Analytical chemistryNov 25, 2025

A Two-Part CRISPR System That Improves Pathogen Detection Without Pre-Amplification

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 3, 2026

Abstract

A modular dual-CRISPR approach achieves attomolar (aM) level sensitivity for pathogen nucleic acid detection without pre-amplification.

The dual-CRISPR system significantly improves sensitivity, providing over six orders of magnitude higher detection capability than traditional single-CRISPR/Cas12a methods.
One module specifically recognizes pathogen targets and releases an activating agent, while another module initiates a reaction to generate a strong fluorescent signal.
The system demonstrates versatility in detecting various pathogens, including African swine fever virus, severe fever with thrombocytopenia syndrome virus, and human papillomavirus type 16 DNA.
Practical application was confirmed through testing of quality control samples in complex environments.

Simplified

CRISPR/Cas12a is extensively utilized for pathogen detection owing to its high specificity and efficiency. However, traditional single-CRISPR/Cas12a encounters challenges due to its limited sensitivity, requiring pre-amplification of nucleic acids. This increases the complexity of the procedure and the potential for cross-contamination and false positives. Herein, a modular dual-CRISPR approach was developed coupled with hybridization chain reaction (HCR) for the universal and sensitive detection of pathogen nucleic acids without the need for pre-amplification. The system comprises two core modules: the first CRISPR/Cas12a recognition module specifically identifies pathogen targets and releases the activating agent, while the second CRISPR/Cas12a signal module is activated by this agent to initiate the HCR reaction for generating a strong fluorescent signal through DNA nanostructure self-assembly. Through rational design, we demonstrate the ability of this dual-CRISPR system to achieve attomolar (aM) level sensitivity for pathogen nucleic acid detection without pre-amplification, showing over six-order-of-magnitude higher sensitivity than a traditional single-CRISPR/Cas12a system. Additionally, the flexibility and versatility of the modular dual-CRISPR system have been confirmed for diverse pathogen targets, such as African swine fever virus (ASFV), severe fever with thrombocytopenia syndrome virus (SFTSV), and human papillomavirus type 16 (HPV-16) DNA. The system's practicality was demonstrated by examining ASFV quality control samples in complex environments. The exploration of the pre-amplification-free dual-CRISPR system offers a new perspective on enhancing pathogen nucleic acid detection systems.

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A Two-Part CRISPR System That Improves Pathogen Detection Without Pre-Amplification

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