DithiothreitolFacilitates LbCas12a with ExpandedPAM Preference for Ultrasensitive Nucleic Acid Detection

Clustered regularly interspaced short palindromic repeats-associated (CRISPR/Cas) proteins have been used for a growing class of in vitro molecular diagnostics due to their modularity and high specificity in targeting nucleic acid. However, the requirement of a protospacer adjacent motif (PAM) for Cas protein-catalyzed trans-cleavage poses a challenge for random nucleic acid detection. Here, we demonstrate that dithiothreitol (DTT) enables LbCas12a to adopt a relaxed preference for PAM base pairing, thereby expanding the target sequence space. Accordingly, we propose a DTT-mediated CRISPR/Cas12a toolbox (DTT-deCRISPR) that exhibits relaxed PAM specificity and is readily compatible with nucleic acid amplification techniques including recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR). As a proof of concept, we integrate DTT-deCRISPR with frequently used PCR for sensitively and selectively detecting high-risk human papillomavirus (HPV) 16 and 18. The platform demonstrates the ability to detect synthesized HPV 16 and 18 plasmids down to 1 aM within 60 min. Based on the receiver operating characteristic curve analysis, the clinical sensitivities of the developed method for detecting HPV 16 and 18 are 93.75% and 80.00%, respectively. We further incorporate it into a lateral flow assay (LFA) for point-of-care detection, and the HPV 16 and HPV 18 abundances determined by LFA for clinical samples are consistent with the fluorescence analysis results. Together, this work uncovers an unexpected connection between DTT and PAM preferences of LbCas12a, promoting the universality and flexibility of CRISPR technology in molecular diagnostics.