Development of a CRISPR/dCas9-based membrane-assisted colorimetric assay for detection of high-risk HPV16 and HPV18: a proof-of-concept study.

BACKGROUND AND AIMS: Persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, is the leading cause of cervical cancer. While molecular diagnostics offer high sensitivity, their deployment in decentralized settings remains limited. This study presents a proof-of-concept CRISPR/dCas9-based membrane-assisted detection platform for HR-HPV genotyping.

METHODS

A membrane-based assay integrating recombinase polymerase amplification (RPA) with CRISPR/dCas9-mediated sequence-specific recognition was developed. FAM-labeled amplicons were captured by immobilized dCas9-sgRNA ribonucleoprotein complexes and detected via antibody-mediated colorimetric readout.

RESULTS

The assay enabled specific detection of HPV16 and HPV18 using genotype-specific sgRNAs, producing visually interpretable signals on a nitrocellulose membrane. No signal was observed in negative controls, demonstrating high analytical specificity. Semi-quantitative signal assessment confirmed clear differentiation between positive and negative samples.

CONCLUSION

This study demonstrates the feasibility of a CRISPR/dCas9-based membrane-assisted detection system for HR-HPV genotyping. While not yet configured as a fully integrated lateral flow device, the platform provides a foundation for future development of simplified, point-of-care molecular diagnostics.