Dz-SiG CRISPR: A DNAzyme-Switched G-quadruplex-lock CRISPR system for isothermal and rapid detection of lead ions

Nov 21, 2025Talanta

A DNA-based CRISPR system switched by a special DNA structure for fast, room-temperature detection of lead ions

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Abstract

The Dz-SiG CRISPR system achieves an ultralow limit of detection of 18.91 fM for lead (Pb).

A RNA G-quadruplex structure serves as a conformational lock to suppress Cas12a's activity until Pb binding occurs.
Pb binding activates the GR-5 DNAzyme, which releases the RNA G-quadruplex and triggers Cas12a-mediated cleavage.
The system produces a sharp fluorescent signal, indicating the presence of Pb.
Recovery rates for lead detection in real water and soil samples range from 94.44% to 99.03%.
The assay can be completed within 30 minutes, making it suitable for on-site detection.
The modular design allows for reprogramming to detect other small molecules by changing the DNAzyme module.

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Lead (Pb) poses serious risks to health and ecosystems, necessitating rapid, ultrasensitive detection. CRISPR/Cas12a systems offers exceptional specificity and intrinsic signal amplification. Nevertheless, their adaptation to Pbis hindered by the lack of programmable interfaces to convert small-molecule binding into Cas12a activation. Herein, we report a DNAzyme-Switched G-quadruplex-locked CRISPR (Dz-SiG CRISPR) strategy, enabling ultrasensitive, femtomolar-level detection of Pb. A RNA G-quadruplex (RG4) structure is conjugated to the 5' end of the crRNA, serving as a conformational lock that suppresses Cas12a's trans-cleavage activity. Upon Pbbinding, the GR-5 DNAzyme catalyzes a hydrolytic cleavage that acts as a molecular switch, releasing the RG4 domain and unleashing active crRNA to trigger Cas12a-mediated cleavage of a fluorogenic reporter, yielding a sharp "off-to-on" fluorescent signal. The Dz-SiG CRISPR system achieves an ultralow limit of detection of 18.91 fM for Pband demonstrates outstanding performance in real water and soil samples, with recovery rates ranging from 94.44 % to 99.03 %. The assay can be completed within 30 min, making it highly suitable for rapid on-site lead ion detection. Importantly, the modular Dz-SiG CRISPR framework can be readily reprogrammed for other small molecules by simply substituting the DNAzyme module, offering a generalizable strategy for rapid, ultrasensitive environmental monitoring. 2+ 2+ 2+ 2+ 2+

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Dz-SiG CRISPR: A DNAzyme-Switched G-quadruplex-lock CRISPR system for isothermal and rapid detection of lead ions

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