A sensitive single-molecule assay based on the CRISPR/Cas13 system

Molecular diagnostic technology plays an important role in pathogenic microorganism detection, epidemic prevention and control, disease diagnosis and precision medicine, but it has the disadvantages of long turnaround times, low sensitivity and poor specificity. Therefore, there is an urgent need to develop rapid, sensitive and specific molecular diagnostic techniques. In this study, a single-molecule detection method without amplification was developed by combining the CRISPR/Cas13 system and total internal reflection fluorescence microscopy (TIRF). The conserved 2×HEPN domain of Cas13 protein mutated into dCas13 (deactivated Cas13), resulting in the loss of nuclease activity but retaining the activity of the conjugating enzyme, allowing the dCas13 protein to specifically recognize and bind RNA molecules. Then, the trisomy complex formed by the dCas13 protein, the sgRNA (fluorescent group labeled) and the S gene (target RNA) of the SARS-CoV-2 virus in the reaction system was captured using the capture probe, and the target RNA molecules were detected by TIRF. The experimental results show that, under the condition of no target amplification, the established single-molecule detection method had a detection sensitivity of 1 pmol/L for the target RNA. Compared with the inherent accessory cleavage activity of the CcaCas13b protein, the sensitivity shows a 1000-fold increase. This detection system has high specificity and can effectively distinguish the S gene of the SARS-CoV-2 virus and its common mutants (N501Y and D614G). In addition, the genomic RNA of the SARS-CoV-2 virus (158 ng/µL) was successfully detected using this method. The single-molecule detection technology established in this study affords high sensitivity and strong specificity, and does not require additional nucleic acid amplification steps. Our work provides new ideas for the development of subsequent rapid diagnostic methods and has potential application value.