A Pre-Amplification-Free Modular Dual-CRISPR System for Enhanced Pathogen Detection Sensitivity.

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• CRISPR/Cas12a systems offer high specificity for pathogen detection but often require pre-amplification, increasing complexity and risk of errors.
• Limited sensitivity of single CRISPR/Cas12a systems necessitates nucleic acid amplification, posing challenges like cross-contamination and false positives.

Purpose of the Study:

• To develop a universal and highly sensitive pathogen nucleic acid detection method without pre-amplification.
• To enhance the sensitivity and reduce the complexity of CRISPR/Cas12a-based diagnostics.

Main Methods:

• A modular dual-CRISPR/Cas12a system was designed, incorporating a recognition module and a signal module.
• The system was coupled with hybridization chain reaction (HCR) and DNA nanostructure self-assembly for signal amplification.
• The system was tested for detecting various pathogen nucleic acids, including African swine fever virus (ASFV), severe fever with thrombocytopenia syndrome virus (SFTSV), and human papillomavirus type 16 (HPV-16).

Main Results:

• The dual-CRISPR system achieved attomolar (aM) sensitivity for pathogen nucleic acid detection, surpassing traditional single CRISPR/Cas12a systems by over six orders of magnitude.
• The system demonstrated high sensitivity and specificity without the need for pre-amplification.
• The modular system proved versatile, successfully detecting diverse pathogen targets and showing practicality in complex sample environments (e.g., ASFV quality control).

Conclusions:

• The developed pre-amplification-free dual-CRISPR system offers a significant advancement in sensitive and universal pathogen nucleic acid detection.
• This approach provides a flexible and robust platform for developing next-generation diagnostic tools with improved performance and reduced complexity.
• The findings open new avenues for enhancing nucleic acid detection technologies in various applications, including infectious disease surveillance.