In vivo CRISPR screens identify dual functions of DNMT3A in mediating tumor immune evasion.

Cancer immunotherapy has achieved remarkable clinical efficacy across multiple cancers but remains ineffective for most patients, and the mechanisms underlying resistance remain incompletely understood. Here, we perform in vivo CRISPR-Cas9 screens in female mice and identify DNA methyltransferase 3 A (DNMT3A) as a key driver of tumor immune evasion through both methyltransferase-dependent and -independent mechanisms. DNMT3A represses TNFRSF1A and TNFRSF1B expression, enabling tumor cells to evade TNFα-induced killing. Independently of its methyltransferase activity, DNMT3A interacts with NFYA to activate cholesterol biosynthesis, leading to zymosterol accumulation in tumor interstitial fluid. Zymosterol impairs SREBP2 cleavage and promotes HMGCR degradation in CD8⁺ T cells, thereby suppressing cholesterol biosynthesis and compromising their antitumor effector functions. Targeting DNMT3A with antisense oligonucleotides overcomes resistance to immune checkpoint blockade. Collectively, these findings establish DNMT3A as an integrated epigenetic and metabolic checkpoint in tumor immune evasion and a potential target for immunotherapy.