GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE272nnn/GSE272885/primaryOK200TranscriptomicsHomo sapiensExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE272885GEOGSEfalseIn vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity [bulk RNA-seq]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.2026/06/17GSE272885GSM8414084GSM8414062GSM8414085GSM8414063GSM8414041GSM8414042GSM8414086GSM8414064GSM8414065GSM8414043GSM8414087GSM8414080GSM8414081GSM8414060GSM8414082GSM8414083GSM8414061GSM8414048GSM8414049GSM8414088GSM8414066GSM8414044GSM8414045GSM8414067GSM8414068GSM8414046GSM8414069GSM8414047GSM8414051GSM8414073GSM8414074GSM8414052GSM8414075GSM8414053GSM8414054GSM8414076GSM8414070GSM8414071GSM8414072GSM8414050GSM8414059GSM8414077GSM8414055GSM8414078GSM8414056GSM8414057GSM8414079GSM841405824676272885Homo sapiens