In vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies. However, its efficacy is hindered by multifaceted negative regulatory mechanisms intrinsic to T cells. Here we perform a large-scale high-throughput in vivo double-knockout (DKO) CRISPR screen in human CAR-T cells, and identify multiple DKO gene pairs that are both effective and synergistic. The top pair NR4A1_SOCS3 DKO has multiple favorable immunological features. Whole-transcriptome profiling and in vivo single cell RNA sequencing reveal that NR4A1_SOCS3 DKO specifically upregulated metabolic pathways in CAR-T cells, which was demonstrated to have significant higher metabolic fitness and mitochondria activities. NR4A1_SOCS3 DKO CAR-T or T cells exhibit exceptionally potent anti-tumor efficacy long-term tumor control across different tumor models, without compromising safety features. NR4A1_SOCS3 DKO outperforms other T/CAR-T cell genetic modifications such as REGNASE1 KO, MED12 KO, RASA2 KO, TRAF6 KO and PD1_CTLA4 DKO. These data demonstrate high-throughput DKO screening for rapid discovery of combination targets, and establish NR4A1_SOCS3 as promising joint intracellular checkpoints to engineer high-performance CAR-T therapies against cancer.