Bis-enzyme cascade CRISPR-Cas12a platform for miRNA detection

MicroRNA (miRNA) play a vital role in the pathological development of many diseases. It is considered to be the diagnosis and potential biomarkers of prognosis. Herein, we proposed Bis-enzyme cascade Platform by combining T7 RNA polymerase and CRISPR-Cas12a (BPTC) for a miRNA detection. In the proposed BPTC, the RNA to DNA conversion ability of phi29 amplification and trans -cleavage of CRISPR-Cas12a are combined. The target miRNA can be amplified after binding to the recognizer ssDNA, and then transcribed the CRISPR-derived RNA (crRNA) by T7 RNA polymerase. The produced crRNA can thereby be assembled by CRISPR-Cas12a and recognized with its target dsDNA, thus triggered its trans -cleavage towards surrounding fluorescent reporters, labeled with a fluorophore and a corresponding quenching group. Based on the bis-enzyme cascade system, the biosensor shows highly sensitivity and excellent specificity. Moreover, this study provided a novel all-in-one detect strategy for miRNA and may open a new idea for the design of CRISR-Cas-based miRNA biosensing platforms.

Automated summary

MicroRNA (miRNA) plays a crucial role in the development of various diseases, making it important to have a reliable detection method. In this study, a Bis-enzyme cascade Platform (BPTC) combining T7 RNA polymerase and CRISPR-Cas12a was proposed for miRNA detection. The BPTC showed high sensitivity and excellent specificity, providing a new strategy for the design of CRISPR-Cas-based miRNA biosensing platforms.