In vivo CAR-T double knockout screen maps synergistic antitumour gene pairs.

NR4A1–SOCS3 double-knockout CAR-T cells show exceptionally potent antitumour efficacy and long-term tumour control across different tumour models, with higher metabolic fitness and mitochondrial activity.

• Why it matters: CAR-T efficacy in haematological malignancies is limited by multiple intrinsic T-cell negative regulatory mechanisms, creating a need to rapidly find effective synergistic gene combinations.
• What they did: The study used a high-throughput in vivo CAR-T double-knockout CRISPR screen, then applied whole-transcriptome profiling and in vivo single-cell RNA sequencing to characterize NR4A1–SOCS3 DKO CAR-T cells.
• The result: NR4A1–SOCS3 DKO specifically upregulates metabolic pathways, enabling stronger antitumour performance and long-term tumour control without compromising safety features, and it outperforms several benchmark single- and dual-gene modifications.

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