Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.