GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE272nnn/GSE272884/primaryOK200Othersynthetic construct Homo sapiensOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE272884GEOGSEfalseIn vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity [crispr_screen]Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies. However, its efficacy against solid tumors is hindered by multifaceted negative regulatory mechanisms intrinsic to T cells. Although single gene screens have been performed in T cells, such studies cannot provide causality for the complex genetic interactions governing T cell function. To systematically discover genetic interactions that are critical for CAR-T cell’s anti-tumor immunity, here, we perform a 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. All five top-scoring DKO hits are validated to promote CAR-T cell activation, cytotoxicity, degranulation, effector cytokine production, and memory formation, and reduce exhaustion. Mechanistically, these DKOs enhance calcium influx, phospho-ERK, NF-κB and NFAT/AP-1 signaling. Among these hits, NR4A1_SOCS3 DKO has multiple favorable immunological features and showcases the most robust phenotypes. NR4A1_SOCS3 DKO CAR-T cells show potent in vivo anti-tumor efficacy and markedly enhanced infiltration as compared to both single gene knockouts, without increase in safety risk. Whole-transcriptome profiling and single cell RNA sequencing of tumor-infiltrating CAR-T cells reveal that NR4A1_SOCS3 DKO specifically enhanced metabolic fitness of CAR-T cells, rendering them exceptionally potent in long-term tumor control in an orthotopic model, superior to both single gene knockouts and the PD1_CTLA4 dual-checkpoint DKO benchmark. These data together demonstrate the efficacy of high-throughput DKO screening to rapidly uncover critical genetic interactions within T cells, and discover NR4A1_SOCS3 as promising joint intracellular checkpoints to engineer high-performance CAR-T therapies against solid tumors.2026/06/17GSE272884GSM8414040GSM8414030GSM8414031GSM8414032GSM8414037GSM8414026GSM8414027GSM8414038GSM8414039GSM8414028GSM8414029GSM8414033GSM8414034GSM8414035GSM84140362467626526272884synthetic construct Homo sapiens