Genome-wide CRISPR screens identified C18orf32 as a novel regulator of lipid metabolism that mediates PFOA-induced toxicity

Perfluorooctanoic acid (PFOA) remains a public health concern due to its persistence in the environment, despite global production bans. Epidemiological, animal, and in vitro studies have consistently linked PFOA exposure to hepatotoxicity characterized by lipid dysregulation; however, the molecular mechanisms underlying these adverse effects in humans remain poorly understood. To address this gap, we employed an integrated functional toxicogenomics framework combining CRISPR screening (for gene target identification), target validation, and assessment of target-derived cellular and molecular phenotypes. From genome-wide CRISPR screens in HepG2/C3A human liver cells, we identified 319 candidate genes (140 sensitive and 179 resistant) that modulate PFOA toxicity. Among these, C18orf32--encoding a lipid droplet (LD)-associated protein and the top resistant candidate--was selected for further mechanistic investigation based on its potential functional involvement in lipid metabolism. Targeted knockout (KO) of C18orf32 conferred marked cellular resistance to PFOA, accompanied by reduced LD and Triglyceride accumulation in cells under the exposure condition, suggesting a functional role of C18orf32p in lipid dysregulation. To elucidate molecular mechanisms underlying the cellular phenotypes, we conducted systematic transcriptomics profiling of wild-type (WT) and C18orf32 KO cells in the absence and presence of PFOA exposure. C18orf32 KO caused extensive gene expression reprogramming, with over one-third of coding genes differentially expressed. Notably, a range of lipid metabolism pathways including cholesterol metabolism, PPAR signaling, fatty acid metabolism, and peroxisome {beta}-oxidation--known mediators of PFOA-induced hepatotoxicity--were significantly downregulated in C18orf32 KO cells, contrasting to upregulation of these pathways in PFOA exposed WT cells. Collectively, our results identify C18orf32p as a previously unrecognized regulator of hepatic lipid metabolism and a key genetic determinant of PFOA-induced lipid dysregulation and hepatotoxicity in humans.