High-content CRISPR activation screens identify synthetically lethal RNA-based mechanisms to sensitize cancer cells to targeted T cell cytotoxicity.

T cells recognize their target cells through the T cell receptor (TCR). Combining gain-of-function, single-cell and optical high-content screens, we identified RNA-based mechanisms that selectively sensitize target cells to TCR-specific T cell cytotoxicity. First, CRISPR activation screens in melanoma cells identify functionally diverse regulators of TCR-specific cytotoxicity, including SAFB, KHDRBS1, MYC, CD44, WNT3A, WNT1 and others. Expressing sensitizing hits in cancer and virally infected cells restores TCR-specific cytotoxicity. Next, we developed in situ Perturb-seq for optical pooled genetic screens with in situ detection of perturbations and spatial transcriptomic readouts. Perturb-seq and in vivo-in situ Perturb-seq show that the hits converge on shared cell-autonomous and intercellular mechanisms, map gene-environment interactions and reveal that Wnt ligands activate T cells. Introducing a scalable approach to decode gene function at the cell and tissue level, the study uncovered context-specific gene functions to restore targeted T cell-based elimination of dysfunctional cells via synthetically lethal, RNA-based interventions.