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

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.