An innovative "double-locked" CRISPR/Cas12a system based on DNAzyme for the precise imaging of microRNAs in living...

We constructed a "double-locked" CRISPR/Cas12a system based on DNAzyme for miRNA detection and precise imaging. One lock function to close the catalytic activity of DNAzyme, while the other lock serves to inhibit the cleavage function of CRISPR-induced RNA (crRNA). This "double-lock" mechanism ensures that the system is inhibited in the absence of the target molecule miRNA-141, effectively reducing the background signal. When the target miRNA-141 is present, the "lock" of DNAzyme is opened, and DNAzyme further opens the "lock" of crRNA, which activates the trans-cleavage ability of Cas12a on F-Q probe, and the fluorescence signal is restored. The linear range of miRNA-141 was 50 pmol/L ~ 15 nmol/L, and the detection limit was 47 pmol/L (S/N = 3). The system has been successfully applied to detect miRNA-141 expression levels in cell lysates. Meanwhile, this method can be applied for intracellular miRNA-141 fluorescence imaging and fluctuations in intracellular miRNA-141 expression. Overall, this strategy not only offers new prospects for programmable Cas12a detection systems, but also provides new insights for early diagnosis and screening of diseases by combining this in vitro assay with live cell imaging analysis for the detection of cancer markers.