Chemical RNA modification has emerged as a flexible approach for post-synthetic modifications in chemical biology research. Guide RNA (gRNA) plays a crucial role in the clustered regularly interspaced short palindromic repeats and associated protein system (CRISPR-Cas). Several toolkits have been developed to regulate gene expression and editing through modifications of gRNA. However, conditional regulation strategies to control gene editing in cells as required are still lacking. In this context, we introduce a strategy employing a cyclic disulfide-substituted acylating agent to randomly acylate the 2'-OH group on the gRNA strand. The CRISPR-Cas systems demonstrate off-on transformation activity driven by redox-triggered disulfide cleavage and undergo intramolecular cyclization, which releases the functionalized gRNA. Dithiothreitol (DTT) exhibits superior reductive capabilities in cleaving disulfides compared to glutathione (GSH), requiring fewer reductants. This acylation method with cyclic disulfides enables conditional control of CRISPR-Cas9, CRISPR-Cas13a, RNA hybridization, and aptamer folding. Our strategy facilitates precise in vivo control of gene editing, making it particularly valuable for targeted applications.