G-quadruplex molecular beacon: A versatile CRISPR/Cas12a reporter for rapid and label-free biosensing

CRISPR/Cas12a has been widely used in molecular diagnostic due to its excellent trans-cleavage activity. However, its trans-cleavage efficiency on G-quadruplex (G4) is highly correlated with the stability of G4, which causes long cutting time and limits the wide application of G4-probing CRISPR/Cas12a assays. To address these challenges, we utilize the G-quadruplex molecular beacon (G4MB) with the stem-loop structure incorporating split G-quadruplex as the reporter to construct a CRISPR/Cas12a-based biosensor. In comparison with the previously reported G4 reporter, the cleavage speed of Cas12 on G4MB reporter is faster because of the high susceptibility of Cas12a to ssDNA (ssDNA). More importantly, the formation or disassembly of G4MB can be regulated directly by its stem-loop structure, thus the trans-cleavage rate of Cas12a on G4MB was not affected by the stability of G-quadruplex, offering great extent of freedom to choose suitable G4 sequence in CRISPR/Cas12a system. In addition, this biosensor is label-free by the utilization of G4 binding dye (ThT) and has high selectivity due to the high fidelity of Cas12a. This work can broaden the application range of G4 in CRISPR/Cas12a-based biosensors and hold enormous promise in future research on medical diagnoses and biology science.