Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

CAR T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction is a common cause of treatment failure. 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 method in a xenograft model of human leukemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered RHOG knockout as 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 patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify effective knockout 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.