CRISPR-Cas12a coupled with cyclic reverse transcription for amplified detection of miRNA

Abstract

In this work, we present a highly sensitive, specific, and versatile method to quantify miRNA expression by coupling CRISPR-Cas12a with cyclic reverse transcription (CRT), termed as CRISPR-CRT. Each miRNA target was first converted and amplified into multiple hairpin RT products via CRT. Afterward, the hairpin RT products could serve as activators to initiate the collateral cleavage activity of CRISPR-Cas12a. Due to the above two-stage amplification, this assay could detect miRNA at sub-femtomolar level (LOD, 0.201 fM). Since the sequence of target miRNA is double checked: first in the CRT and then in the CRISPR system, the proposed assay also shows an excellent specificity in detecting miR-21. Finally, with the usage of this assay, the sensitive assessment of miR-21 levels in human serum samples has been achieved and the disease human serum has been detected. Conclusively, CRISPR-CRT holds a great application prospective in the field of clinical molecular diagnosis.