Genome-wide CRISPR screen identifies ATAD3A promotes acetaminophen-induced liver injury by driving ferroptosis

Abstract Acetaminophen (APAP) overdose is a major cause of acute liver injury, yet effective therapeutic targets remain scarce. This study investigated the genes regulating hepatocyte vulnerability to APAP toxicity and the underlying mechanisms. Genome-wide CRISPR loss-of-function screening was performed in hepatocytes exposed to APAP to uncover candidate regulators. The loss of the ATAD3A gene protected against APAP-induced liver injury. Mechanistically, ATAD3A promoted ferroptosis through two routes: enhancing mitochondrial reactive oxygen species (ROS) release by facilitating VDAC1 oligomerization via the IP3R–HSPA9–VDAC1 complex, and increasing intracellular iron levels by limiting HSPA9-dependent lysosomal degradation of transferrin receptor. Loss of ATAD3A lowered ROS and iron levels and improved hepatocyte survival. In conclusion, genome-wide CRISPR screening identified ATAD3A as a contributor to APAP hepatotoxicity. ATAD3A promotes mitochondrial ROS release and iron accumulation, thereby driving ferroptosis. ATAD3A deficiency protects against APAP-induced liver injury by suppressing ferroptosis.