Bulge DNA-driven CRISPR/Cas12a dynamic activation circuit enables highly sensitive and versatile biosensing

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• CRISPR/Cas12a systems offer innovative biosensing capabilities.
• Linear signal accumulation in traditional CRISPR/Cas12a limits sensitivity.
• A need exists for more sensitive and versatile biosensing platforms.

Purpose of the Study:

• To develop a highly sensitive and versatile biosensing platform using CRISPR/Cas12a.
• To overcome the limitations of linear signal accumulation in CRISPR/Cas12a systems.
• To enable detection of both nucleic acid and non-nucleic acid targets.

Main Methods:

• Engineered a bulge DNA (BD)-driven CRISPR/Cas12a dynamic activation circuit (CBD).
• Designed BD for programmable structural transformation upon degradation, initiating self-amplifying Cas12a activation.
• Developed strategies for detecting nucleic acids and non-nucleic acid targets (pesticides, mycotoxins) using the CBD platform.
• Constructed an "OR" logic gate for simultaneous dual mycotoxin detection.

Main Results:

• Achieved highly sensitive detection of methicillin-resistant Staphylococcus aureus down to 14 CFU/mL.
• Enabled picogram-per-milliliter level detection of pesticides and mycotoxins.
• Demonstrated successful simultaneous detection of dual mycotoxins using an "OR" logic gate.
• CBD system showed exponential, self-amplifying activation of the Cas12a circuit.

Conclusions:

• The bulge DNA (BD)-driven CRISPR/Cas12a dynamic activation circuit (CBD) significantly enhances biosensing sensitivity and versatility.
• CBD platform effectively detects both nucleic acid and non-nucleic acid targets.
• CBD represents a promising new paradigm for next-generation biosensing technologies, including multiplexed hazard monitoring.