Prime editing enables isogenic modeling of PNPLA3-I148M-associated lipotoxicity in hepatocytes.

The PNPLA3-I148M (rs738409 C>G) variant is a well-established genetic determinant of hepatic steatosis, fibrosis, and hepatocellular carcinoma. However, its precise functional impact on hepatocyte physiology remains incompletely understood. Here, we employed a GFP-linked all-in-one prime editing system to generate isogenic HepG2 clones representing all PNPLA3 genotypes in an identical genetic background. Following palmitic acid (PA) treatment, we evaluated genotype-specific responses to lipotoxic stress. Lipid droplet accumulation and cell viability assays showed that clones harboring the G allele (GG, GC) exhibited enhanced lipid storage and reduced sensitivity to PA-induced toxicity compared to wild-type (CC) clones. Transcriptomic profiling revealed distinct expression patterns among genotypes, consistent with differential regulation of lipid handling and stress adaptation programs. Heterozygous clones displayed partially intermediate phenotypes, suggesting allele-associated effects of PNPLA3-I148M on hepatocellular lipotoxic responses. This study demonstrates that prime editing enables a precise approach for modeling disease-associated genetic variants in human hepatocytes and provides a platform to investigate genotype-associated cellular responses. By combining precise genome editing with human cell modeling, this study establishes a useful system for dissecting PNPLA3-I148M-related genotype-phenotype relationships and for future mechanistic studies of genetically defined liver disease phenotypes.