In vivo genome-wide CRISPR screens in human T cells to enhance T cell therapy for solid tumors

Large-scale CRISPR screening in human T cells holds significant promise for identifying genetic modifications that can enhance cellular immunotherapy. However, many genetic regulators of T cell performance in solid tumors may not be readily revealed in vitro. In vivo screening in tumor-bearing mice offers greater physiological relevance but has historically been limited by low intratumoral T cell recovery. Here, we developed a new model system that achieves substantially higher human T cell recovery from tumors, enabling genome-wide in vivo screens with relatively small numbers of mice. Tumor-infiltrating T cells in this model exhibit hallmarks of dysfunction compared to matched splenic T cells, creating an ideal context for screening for genetic modifiers of T cell activity in the tumor microenvironment. Using this platform, we performed two genome-wide CRISPR knockout screens to identify genes regulating T cell intratumoral abundance and effector function, as measured by IFN-γ production. The intratumoral abundance screen uncovered the P2RY8-Gα13 GPCR signaling axis as a negative regulator of human T cell infiltration into tumors. The effector function screen identified GNAS as a key regulator of T cell dysfunction in tumors. Its protein product, Gαs, acts as a convergent signaling node downstream of multiple GPCRs that sense different suppressive ligands. Targeted GNAS knockout rendered T cells resistant to multiple suppressive cues and significantly improved therapeutic performance across diverse solid tumor models in both CAR and TCR systems. Moreover, combinatorial knockout of P2RY8 and GNAS further enhanced overall tumor control, demonstrating that complementary screens with distinct in vivo readouts can nominate gene targets addressing orthogonal barriers, and that combining such edits can improve therapeutic potency. This flexible and scalable platform can be adapted to diverse tumor models and screening modalities, enabling systematic discovery of genetic strategies to equip T cell therapies for solid tumors.