Journal: bioRxiv
Article Title: Genome-wide CRISPR screens identified C18orf32 as a novel regulator of lipid metabolism that mediates PFOA-induced toxicity
doi: 10.64898/2025.12.23.696201
Figure Lengend Snippet: (A) Schematic overview of the genome-wide CRISPR/Cas9 loss-of-function screen. HepG2/C3A cells stably expressing Cas9 were transduced with the MiniLibCas9 sgRNA library (37,843 sgRNAs; MOI = 0.3) and selected with puromycin. Cells were then split into control and PFOA-exposed groups and cultured for ∼10 doublings. PFOA treatment (IC₅ concentration) selectively depleted sgRNAs from sensitive gene knockouts and enriched sgRNAs from resistant gene knockouts. Genomic DNA was isolated, sgRNA cassettes were PCR-amplified, and next-generation sequencing (NovaSeqX) was performed. sgRNA abundance was quantified and analyzed using MAGeCK to rank gene-level enrichment or depletion. MOI, Multiplicity of Infection (MOI); IC 25 , Inhibitory Concentration of 25 %. (B) Schematic depiction of bioinformatics flow to identify candidates in the screen: sensitive genes (blue), whose loss increases PFOA toxicity, and resistant genes (red), whose loss confers resistance to PFOA. (C) Volcano plot showing gene-level log₂ (fold-change) versus –log(p-value) from MAGeCK analysis. A total of 140 sensitive genes and 179 resistant genes were significantly altered under PFOA exposure. C18orf32 is highlighted as the top candidate resistant gene. (D) Ranked −log(αRRA) resistant gene scores from the PinAPL-Py analysis. Each point represents one gene in the CRISPR library. C18orf32 (red) ranks among the most significant resistant hits, well above the distribution of background or non-targeting control guides.
Article Snippet: Cytostasis/Cytotoxicity of PFOA (purity≥95%, Santa Cruz Biotechnology-sc250662, CAS# 335-67-1) exposure in HepG2/C3A cells exposed to a range of nominal concentrations (0–400 μM) in triplicate for 6 days was assessed by measuring ATP levels using the CellTiter-Glo2.0 cell viability assay kit (Promega, Madison, WI) following the manufacturer’s instruction and our previous study.
Techniques: Genome Wide, CRISPR, Stable Transfection, Expressing, Transduction, Control, Cell Culture, Concentration Assay, Isolation, Amplification, Next-Generation Sequencing, Infection
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