Identification of Functional Genetic Components modulating Cellular Toxicity Response to PFOS using Genome-wide CRISPR Screens
Per- and polyfluoroalkyl substances (PFAS) are a group of chemicals that pose significant health and environmental risks due to their persistence and widespread use. These substances, known for their strong chemical bonds, are linked to various health issues such as liver damage, developmental disorders, and immune dysfunction. However, the molecular mechanisms behind their toxicity are not fully understood. Although several studies have identified molecular responses to PFAS exposure, there remains considerable uncertainty about the underlying mechanisms leading to PFAS-associated health effects. To help bridge this gap, we conducted genome-wide CRISPR-based screens in HepG2/C3A human liver cells to identify genes and pathways influencing cellular toxicity upon PFAS exposure. We focused on perfluorooctane sulfonate (PFOS), a widely used and studied PFAS compound with known toxic effects, yet unclear molecular mechanisms underlying PFOS-induced adverse outcomes. Using a genome-wide CRISPR knockout library targeting 18819 genes, we identified 340 candidate genes which modulate the cellular toxicity of PFOS when disrupted. Pathway and enrichment analyses of the corresponding gene products suggest a role for cell cycle, DNA damage response, and Wnt/?-catenin pathways in mediating PFOS cytotoxicity, while further gene-phenotype association analysis using the comparative toxicogenomics database indicated enrichment of gene products previously associated with liver disease and neoplasia. We carried out individual targeted disruption of two candidate genes, SLC6A9 and CPSF2, in HepG2/C3A to confirm their roles in resistance to PFOS exposure. Additionally, molecular docking analysis predicts that PFOS may bind directly to GlyT1, the SLC6A9 gene product. Lastly, cross-species relevance analysis using gene targets identified in our screens suggested that molecular pathways of PFOS toxicity are highly conversed in several environmentally relevant species. Overall design: Genome-wide CRISPR screen experiments using HepG2/C3A cells followed by sgRNA abundance profiling in control and PFOS exposure (192 uM) samples after 10 doubling times (= at Day 18)