A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR/Cas systems offer significant potential for biomedical applications.
• Precise control over CRISPR/Cas activity is crucial for diagnostics and therapeutics.
• Existing methods for CRISPR/Cas control can be complex or lack fine-tuning capabilities.

Purpose of the Study:

• To develop a photo-controllable CRISPR/Cas12a system for enhanced diagnostic applications.
• To demonstrate the precise regulation of CRISPR/Cas12a activity using a light-responsive crRNA.
• To evaluate the system's efficiency and potential for clinical diagnostics.

Main Methods:

• Conjugation of a G-quadruplex (G4) block to the 3' end of CRISPR RNA (crRNA) via a photocleavable linker.
• Photo-irradiation to remove the G4 block, enabling crRNA-target DNA hybridization and Cas12a processing.
• Detection of human papillomavirus 16 DNA in clinical samples using the developed one-pot strategy.

Main Results:

• The photo-controllable CRISPR/Cas12a system demonstrated efficient detection of HPV16 DNA.
• The system achieved high sensitivity (95.7%) and specificity (100%) compared to qPCR.
• The method was faster (25 min vs. 60 min), simpler, and cost-effective, suitable for lateral flow tests.

Conclusions:

• A novel, photo-controllable CRISPR/Cas12a system was successfully developed.
• This system offers precise control over CRISPR/Cas activity for diagnostic purposes.
• The technology holds significant promise for next-generation clinical diagnostics, particularly for infectious diseases.