Synthetic and systems biotechnologyDec 1, 2025

CRISPR-based biosensor using two fluorescent signals to detect specific DNA methylation without labels

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 3, 2026

Abstract

A limit of detection of 152 pM was achieved using a novel label-free dual-fluorescent functional nucleic acid (DFFNA) biosensor for detecting DNA methylation.

The DFFNA was engineered to include an aptamer for auramine O recognition and a DNA duplex for binding 5,6,7-trimethyl-1,8-naphthyridin-2-amine.
Fluorescence intensity ratios between ATMND and AO increased significantly after DFFNAs were cleaved by activated Cas12a.
The developed biosensor effectively detects site-specific DNA methylation with notable specificity and sensitivity.
High resolution and cleavage activity of Cas12a contributed to the biosensor's performance.
The system supports portable, point-of-care testing with observable color changes and smartphone imaging capabilities.
The biosensor successfully differentiated colorectal cancer patients from healthy individuals.

Simplified

The CRISPR/Cas12a system holds significant promise for biomedical applications. Nevertheless, the commonly used reporter, fluorophore-quencher-labeled substrates, is hindered by labor-intensive synthesis procedures and high costs, while also relying on a single-photon method and being vulnerable to environmental interference. Herein, a label-free dual-fluorescent functional nucleic acid (DFFNA) was engineered, comprising an aptamer domain for auramine O (AO) recognition and a dSpacer-integrated DNA duplex region for 5,6,7-trimethyl-1,8-naphthyridin-2-amine (ATMND) binding. The fluorescence of AO and ATMND can be enhanced and quenched, respectively, when bound to DFFNAs. The fluorescence intensity ratio between ATMND and AO increased significantly following the cleavage of DFFNAs by activated Cas12a, thus offering a universal, label-free, ratiometric fluorescent reporter for the CRISPR/Cas12a system. To explore the application of the DFFNA-based CRISPR/Cas12a system, a novel biosensor was developed to detect site-specific DNA methylation. It employs a methylation-sensitive restriction enzyme to recognize methylation sites, Cas12a for site-specific DNA identification and signal amplification, and DFFNAs to produce ratiometric fluorescence. The assay demonstrated remarkable specificity and sensitivity, with a limit of detection of 152 pM, due to the high resolution and-cleavage activity of Cas12a. The rationally designed and label-free DFFNAs enhance stability, increase flexibility, and reduce cost. The observable color change and smartphone imaging capability facilitate portable, point-of-care testing. Specifically, the biosensor demonstrated excellent specificity by differentiating colorectal cancer patients from healthy individuals. Consequently, this work presents a superior label-free and ratiometric fluorescent reporter for the CRISPR/Cas12a system, which offers a promising strategy for DNA methylation detection in clinical settings. trans

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CRISPR-based biosensor using two fluorescent signals to detect specific DNA methylation without labels

Abstract

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