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Background CAR-NK therapy holds great promise as a safe and effective strategy for hematologic malignancies; however, its efficacy in solid tumors is hindered by the immunosuppressive tumor microenvironment (TME), which rapidly induces NK cell dysfunction. To overcome these challenges, we utilized a novel non-viral mini-circular single-stranded DNA (mcssDNA)-based CRISPR/Cas9 targeted genome editing (mcssDNA/CRISPR/Cas9) technology to develop CISH-knockout and locus-specific integrated IL-15-armored CAR-NK cells to enhance CAR-NK cell function against hepatocellular carcinoma (HCC).Methods A GPC3-targeting nanobody was first identified via a yeast surface display screening platform and incorporated into a second-generation CAR construct ( figure 1A). We then utilized a single-step electroporation approach to co-deliver CRISPR/Cas9 ribonucleoprotein (RNP) complexes and a mini-circular single-stranded DNA (mcssDNA) donor template to site-specifically integrate a GPC3-targeting IL-15-armored CAR into the CISH locus of primary human NK cells (figure 1B-C). Editing efficiency and precision were assessed by Sanger sequencing and whole-genome sequencing (WGS). The proliferation, cytotoxicity, and cytokine secretion of the generated CAR-NK cells were analyzed in vitro. Single-cell RNA sequencing (scRNA-seq) was performed to assess transcriptional phenotypes. In vivo efficacy was evaluated in a lung metastasis model of HCC.Results The non-viral CISH locus-targeted CAR-NK cells were generated efficiently using the mcssDNA/CRISPR/Cas9 platform (figure 1D-F). WGS confirmed high editing specificity, with no detectable off-target events observed at the current sequencing depth (figure 1G-I). Compared to lentiviral-transduced CAR-NK cells, CISH knockout CAR-NK cells exhibited significantly enhanced proliferation, IFN-γ secretion, and cytotoxicity against GPC3+ HCC targets (figure 1J-K). ScRNA-seq revealed a pronounced enrichment of adaptive-like NK subsets in CISH-KO CAR-NK cells, with upregulation of genes involved in JAK/STAT signaling, metabolic pathways, and activating receptors (figure 1L-N). To further enhance their functional persistence, we incorporated IL-15 into the CAR construct via site-specific integration at the CISH locus. The resulting IL-15-armored CISH-knockout CAR-NK cells showed enhanced proliferation and retained potent cytotoxicity against GPC3+ tumor cells in vitro (figure 1O-P). In vivo, IL-15-armored CISH-knockout CAR-NK cells induced sustained tumor regression and improved survival in a lung metastatic HCC model. (figure 1Q-S).Conclusions Our results demonstrate that non-viral integration of IL-15-armored CAR into the CISH locus via a simplified one-step genome editing process can robustly enhance NK cell function and persistence in solid tumors. This strategy offers a clinically translatable platform for effective CAR-NK therapy against solid tumor.Acknowledgements This work was supported by Hospital-Industry Collaboration Project (GRGZ2022330, GRGZ20230520, and GRGZ20250104), the National Natural Science Foundation of China Grants (82373302), Science and Technology Projects of Guangzhou (2024A04J6613), and Science and Technology Plan Project of Guangdong (2024A0505040015). We are thankful for the cloning and sequencing services by Quintara Bioscience Inc., we thank Danna Lee from Full Circles Therapeutics Inc. for the technical help for DNA vector purification.Ethics Approval Studies involving human participants were approved by the Ethics Committee of Nanfang Hospital (Approval ID: NFEC-202209-K14), and written informed consent was obtained from all healthy volunteers. Animal experiments related to CAR-NK cell therapy for hepatocellular carcinoma were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanfang Hospital (Approval ID: IACUC-LAC-20240723-001).Abstract 278 Figure 1