RNase H-Assisted, Split-crRNA-Based CRISPR/Cas12a Lateral Flow Assay for Amplification-Free, Multi-Site Detection of Bacterial 16S rRNA

Abstract With the growing demand for on-site testing of foodborne pathogens, rapid and sensitive detection methods are urgently needed. Here, we developed a split-crRNA-based CRISPR/Cas12a assay integrated with a lateral flow assay (sCR-LFA) for rapid, sensitive detection of bacterial 16S rRNA within 1 h, without pre-amplification. To detect highly structured 16S rRNA, we used RNA fragmentation via DNA-helper-mediated RNase H pretreatment. When short RNA targets interact with corresponding RNA blockers, unblocked RNA spacers hybridize with a DNA activator to form spacer/activator duplexes, which activate split-crRNA/Cas12a complexes. The sCR-LFA achieved a detection limit of 104 CFU/mL for single-site targeting. Using an enhanced sCR-LFA that integrates a multi-site targeting strategy, we achieved a lower detection limit of 102 CFU/mL. Furthermore, the enhanced sCR-LFA was successfully validated in simulated milk and mushroom samples spiked with bacteria. Collectively, this work presents a promising approach for effective food safety monitoring and prevention of infectious diseases.