Spectrochimica acta. Part A, Molecular and biomolecular spectroscopyMay 2, 2026

Direct detection of microRNA using a specially designed CRISPR/Cas12a amplification method

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated May 3, 2026

Abstract

A biosensor detects microRNA-21 with a limit of 2.88 pM and high accuracy.

Direct detection of microRNA-21 is achieved without the need for target amplification.
The method utilizes a molecular switch probe that activates Cas12a activity in the presence of miRNA-21.
A self-driven cascade signal amplification is established by an amplifier probe.
Detection accuracy ranges from 95.5% to 108.6%, with precision indicated by a relative standard deviation of 2.35%-7.18%.
Elevated levels of miRNA-21 are quantified in lung squamous carcinoma cells compared to normal bronchial epithelial cells.
A comparison with RT-qPCR shows a similar trend in quantifying miRNA-21 levels.

Simplified

The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its high specificity, sensitivity and programmability. However, direct RNA detection is hindered by its inherent DNA-targeting trans-cleavage activity, which typically necessitates a reverse transcription amplification to convert RNA into DNA. Herein, we report a one-pot assay that enables direct detection of microRNA-21 without target amplification. The method employs a molecular switch probe (MSP) that recognizes miRNA-21 and activates Cas12a activity, along with an amplifier probe (AMP) that establishes a self-driven cascade signal amplification. This biosensor achieves a detection limit of 2.88 pM, with high accuracy (recovery of 95.5%-108.6%) and good precision (RSD: 2.35%-7.18%), and exhibits excellent specificity against homologous miRNAs. Using this assay, we successfully quantified the elevated levels of miRNA-21 in lung squamous carcinoma (H520) cells compared to normal bronchial epithelial cells (BEAS-2B). Furthermore, a methodological comparison with RT-qPCR revealed a similar trend between the two methods. This study provides a simple and reliable strategy for direct RNA detection using CRISPR/Cas12a.

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PubMed

Direct detection of microRNA using a specially designed CRISPR/Cas12a amplification method

Abstract

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Competing interests

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Abstract

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Related papers

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