Poly(vinylpyrrolidone)-Enhanced CRISPR-Cas System for Robust Nucleic Acid Diagnostics

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Technology

Background:

• CRISPR technology offers novel molecular diagnostics via RNA-programmed trans-cleavage.
• Current CRISPR systems lack the sensitivity required for widespread clinical diagnostics.

Purpose of the Study:

• To investigate the impact of surfactants on CRISPR-Cas12a system sensitivity.
• To enhance the accessibility and sensitivity of CRISPR-based molecular diagnostics for clinical use.

Main Methods:

• Systematic screening of various surfactants for their effect on the CRISPR-Cas12a system.
• Testing the compatibility and versatility of the optimal surfactant (PVP) with CRISPR-Cas12b and Cas13a systems.
• Integration of the PVP-enhanced CRISPR system with isothermal nucleic acid amplification for human papillomavirus (HPV) detection.

Main Results:

• Poly(vinylpyrrolidone) (PVP) demonstrated the highest enhancement effect among tested surfactants for CRISPR-Cas12a.
• PVP enhanced the sensitivity of CRISPR-Cas12a, Cas12b, and Cas13a systems by 1-2 orders of magnitude for synthetic targets.
• PVP-enhanced CRISPR assays, combined with isothermal amplification, achieved sensitivity and specificity comparable to quantitative polymerase chain reaction (qPCR) for clinical HPV samples.

Conclusions:

• PVP significantly enhances the sensitivity of CRISPR-based molecular diagnostic systems.
• The PVP-enhanced CRISPR system holds significant promise for advancing sensitive and accessible clinical diagnostics.
• This approach could revolutionize molecular diagnostics and biomedical research, particularly for infectious disease detection.