Breakthrough in LbCas12a Chemical Inhibitor Screening: The Firefly Probe and Its Application in Dual-Mode Biosensors

Area of Science:

• Biochemistry
• Molecular Biology
• Biosensor Technology

Background:

• Anti-CRISPR (Acr) proteins face limitations in biosensor applications due to high manufacturing costs and stringent storage requirements.
• Chemical inhibitors present a more accessible, cost-effective, and stable alternative for industrial-scale production and application.
• CRISPR/Cas12a systems are crucial in molecular biology and diagnostics, necessitating effective control mechanisms.

Purpose of the Study:

• To develop a novel method for screening chemical inhibitors of CRISPR/Cas12a.
• To identify and characterize small molecule inhibitors of LbCas12a.
• To establish a versatile biosensing platform utilizing these inhibitors.

Main Methods:

• Development of a label-free fluorescent probe (firefly probe) for inhibitor screening.
• Identification of inhibitors using the firefly probe assay.
• Characterization of inhibitory mechanisms via electrophoresis and molecular docking.
• Construction of a universal sensing platform for enzyme detection.

Main Results:

• Three effective chemical inhibitors (H2O2, catechol, hydroquinone) against LbCas12a were discovered.
• These inhibitors bind to LbCas12a, disrupting complex formation and inhibiting both cis- and trans-cleavage activities.
• A sensing platform using H2O2 enabled sensitive fluorescence and visual detection of catalase and glucose.
• Low limits of detection were achieved for catalase (0.080 U/mL) and glucose (0.0059 mM).

Conclusions:

• Small molecule inhibitors of LbCas12a were successfully identified and their inhibitory mechanisms elucidated.
• These chemical inhibitors offer a practical and scalable alternative to protein-based inhibitors for CRISPR/Cas12a systems.
• The developed sensing platform demonstrates the potential of LbCas12a inhibitors in advanced biosensor applications.