Enhanced CRISPR/Cas12a Fluorimetry via a DNAzyme-Embedded Framework Nucleic Acid Substrate

Area of Science:

• Molecular Biology
• Biosensing Technology
• Nucleic Acid Chemistry

Background:

• CRISPR/Cas12a fluorimetry offers high selectivity, simplicity, and rapidness for biosensing.
• Typical CRISPR/Cas12a methods face limitations in sensitivity due to suboptimal Cas12a trans-cleavage efficiency, requiring preamplification.
• There is a need for improved CRISPR/Cas12a-based detection systems with enhanced sensitivity and simplified workflows.

Purpose of the Study:

• To develop an enhanced CRISPR/Cas12a fluorimetry with improved sensitivity and signal-to-noise ratio.
• To create a novel DNAzyme-embedded framework nucleic acid (FNAzyme) substrate for CRISPR/Cas12a applications.
• To demonstrate the application of the enhanced system for detecting methicillin-resistant Staphylococcus aureus in food matrices.

Main Methods:

• Designed a framework nucleic acid (FNAzyme) by embedding four CLICK-17 DNAzymes into a tetrahedral scaffold.
• Integrated the FNAzyme substrate with CRISPR/Cas12a trans-cleavage activity and a functional nucleic acid recognition module.
• Evaluated the detection performance for methicillin-resistant Staphylococcus aureus, including sensitivity and recovery in food samples.

Main Results:

• The developed FNAzyme substrate significantly enhanced Cas12a trans-cleavage efficiency and signal-to-noise ratio.
• Achieved a limit of detection as low as 18 CFU/mL for methicillin-resistant Staphylococcus aureus, a 54-fold improvement over TaqMan probe-mediated methods.
• Demonstrated satisfactory recoveries (80-101%) in various food matrices, indicating practical applicability.

Conclusions:

• The FNAzyme-enhanced CRISPR/Cas12a fluorimetry provides a highly sensitive and specific detection platform.
• The method eliminates the need for DNA extraction and preamplification, simplifying the detection workflow.
• This advanced biosensing tool holds significant potential for next-generation microbial detection and diagnostics.