Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

Paul Datlinger,  Eugenia V. Pankevich, Cosmas D. Arnold, Nicole Pranckevicius, Jenny Lin, Daria Romanovskaia, Moritz Schaefer, Francesco Piras, Anne-Christine Orts, Amelie Nemc, Paulina N. Biesaga, Michelle Chan, Teresa Neuwirth, Artem V. Artemov, Wentao Li, Sabrina Ladstätter, Thomas Krausgruber & Christoph Bock

Nature | 2025 | 10.1038/s41586-025-09507-9

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 failure1. 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-seq2 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.