Targeted PLK1 suppression through RNA interference mediated by high‐fidelity Cas13d mitigates osteosarcoma progression via TGF‐β/Smad3 signalling

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Overexpression of polo‐like kinase 1 (PLK1) is frequent in osteosarcoma and drives disease progression and metastasis, making it a promising therapeutic target. In this study, we explored PLK1 knockdown in osteosarcoma cells using RNA interference mediated by high‐fidelity Cas13d (hfCas13d). PLK1 was found to be significantly upregulated in osteosarcoma tumour tissues compared to normal bone. sgRNA‐mediated PLK1 suppression via hfCas13d transfection inhibited osteosarcoma cell proliferation, induced G2/M cell cycle arrest, promoted apoptosis, reduced cell invasion and increased expression of the epithelial marker E‐cadherin. Proximity labelling by TurboID coupled with co‐immunoprecipitation identified novel PLK1 interactions with Smad3, a key intracellular transducer of TGF‐β signalling. PLK1 knockdown impaired Smad2/3 phosphorylation and modulated TGF‐β/Smad3 pathway inactivation. Finally, in vivo delivery of hfCas13d vectors targeting PLK1 substantially attenuated osteosarcoma xenograft growth in nude mice. Taken together, this study highlights PLK1 as a potential therapeutic target and driver of disease progression in osteosarcoma. It also demonstrates the utility of hfCas13d‐mediated gene knockdown as a strategy for targeted therapy. Further optimization of PLK1 suppression approaches may ultimately improve clinical outcomes for osteosarcoma patients.