Saronio, G.; Antonini, G.; Jain, A.; Vogel, I.; Teneggi, R.; Neumann, J.; Zoppi, G.; Ding, Y.; Pecoraro, M.; Langner, M.; Mirtschink, P.; Stavrakis, S.; deMello, A.; Geiger, R.

Cytotoxic CD8 T cells kill target cells through brief cell-cell encounters, but pooled genetic screens cannot readily link perturbations in individual T cells to the fate of the target cells they engage. We developed droplet single-cell CRISPR screening to pair individual primary human CD8 T cells with cancer cells, measure rapid target-cell death, and recover sgRNAs from phenotype-defined droplets. Applied across primary T cells from multiple donors, the platform recovered regulators of T cell receptor signaling, synapse formation, granule exocytosis and cytotoxic differentiation, and identified negative regulators of killing, including established inhibitory nodes such as PTEN, RASA2 and FOXO1, together with AFAP1L2 and components of the mTORC1 pathway. Validation across bispecific engager and TCR-engineered settings showed that selected hits modulate target-cell killing across recognition modalities and tumor models. Unexpectedly, perturbation of RPTOR or RHEB enhanced cytotoxic execution while reducing mTORC1 output, increasing AKT phosphorylation, and attenuating anabolic programs. Transient pharmacologic mTORC1 inhibition reproduced this rapid-killing state and improved antitumor activity after adoptive transfer. These results establish an interaction-resolved pooled genetic strategy for mapping cytotoxicity regulators and reveal that transient modulation of mTORC1 can shift T cells from anabolic growth toward rapid cytotoxic execution to enhance antitumor activity.