CRISPR-Based Multiplex Detection of Human Papillomaviruses for One-Pot Point-of-Care Diagnostics

Abstract The World Health Organization’s global initiative toward eliminating high-risk Human Papillomavirus (hrHPV)-related cancers recommends DNA testing over visual inspection in all settings for primary cancer screening and HPV eradication by 2100. However, multiple hrHPV types cause different types of cancers, and there is a pressing need for a, easy-to-use, multiplex point-of-care diagnostic platform for detecting different hrHPV types. Recently, CRISPR-Cas systems have been repurposed for point-of-care detection. Here, we established a CRISPR -Cas multiplexed d iagnostic assay (CRISPRD) to detect cervical cancer-causing hrHPVs in one reaction (one-pot assay). We harnessed the compatibility of thermostable AapCas12b, TccCas13a, and HheCas13a nucleases with isothermal amplification and successfully detected HPV16 and HPV18, along with an internal control in a single-pot assay with a limit of detection of 10 copies and 100% specificity. This platform offers a rapid and practical solution for multiplex detection of hrHPVs, facilitating large-scale hrHPV point-of-care screening, guiding effective treatment and policy development, and supporting global health initiatives. Furthermore, the CRISPRD platform’s programmability enables it to be adapted for simultaneously detecting hrHPVs-causing cancer, strengthening the scope of early diagnosis of HPV in the fight against infectious disease.