CRISPR-Cas12a2-based rapid and sensitive detection system for target nucleic acid

Infectious diseases are extremely important public health issues, where the design of effective, rapid, and convenient detection platforms is critical. In this study, we coupled SuCas12a2, a novel Cas12 family RNA-targeting nuclease, with conventional PCR and recombinase polymerase amplification (RPA), respectively, to develop novel detection approaches, named PCR-SuCas12a2 and RPA-SuCas12a2. SuCas12a2 possesses collateral cleavage activity and cuts the additional single-stranded RNA (ssRNA) added to the reaction system once the ternary complex RNA-SuCas12a2-CRISPR RNA (crRNA) is formed. SuCas12a2 is specifically activated, where the cleaved fluorescent-labeled probes release fluorescent signals, with the strength of the fluorescent signal being proportional to the concentration of nucleic acids specifically bound to crRNA. Simultaneous transcription and SuCas12a2 detection can be performed in a single tube by introducing the T7 promoter sequence into the forward primer. Entamoeba histolytica (E. histolytica) and Mycoplasma pneumoniae (M. pneumoniae) were used as proof specimens to evaluate the performance of the platform. PCR-SuCas12a2 has excellent capabilities, including high specificity with no cross-reactivity from other species and ultra-sensitivity that achieves a detection of one copy per reaction for E. histolytica and M. pneumoniae. However, the sensitivity of the RPA-SuCas12a2 assay was 10² copies per reaction, which was inferior to PCR-SuCas12a2. Clinical samples were obtained from suspected infection patients of E. histolytica and M. pneumoniae, and used to evaluate the systems demonstrated 100 % specificity. The technique shows robust performance and suggests great potential for point-of-care testing of other pathogens to facilitate effective management and control of the spread of diseases.