Systematic discover of CRISPR-boosted CAR T cell immunotherapies

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction remains a common cause of treatment failure[1](https://www.nature.com/articles/s41586-025-09507-9#ref-CR1 "Ruella, M., Korell, F., Porazzi, P. & Maus, M. V. Mechanisms of resistance to chimeric antigen receptor-T cells in haematological malignancies. Nat. Rev. Drug Discov. 22, 976–995 (2023)."). Here we present CELLFIE, a CRISPR screening platform for enhancing CAR T cells across multiple clinical objectives. We performed genome-wide screens in human primary CAR T cells, with readouts capturing key aspects of T cell biology, including proliferation, target cell recognition, activation, apoptosis and fratricide, and exhaustion. Screening hits were prioritized using a new in vivo CROP-seq[2](https://www.nature.com/articles/s41586-025-09507-9#ref-CR2 "Datlinger, P. et al. Pooled CRISPR screening with single-cell transcriptome read-out. Nat. Methods 14, 297–301 (2017).") method in a xenograft model of human leukaemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered that RHOG knockout is a potent and unexpected CAR T cell enhancer, both individually and together with FAS knockout, which was validated across multiple in vivo models, CAR designs and sample donors, and in patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify synergistic gene pairs and saturation base-editing screens to characterize RHOG variants. In summary, we discovered, validated and biologically characterized CRISPR-boosted CAR T cells that outperform standard CAR T cells in widely used benchmarks, establishing a foundational resource for optimizing cell-based immunotherapies.

Main

CAR T cell therapy is a groundbreaking advance in cancer treatment and a convincing proof of concept for genetically engineered cells as therapeutics. Beyond clinically approved products for blood cancers[3](https://www.nature.com/articles/s41586-025-09507-9#ref-CR3 "Maude, S. L. et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N. Engl. J. Med. 378, 439–448 (2018)."),[4](https://www.nature.com/articles/s41586-025-09507-9#ref-CR4 "Neelapu, S. S. et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N. Engl. J. Med. 377, 2531–2544 (2017)."), CAR T cells hold great potential for solid tumours[5](https://www.nature.com/articles/s41586-025-09507-9#ref-CR5 "Choi, B. D. et al. Intraventricular CARv3-TEAM-E T cells in recurrent glioblastoma. N. Engl. J. Med. 390, 1290–1298 (2024)."),[6](https://www.nature.com/articles/s41586-025-09507-9#ref-CR6 "Bagley, S. J. et al. Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results. Nat. Med. 30, 1320–1329 (2024)."), autoimmune diseases[7](https://www.nature.com/articles/s41586-025-09507-9#ref-CR7 "Müller, F. et al. CD19 CAR T-cell therapy in autoimmune disease — a case series with follow-up. N. Engl. J. Med. 390, 687–700 (2024).") and tissue regeneration[8](https://www.nature.com/articles/s41586-025-09507-9#ref-CR8 "Rurik, J. G. et al. CAR T cells produced in vivo to treat cardiac injury. Science 375, 91–96 (2022)."). However, clinical trials have uncovered major obstacles to CAR T cell efficacy[9](https://www.nature.com/articles/s41586-025-09507-9#ref-CR9 "Larson, R. C. & Maus, M. V. Recent advances and discoveries in the mechanisms and functions of CAR T cells. Nat. Rev. Cancer 21, 145–161 (2021)."), including limited T cell fitness and proliferation, insufficient T cells in pretreated patients with cancer, exhaustion from chronic CAR stimulation, immunosuppressive and nutrient-poor tumour environments, and fratricide when CAR T cells acquire antigen from their target cells via trogocytosis[10](https://www.nature.com/articles/s41586-025-09507-9#ref-CR10 "Hamieh, M. et al. CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature 568, 112–116 (2019).").