ACS applied materials & interfacesJul 3, 2026

Improved One-Step RNA Testing Using DNAzyme-Boosted CRISPR Technology at Constant Temperature

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jul 4, 2026

Abstract

An RNA-triggered DNAzyme circuit integrated with CRISPR/Cas12a can enhance nucleic acid detection sensitivity by converting a single RNA molecule into numerous initiators.

The system utilizes a DNAzyme that, once activated by the target analyte, cleaves a substrate to release multiple activators.
This cascade reaction triggers a secondary amplification process through CRISPR/Cas12a, resulting in a visible signal on a lateral flow assay.
The detection method is designed to be one-pot and homogeneous, simplifying the workflow compared to traditional approaches.
The DNAzyme circuit demonstrates high programmability, allowing easy customization for various target sequences.
This strategy breaks the one-to-one activation relationship, significantly improving detection sensitivity.

Simplified

While integrating DNAzymes with Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems offers a promising route to enhance CRISPR/Cas12a-based molecular diagnosis via enzyme-coupled cascade amplification, their implementation in simple, specific, and sensitive nucleic acid detection remains challenging, largely due to reliance on complex, multistep workflows. Here, we report an RNA-triggered DNAzyme circuit integrated with CRISPR/Cas12a that serves as a universal nucleic acid preamplifier, enabling one-pot and homogeneous detection. The catalytic activity of DNAzyme, initially suppressed by a complementary blocker strand, was restored upon the recognition of the target analyte. The activated DNAzyme then cleaved a hairpin-shaped substrate, liberating multiple activators that triggered a secondary CRISPR/Cas amplification reaction. This cascade generated a visible red band signal on a lateral flow assay via the collateral cleavage of a reporter. By employing the DNAzyme as a signal amplifier, the system efficiently converted a single RNA molecule into numerous initiators, breaking the one-to-one activation relationship between the target and Cas12a ribonucleoprotein and thereby greatly enhancing the detection sensitivity. Additionally, the system exhibited high programmability and universality, as a biosensor for a given target could be easily constructed by simply customizing the corresponding region of the blocker strand that is complementary to the target sequence. This integrated cascade system enables efficient signal amplification within a simple one-pot format and holds significant promise for practical applications.

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Improved One-Step RNA Testing Using DNAzyme-Boosted CRISPR Technology at Constant Temperature

Abstract

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