Cas12a-Programmed Modular CRISPR Cascade Reaction on Paper Supports for Dual-Mode Detection of Pathogenic Genomes

Area of Science:

• Biotechnology
• Biosensing
• Molecular Diagnostics

Background:

• Paper-based biosensors offer a low-cost, scalable platform for diagnostics.
• CRISPR-Cas12a systems provide precise nucleic acid detection capabilities.
• Accurate identification of bacterial pathogens like Campylobacter jejuni, E. coli, and Listeria monocytogenes is crucial for public health.

Purpose of the Study:

• To develop a paper-based optical biosensing platform for detecting three major bacterial pathogens.
• To utilize fluorescent DNA-templated silver nanoclusters (FNPs) and a CRISPR-Cas12a cascade reaction for enhanced detection.
• To demonstrate a scalable and cost-efficient method for pathogen identification in resource-limited settings.

Main Methods:

• Fabrication of letter-shaped paper cutouts as sensors, each representing a specific pathogen.
• Utilizing an ON-to-OFF FNP degradation mechanism triggered by CRISPR-Cas12a recognition.
• Developing a two-step CRISPR-Cas12a cascade reaction for an intuitive ON signal output (ON-retention).
• Detection of whole Listeria genome amplified by isothermal recombinase polymerase amplification.

Main Results:

• Successful detection of individual and multiple targets (seven combinations) using the paper-based platform.
• Demonstrated both ON-to-OFF and ON-retention detection modes with clear fluorescence signals.
• Achieved reliable detection of as few as 40 full Listeria genomic copies using fluorescent images on paper.
• Validated the platform's capability for multistep biochemical reactions directly on paper support.

Conclusions:

• The developed paper-based biosensing platform offers a promising solution for low-cost, scalable pathogen detection.
• The innovative CRISPR-Cas12a cascade reaction enhances diagnostic clarity and reporting.
• This technology holds significant potential for application in resource-limited settings, improving public health surveillance.