Self-Priming Cyclic Amplification Accelerating CRISPR Sensor for Sensitive and Specific MicroRNA Analysis with No Background
Dayong Li, Yanheng Yao, Wenting Cheng, Zhiqiang Hou, Zhongyun Wang, Yang Xiang
Abstract
In this work, we demonstrate for the first time the application of the phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction for miRNA assays. A self-priming amplification accelerating CRISPR sensor was well-established for sensitive and specific miRNA detection by integrating the PS-THSP reaction and CRISPR/Cas12a system. The sensor consists of three steps: (1) the formation of a complete PS-THSP template in the presence of target miRNA and ligase; (2) the exponential isothermal amplification of the PS-THSP reaction under the action of DNA polymerase; (3) the activation of the CRISPR/Cas12a fluorescence system to generate signals. We used miR-21 as a model target. The sensor can achieve sensitive detection of miR-21 without the involvement of any primers, and the special design of the CRISPR proto-spacer neighbor motif (PAM) sequence effectively avoids the interference of the background signal. In addition, the sensor can not only identify single-base mutant homologous sequences but also show stable performance in complex biological matrices. We have successfully used this sensor to accurately analyze miR-21 in different cell lines and real clinical samples, demonstrating its great potential in clinical diagnosis.