Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection

Some non-coding RNAs are abnormally expressed during the occurrence and development of diseases, so it is necessary to develop analytical methods that can specifically and sensitively detect them. In typical CRISPR/ Cas12a system, a complete crRNA that can recognize single-stranded or double-stranded DNA is necessary to activate its trans-cleavage activity, which limits its direct application in RNA detection. Here, we prospectively find that slicing the facilitated crRNA in the typical CRISPR/Cas12a system at a fitted site did not affect its trans- cleavage activity, and a mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) was proposed based on this. This system can detect non-coding RNA to pM-level (10 pM for miRNA-21). To expand the application of this system, we combined it with HCR and CHA to establish a detection platform for non-coding RNA. The results show that the proposed method can specifically detect RNA to fM-level (2.5 fM for miRNA-21, 8.98 fM for miR-128-3p, and 81.6 fM for lncRNA PACER). The spiked recovery rates of miRNA-21, miR-128-3p, and lncRNA PACER in normal human serum were in range from 104.7 to 109.4 %, indicating the proposed method owns good applicability. In general, this MCM-CRISPR/Cas12a system further breaks the limitations of the typical CRISPR/Cas12a system that cannot be directly used for non-coding RNA detection. Besides, its combination with HCR and CHA achieves highly sensitive detection of non-coding RNA.