Reprogramming endogenous NK circuits by highly efficient nonviral genome editing.

Natural killer (NK) cells are promising platforms for off-the-shelf immunotherapy, yet nonviral precision engineering remains limited by poor HDR efficiency, DNA toxicity, and manufacturing challenges. The aim of this study was to establish a high-yield, nonviral knock-in platform. Through extensive in-depth rational screens, we achieved ∼90% HDR insertion of therapeutic payloads while maintaining 100% postediting recovery. By hijacking endogenous transcriptional programs, we installed genetic circuits into defined genomic loci to tune transgene expression. To enable context-dependent therapeutic responses, we integrated a synthetic positive feedback circuit at the CISH locus, which enhanced NK cell persistence and drove strong expression of anti-CD22/19 dual CAR. A hypoxia-responsive IL-12 circuit gated by the PFKFB4 promoter restored cytotoxicity under environmental stress. Finally, we showed this platform is compatible with GMP manufacturing and supports clinical-scale expansion. These findings provide a scalable framework for programmable, nonviral editing of NK cell effector functions for therapeutic and research applications.