Engineering OMEGA-TnpB system for in vitro targeted knockout of MDM2 gene to suppress malignant behaviors in osteosarcoma cells

Background Conventional treatments have limited effectiveness in patients with advanced, recurrent, or metastatic osteosarcoma. Gene therapy offers a promising therapeutic option for osteosarcoma. However, the clinical application of current gene-editing tools (e.g. CRISPR-Cas9) is limited by their large size and high immunogenicity. The newly discovered OMEGA-TnpB system, with its compact size (~ 400 aa), shows great potential for tumor gene editing.Objective To develop efficient and compact gene-editing tools by engineering the OMEGA-TnpB system, and validate their potential for osteosarcoma gene therapy.Methods Through rational design, TnpB mutants were obtained. A single-strand annealing (SSA) reporter system was employed to screen mutants, and their editing efficiency was validated in HEK293T and osteosarcoma cell lines (143B). The optimized TnpB mutant (ISYmu1-L225K) was used to knock out the osteosarcoma gene MDM2. The effects on cell proliferation, apoptosis, migration, and invasion were assessed by RT-qPCR, flow cytometry, CCK-8, wound-healing assay, and Transwell assay.Results Several highactivity TnpB mutants were identified, among which ISYmu1-L225K exhibited significantly higher editing efficiency than enOsCas12f1 in both HEK293T (P<0.01) and 143B cells (P<0.01). In HEK293T cells, its efficiency at certain loci showed no significant difference compared to SpCas9. Knockout of MDM2 reduced osteosarcoma cell proliferation (P<0.01), increased apoptosis rates (P<0.01), and inhibited both migration (P<0.01) and invasion (P<0.05). Conclusion This study successfully develops the TnpB mutant ISYmu1-L225K, which exhibits enhanced gene editing efficiency in mammalian cells. ISYmu1-L225K suppresses the malignant behavior of osteosarcoma cells by targeting MDM2, providing a novel strategy for precision therapy in osteosarcoma.