CrRNA Conformation-Engineered CRISPR-Cas12a System for Robust and Ultrasensitive Nucleic Acid Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• CRISPR-Cas12a is widely used in diagnostics for its programmability and trans-cleavage activity.
• Linear CRISPR RNA (crRNA) is prone to degradation and sensitive to environmental conditions, limiting practical applications.
• Improving the stability and robustness of crRNA is crucial for enhancing CRISPR-based diagnostic systems.

Purpose of the Study:

• To engineer a more stable and robust crRNA component for the CRISPR-Cas12a system.
• To develop a novel CRISPR molecular diagnostic toolkit based on engineered crRNA.
• To evaluate the performance of the new system for pathogen detection in clinical samples.

Main Methods:

• Engineered covalently closed circular crRNA (C-crRNA) to replace linear crRNA in Cas12a complexes.
• Integrated the C-crRNA-Cas12a system with nucleic acid amplification for molecular diagnostics.
• Validated the system's sensitivity and specificity using synthesized Human Papillomavirus type 16 (HPV-16) plasmids.
• Applied the developed CRISPR-Cas12a system for ultrasensitive detection of HPV-16 and influenza A viruses in clinical samples.

Main Results:

• The C-crRNA formed functional complexes with Cas12a, significantly enhancing anti-interference ability while maintaining sensitivity and specificity.
• The circular crRNA-mediated CRISPR molecular diagnostic (CRCD) toolkit achieved 10 aM sensitivity for synthesized HPV-16 plasmids.
• Clinical sample analysis demonstrated consistent results with established methods like PANTHER and qRT-PCR for HPV-16 and influenza A virus detection.
• The CRCD system showed high sensitivity and specificity in detecting target pathogens in real-world samples.

Conclusions:

• Engineered C-crRNA offers a stable and effective alternative to linear crRNA for CRISPR-Cas12a diagnostics.
• The CRCD toolkit represents a significant advancement in CRISPR-based molecular diagnostics, enabling ultrasensitive pathogen detection.
• This strategy provides a novel paradigm for crRNA engineering with broad potential to revolutionize CRISPR-based molecular diagnostics.