Abstract

Natural killer (NK) cells hold promise for adoptive cell therapy due to their innate cytotoxicity. Early clinical trials confirm their safety and efficacy in cancer and autoimmune disease treatment. Engineering NK cells with chimeric antigen receptors (CARs) enhances target specificity and facilitates their development as off-the-shelf allogeneic therapies. However, both viral and non-viral engineering methods of NK cells present challenges. Here, we introduce CISH locus integrated CAR killer (CLICK), a novel non-viral approach using a mini-circular single-stranded DNA genome editing system. CLICK enables efficient integration of CD19CAR sequences into the metabolic checkpoint CISH locus, simultaneously disrupting CISH and driving stable, progressively increasing CAR expression in peripheral blood-derived NK cells. CLICK-engineered CAR-NK cells exhibit potent cytotoxicity, enhanced anti-tumor activity ex vivo and in vivo , extended persistence, and reduced exhaustion. Together, these findings highlight CLICK as a highly efficient and versatile platform for non-viral CAR-NK cell engineering, offering a scalable approach for next-generation allogeneic immune cell therapies.