A Universal Light-Activated CRISPR-RNA Based on Split Direct Repeat for One-Pot Cas12a Nucleic Acid Detection

Area of Science:

• Molecular Biology
• Biotechnology
• CRISPR-Cas Systems

Background:

• CRISPR-Cas systems offer potential for gene editing and diagnostics.
• Current photochemical CRISPR control lacks a universal approach.
• A modular, light-activated CRISPR-RNA design is needed.

Purpose of the Study:

• To develop a universal, light-activated CRISPR-RNA system for modular applications.
• To create a light-controlled CRISPR system adaptable for various targets.
• To establish a one-pot detection system for enhanced molecular diagnostics.

Main Methods:

• Designed a modular CRISPR-RNA by splitting crRNA into 5' SDR and 3' SDR-Spacer domains.
• Introduced double-stranded extensions at the cleavage site for functional integrity.
• Screened light-sensitive caging groups on the 5' SDR for universal application.

Main Results:

• Developed a novel light-activated CRISPR-RNA system with a universal caged 5' SDR.
• Established the UniLight-CRISPR system for one-pot RAA-Cas12a detection.
• Achieved 95.45% sensitivity and 100% specificity in *Mycoplasma pneumoniae* detection.

Conclusions:

• The universal photo-regulation strategy overcomes limitations in CRISPR diagnostics.
• The developed system demonstrates high performance comparable to conventional assays.
• This approach provides a blueprint for adapting Cas enzymes and broad applications in diagnostics and gene editing.