and the expression of these genes is shown in cyto-STARD7 cells (f) and PARL−/− cells (g) (n = 4 biological replicates). The error bars represent the 95% confidence interval. P values are shown above the graphs. SQLE, squalene epoxidase; INSIG1, insulin-induced gene 1; PARL, presenilin-associated rhomboid-like protein; GPX4, glutathione peroxidase 4.