Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency

Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12 , ARIH2 , and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo . CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced metabolic fitness, increased secretion of proinflammatory cytokines, and expansion of cytotoxic NK cell subsets. Through high-content genome-wide CRISPR screening in NK cells, this study reveals critical regulators of NK cell function and provides a valuable resource for engineering next-generation NK cell therapies with improved efficacy against cancer. • Genome-wide CRISPR screens uncover critical regulators of NK cell function • Orthogonal screens reveal targets to overcome NK cell dysfunction in the TME • Knockout of MED12 , CCNC , or ARIH2 enhances NK cell antitumor potency • Dual KO of ARIH2 and CCNC in CAR-NK cells improves in vivo antitumor efficacy Biederstädt et al. conduct genome-wide CRISPR screens in primary human NK cells, identifying MED12 , ARIH2 , and CCNC as critical regulators of antitumor activity. Targeted knockout of these genes enhances CAR-NK cell cytotoxicity, cytokine release, and metabolic fitness, providing a mechanistic framework for developing next-generation NK cell therapies against treatment-resistant malignancies.