Ordered assembly of Cas12a for better performance of CRISPR/Cas system with application to the diagnosis of dental caries

Abstract Dental caries is the most common chronic infectious disease among the world, which is mainly caused by Streptococcus mutans ( S. mutans ). Herein, a strategy is explored in this work based on tetrahedral DNA frameworks (TDFs)‐mediated Cas12a ordered assembly (TCOA) for the development of a new method to diagnose dental caries. Specifically, TDFs arrange Cas12a into a highly ordered assembly in a controllable manner, thereby conferring high catalytic efficiency to the protein. Meanwhile, the confined TDFs can maintain an appropriate spatial interval on the surface to prevent excessive local crowding of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a, ensuring efficient substrate diffusion across the interface. Thus, by immobilizing Cas12a orderly, the catalytic efficiency can be greatly enhanced through the formation of localized Cas12a hotspots, and the CRISPR/Cas system can be performed more perfectly. Moreover, the TCOA strategy can be used to sensitively analyze S. mutans with a detection limit as low as 16.59 CFU/mL without pre‐amplification steps. Consequently, with high reproducibility and stability, the TCOA strategy‐based method can be successfully applied to detect 120 clinical saliva samples, demonstrating its robust potential to be a simple, rapid, and highly sensitive diagnosis tool for dental caries.