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

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has opened a new path for molecular diagnostics based on RNA programmed <i>trans</i>-cleavage activity. However, their accessibility for highly sensitive clinical diagnostics remains insufficient. In this study, we systematically investigated the impact of various surfactants on the CRISPR-Cas12a system and found that poly(vinylpyrrolidone) (PVP), a nonionic surfactant, showed the highest enhancement effect among these tested surfactants. Additionally, the enhancement effects of PVP are compatible and versatile to CRISPR-Cas12b and Cas13a systems, improving the sensitivity of these CRISPR-Cas systems toward synthetic targets by 1-2 orders of magnitude. By integrating the PVP-enhanced CRISPR system with isothermal nucleic acid amplification, both the two- and one-step assays exhibited comparable sensitivity and specificity to gold-standard quantitative polymerase chain reaction (qPCR) in the assay of clinical human papillomavirus (HPV) samples, thereby holding significant promise for advancing clinical diagnostics and biomedical research.