A Genome-Wide CRISPR Screen Identifies C18orf32 as a Novel Modulator of PFOA-Induced Cytotoxicity and Lipid Dysregulation

Abstract

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-induced cytotoxicity. 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 increased cellular resistance to PFOA and markedly reduced LD and triacylglycerol (TAG) accumulation in PFOA-exposed cells, suggesting a functional role for C18orf32p (protein) in PFOA-mediated lipid dysregulation. Chemical-comparison experiments showed that C18orf32 KO similarly increased resistance to PFOS and HFPO−DA, but not to rotenone and diquat, indicating that the phenotype was not a generalized resistance to cytotoxic stress but extended across the tested PFAS. Transcriptomic analysis revealed that C18orf32 KO induced broad gene expression changes both in the absence and presence of PFOA exposure, indicating extensive metabolic remodeling. Notably, multiple lipid metabolism-related pathways, including cholesterol metabolism, PPAR signaling, fatty acid metabolism, and peroxisome β-oxidation─known mediators of PFOA-induced hepatotoxicity─were significantly decreased in C18orf32 KO cells regardless of exposure condition, consistent with lipid metabolism changes. Lipidomics analysis further demonstrated a marked reduction in TAG levels in C18orf32 KO cells, accompanied by alterations in several additional lipid species. Collectively, our findings support a functional role for C18orf32p in modulating cellular susceptibility to PFOA-induced lipid dysregulation and hepatotoxicity in human liver cells, with additional evidence suggesting that this role extends to other tested PFAS.