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 used conventional PCR coupled with SuCas12a2, a novel Cas12 family RNA-targeting nuclease, to develop a detection approach. 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 was used 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. There were five samples from amoebiasis patients confirmed by indirect immunofluorescence assay that were used as proof specimens, where the PCR-SuCas12a2 assay demonstrated 100% specificity. Furthermore, we replaced conventional PCR with recombinase polymerase amplification (RPA) to simplify the procedure for producing amplicons harboring the T7 promoter sequence. The sensitivity of the RPA-SuCas12a2 assay was 10 ² copies per reaction, which was inferior to PCR-SuCas12a2, and 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.