Multistage‐Responsive Gene Editing to Sensitize Ion‐Interference Enhanced Carbon Monoxide Gas Therapy

Abstract As a promising therapeutic modality targeting cancer, gas therapy still faces critical challenges, especially in enhancing therapeutic efficacy and avoiding gas poisoning risks. Here, a pH/glutathione (GSH) dual stimuli‐responsive CRISPR/Cas9 gene‐editing nanoplatform combined with calcium‐enhanced CO gas therapy for precise anticancer therapy, is established. In the tumor microenvironment (TME), the fast biodegradation of the CaCO 3 layer via pH‐induced hydrolyzation allows glucose oxidase (GO x ) to catalyze glucose for H 2 O 2 production, which further reacts with manganese carbonyl (MnCO) and achieves the precise release of CO gas. Simultaneously, in situ Ca 2+ overload from CaCO 3 degradation disturbs mitochondrial Ca 2+ homeostasis, resulting in Ca 2+ ‐driven reactive oxygen species (ROS) formation and subsequent mitochondrial apoptosis signaling pathway activation. Subsequently, by GSH‐induced cleavage of a disulfide bond, the released Cas9/sgRNA (RNP) can achieve nuclear factor E2‐related factor 2 (Nrf2) gene ablation to sensitize gas therapy by interfering with ROS signaling. This therapeutic modality endows codelivery of CRISPR, ions, and gas with smart control features, which demonstrates great potential for future clinical applications in precise nanomedicine.