Hybridization chain reaction circuit controller: CRISPR/Cas12a conversion amplifier for miRNA-21 sensitive detection

MicroRNA (miRNA) is crucial to the diagnose of various diseases. However, the accurate detection of miRNA has been challenging due to its short length and low abundance. Here, we designed a hybridization chain reaction (HCR) circuit controller to initiate the CRISPR/Cas12a conversion amplifier (HCR-Cas12a controller) for sensitive detection of miRNA-21 (miR-21). In the HCR, pre-crRNA was encapsulated in a hairpin structure until the miR-21 was present. Afterward, Cas12a fully exerted its RNase activity to self-mature pre-crRNA. Then, the trans-cleavage activity of Cas12a was initiated by activator. This results in the conversion of biological signals to fluorescent signal. During HCR-Cas12a controller, the circuit formed quickly, while the Cas12a system worked in a short time. The miR-21 was ultra-sensitively detected with the wide detection range of 1 fM - 100 nM, and the calculated limit of detection was 75.4 aM. The sensitivity was an order of magnitude lower than the standard method. The formation of HCR at room temperature does not require a thermal cycler. Additionally, Cas12a can work without the need for precise or expensive instruments. Therefore, our proposed method was suitable for low-resource settings, and provided a technical basis for sensitive detection of miRNA in low concentration range.