A CRISPR/Cas12a-coupled multiplexed amplification system for ultrasensitive detection of miRNA-155.

miRNA plays an important role in gene regulation and can be an effective biomarker for disease diagnosis. Herein, a new miRNA detection platform based on the CRISPR/Cas12a-coupled multiplexed amplification system is developed. In this strategy, miRNA-155 acts as an intermediary to trigger the recombinase polymerase amplification (RPA). Due to the introduction of endonuclide recognition sites in the amplification template, the resulting double-stranded DNA (dsDNA) can in turn initiate a strand replacement reaction (SDA), generating a great deal of single-stranded DNA (ssDNA). The ssDNA can directly unlock the trans-cleavage activity of CRSIPR/Cas12a, and the process is independent of PAM sites. Subsequently, the activated Cas12a trans-cleaves nearby signaling molecules, outputting a fluorescence/visualization signal. This method achieves miRNA detection as low as 68.69 fM, with a linear range of 200 fM to 1 nM, and shows good selectivity and repeatability. Meanwhile, the target of 10 pM can be distinguished by the naked eye. Moreover, the proposed method can achieve miRNA-155 detection in complicated cell extracts. The excellent detection sensitivity is mainly due to the integration of two amplification techniques, while the CRISPR/Cas12a system enables fast and accurate visual detection. More importantly, the actual detection results are consistent with standard methods (RT-qPCR), indicating that the CRISPR/Cas12a-coupled multiplexed amplification system is reliable and has potential clinical application value.