Genome-wide CRISPR screens in primary human natural killer cells identify countermeasures against immunosuppressive environment

Natural killer (NK) cells are promising effectors for cancer immunotherapy, but their efficacy is limited by immunosuppressive tumor microenvironments. To uncover strategies for enhancing NK cell function, we establish a CRISPR loss-of-function screening platform for primary human NK cells by combining BaEVRless-pseudotyped lentiviral transduction of sgRNA libraries with Cas9 protein electroporation. This platform enables genome-scale interrogation of gene function in non-transformed NK cells. Kinome-focused and genome-wide screens identify key regulators of NK cell proliferation, cytotoxicity, and resistance to prostaglandin E2 (PGE2)-mediated suppression. STK17B deletion enhances NK cell expansion, while loss of CCDC53 boosts degranulation and cytotoxicity. We also uncover the CRL5 complex—including RNF7, UBE2F, and CISH—as critical inhibitors of IL-2 signaling and effector function under PGE2 stress. These findings establish a scalable platform for CRISPR-based functional genomics in primary NK cells and reveal engineering targets to enhance NK cell persistence and efficacy in tumor microenvironments. The mechanisms that restrict NK cell activity in tumor microenvironments remain to be explored. The authors here establish genome-wide CRISPR screening in primary NK cells and uncover intracellular checkpoints that limit proliferation, cytotoxicity, and resistance in tumor microenvironments.