Combining CRISPR/Cas12a with isothermal exponential amplification as an ultrasensitive sensing platform for microRNA detection

Abstract

MicroRNA (miRNA) is considered as a potential biomarker in the diagnosis of cancer. Thus, a new diagnostic technique with high sensitivity and selectivity is crucial for miRNA detection in clinical testing. Herein, we presented a sensing platform based on CRISPR/Cas12a and isothermal exponential amplification reaction (EXPAR) to realize ultrasensitive analysis of miRNA. MiRNA-27a (miR-27a) was chosen as a model target. In this sensing platform, a miR-27a-triggered cascade EXPAR process was introduced to achieve a mass of activators. The resultant activators could hybrid specifically with crRNA of CRISPR/Cas12a system, triggering the multiple-turnover trans-cleavage activity of Cas12a for achieving signal amplification. The sensing platform rendered a wide detection (ranging from 100 aM to 10 nM) and a low limit of detection (90 aM). In particular, this strategy achieved ultrasensitive detection of the other microRNA biomarkers such as let-7a and miR-141 by the flexible design of the region of template, which demonstrated the universal detection ability. Furthermore, an excellent selectivity feature was obtained by the discrimination of microRNA family members and base mismatch analysis. The proposed sensing platform also was able to assess miR-27a levels in breast cancer cell and serum sample, revealing a promising application in clinical molecular diagnostics.