An RNA-based catalytic hairpin assembly circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs.

CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system.