{"database":"GEO","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE272nnn/GSE272886/"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"omics_type":["Transcriptomics"],"species":["Homo sapiens"],"gds_type":["Expression profiling by high throughput sequencing"],"full_dataset_link":["https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE272886"],"repository":["GEO"],"entry_type":["GSE"],"additional_accession":[]},"is_claimable":false,"name":"In vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity [scRNA-seq]","description":"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.","dates":{"publication":"2026/06/17"},"accession":"GSE272886","cross_references":{"GSM":["GSM8414091","GSM8414092","GSM8414090","GSM8414089"],"GPL":["24676"],"GSE":["272886"],"taxon":["Homo sapiens"]}}