CRISPR-Cas12a-activated proximity catalytic hairpin assembly strategy for sensitive gene analysis in Streptococcus mutans

The cnm gene in Streptococcus mutans (S. mutans) mediates bacterial binding to extracellular matrix and invasion within host cells, and has been found to be correlated with the occurrence of brain microbleeds. Hence, it is crucial to develop a reliable method to evaluate cnm-positive S. mutans. Herein, we propose a novel diagnostic platform utilizing Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for accurate identification of the cnm gene, along with proximity catalytic hairpin assembly (CHA) for enhanced signal amplification, for the detection and analysis of the cnm gene in S. mutans. When the cnm gene is present, it activates trans-cleavage activity of Cas12a to trigger the proximity CHA. In contrast to conventional CHA, the hairpin probes in proximity CHA were linked in a sequential manner using a liner ssDNA sequence. Additionally, the CHA process was facilitated by the Duplex-Specific Nuclease (DSN), which enhanced the efficiency and speed of the reaction. The approach exhibits several notable benefits, including i) the target recognition capability of crRNA in CRISPR-Cas system enhances the detection specificity; ii) Cas12a-crRNA is independent on the PAM during recognizing target dsDNA; iii) the use of the DSN and the proximity design of the H1-H2 complex has significantly enhanced the reactive efficiency of proximity CHA. As a result, the method exhibits a low limit of detection of 47 aM and a high selectivity to cnm gene. Moreover, the approach has been effectively employed in clinical settings to accurately differentiate S. mutans from a combination of microorganisms.