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Background Gamma delta (γδ) T cells possess unique innate and adaptive immune properties, notably recognizing stress-induced antigens in an MHC-unrestricted manner, unlike αβ T cells. To fully harness their therapeutic potential for cancer immunotherapy, uncovering the molecular mechanisms that govern cancer cell sensitization and resistance to γδ T cell-mediated killing is essential. γδ T cells recognize BTN2A1-BTN3A1 heterodimer complex that is activated by phosphoantigens abundantly produced by tumor cells. Here, MEDiC employed a genome-wide loss-of-function CRISPR screen to identify tumor-intrinsic genes/pathways that regulate γδ T cell-mediated cytotoxicity with and without BTN3A1 activation.Methods Stable Cas9-expressing MDA-MB-231, SKOV3, and MDA-MB-468 cell lines were transduced with a lentiviral CRISPR library comprising 60,000 sgRNAs targeting 20,000 genes. Pooled cells were then exposed to γδ T cell treatment either alone or in combination with an agonistic BTN3A1 antibody at a concentration that achieved 20–40% growth inhibition relative to controls. Next-generation-sequencing was performed to quantify sgRNA abundance across conditions. Additionally, stable knockout (KO) MDA-MB-231 cell lines for three of the top resistor genes were generated and evaluated for growth under baseline and co-culture conditions.Results Quality control confirmed robust library coverage, and sequencing depth. Data analysis identified sensitizing and resistance factors based on γδ T cell + antibody immune effect score and corresponding p values. Known resistors and senstizers of cancer cell-γδ T cell interactions were identified as few of the top hits, such as, BTN2A1, IFNγ pathway genes, FDPS, and HLA-E. Validation experiments revealed that KO of candidate genes: YTHDF2, EED, or MED12 did not affect baseline cell growth. However, upon co-culture with γδ T cells—with or without BTN3A1 antibody—KO cell lines showed significant reduction in cell growth relative to controls. Notably, the data suggests a distinct role for YTHDF2 in tumor evasion of BTN3A1-dependent γδ T cell killing, while EED may play roles in evading both BTN3A1-dependent and -independent cytotoxicity. Lastly, MED12 may play a role only in BTN3A1-independent killing mechanisms.Conclusions Here, we report a genome-wide CRISPR screening platform that uncovers resistor and sensitizer genes modulating solid tumor –γδ T cell interactions and cytotoxicity, distinct from previously reported studies 1 using a hematological cancer cell line with Zolenderate-enhanced γδ T cell interaction. Furthermore, single gene validation studies confirmed the screening results and uncovered YTHDF2 as a key genetic factor in evading BTN3A1-dependent killing. These findings provide insights that may inform the optimization of γδ T cell-based solid tumor therapeutic strategies.Acknowledgements We would like to thank Bristol Myers Squibb, Princeton, NJ for providing reagents and funding for this study.Reference Mamedov, Murad R., et al. ‘CRISPR screens decode cancer cell pathways that trigger γδ T cell detection.’ Nature. 2023;621:7977:188–195.