A CRISPR-Cas12a-based assay for one-step preamplification-free detection of viral DNA

How to realize fM-level detection of viral DNA without target amplification is a challenge. Traditionally, preamplification of low-abundant DNA targets is a prerequisite, which not only increases the risk of infectious material leakage, enlarges the chance of false positive detection, but also prolongs the overall detection time. Herein, we developed a combinatory CRISPR-Cas12a detection system from three aspects to enhance its limit of detection (LOD) to the femtomolar level which is three orders of magnitude lower than a conventional protocol. Specifically, the detection sensitivity of CRISPR-Cas12a system was enhanced by utilizing multiplex crRNAs for simultaneous recognition of multiple distinct sites of the same viral DNA target, by employing a novel molecular reporter with G-triplex structure that exhibits a significantly enhanced cleaving tendency by Cas12a, by exploring the optimal molecular coexistence reaction environment for maintaining enzyme activity which was the key for sensing. It is worth noting that we have for the first time discovered an environment where sensitive G-triplex reporter forms at a low K+ concentration without damaging CRISPR-Cas12a activity. Using this system, we demonstrated ultrasensitive detection of plasmids containing monkeypox (Mpox) viral DNA sequences in a mimicking physiological scenario. More importantly, we realized sensitive and specific detection and classification of human papillomavirus (HPV) subtypes from clinical samples. Moreover, this design successfully circumvented intricate procedures and the signal readout was not contingent upon the use of unconventional equipment, suggesting its great promise of expanding into a portable, field-deployable test kit for rapid, sensitive, and specific detection of various pathogens.