Rationale: Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells represent a promising modality for cancer immunotherapy, yet their efficacy in solid tumors is limited by immunosuppressive cues from the tumor microenvironment (TME), particularly, transforming growth factor β (TGFβ).

Methods: Primary human NK cells were cytokine-activated (IL-12/15/18) and engineered via a one-step electroporation that delivered Cas9 ribonucleoprotein and a dsDNA donor (1 kb homology arms, SFFV promoter, poly(A), GRE element) to knock out TGFBR2 and knock in a mesothelin CAR. We compared against a two-step AAV method, and treated dexamethasone (Dex) during manufacture. Anti-tumor function was assessed using AsPC-1 cancer killing assays, patient-derived pancreatic cancer organoids (caspase-3/7 imaging, luciferase viability, live/dead FACS), and multi-omics profiling (RNA-seq, ATAC-seq, GSEA) to evaluate metabolic and transcriptional modifications.

Results: We report a streamlined, one-step strategy that simultaneously disrupts the TGFβ receptor II (TGFβRII) and integrates a mesothelin-targeting CAR transgene into primary NK cells via electroporation. By optimizing single-guide RNAs, donor DNA templates incorporating glucocorticoid response elements, and electroporation parameters, we achieved markedly improved knock-in efficiency. Transient Dex treatment during genome editing enhanced CAR expression and cytotoxic function in both electroporation- and AAV-mediated platforms. Dex augmented the cytotoxic activity of CAR-NK cells by promoting oxidative phosphorylation and ATP production.

Conclusions: We designed robust, TGFβ1-resistant allogeneic CAR-NK cells using a virus-free, one-step engineering strategy, establishing a versatile, clinically scalable method for engineering metabolically fortified CAR-NK cells capable of overcoming TME-mediated suppression in solid tumors.