A NanoLock-enabled, Craspase-based strategy for highly sensitive RNA detection

Abstract Rapid and sensitive detection of RNA is important in fields such as biomedical research and clinical diagnostics. However, current methods typically involve an amplification process, require substantial time, and are susceptible to aerosol contamination. Herein, we introduce a NanoLock-powered, amplification-free assay based on the type III-E clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated system for rapid, highly sensitive, and specific RNA diagnostics. This innovative platform, designated CRISPR-guided caspase (Craspase)–NanoLock–Csx30 (CNC), harmoniously integrates the precise protease activity of Craspase with the remarkable luminescent sensitivity of NanoLock, creating a novel and streamlined approach for RNA detection. The CNC platform exhibited exceptional sensitivity in detecting severe acute respiratory syndrome coronavirus-2 N gene RNA through the integration of three guide RNAs, achieving a detection limit of 250 fM in just 10 min without amplification. Preliminary studies further revealed the platform's extended diagnostic potential for detecting influenza A virus and human immunodeficiency virus. These findings collectively establish the CNC platform as an appealing tool for infectious disease detection and significantly broaden the scope of CRISPR-based diagnostic applications.