PAM-Free Activation of CRISPR/Cas12a via Semi-Nested Asymmetric RPA: High Specific Detection of HPV16 dsDNA

Abstract

Early and accurate detection of HPV16 nucleic acids is therefore critical for the effective screening, diagnosis, and prevention of cervical cancer. Although CRISPR/Cas12a-based molecular diagnostics offer a rapid and sensitive approach for HPV16 detection, their application to double-stranded DNA (dsDNA) targets remain constrained by two major limitations: the strict requirement for a protospacer adjacent motif (PAM) site, and the insufficient specificity of current amplification strategies, which can lead to off-target amplification and false-positive results. To address these challenges, we developed a semi-nested asymmetric recombinase polymerase amplification (SNA-RPA) method combined with CRISPR/Cas12a for the detection of HPV16 dsDNA. This strategy employs a semi-nested primer design to significantly enhance target sequence specificity during amplification, while asymmetric primer ratios promote the efficient generation of single-stranded DNA (ssDNA) that directly activates Cas12a without the need for a PAM site. Using this approach, we achieved rapid, highly specific, and detection of HPV16 dsDNA, with a limit of detection as low as 18 aM. Beyond achieving PAM-free detection, our method also substantially improves amplification fidelity, offering a promising solution for precise and reliable HPV diagnostics and cervical cancer screening

Supplementary files

Article information

Article type
Paper
Submitted
03 May 2025
Accepted
14 Jul 2025
First published
16 Jul 2025

Analyst, 2025, Accepted Manuscript

PAM-Free Activation of CRISPR/Cas12a via Semi-Nested Asymmetric RPA: High Specific Detection of HPV16 dsDNA

X. Zou, T. Gu, X. Li, L. Deng, S. Zhu, J. Dong, F. Deng, D. Huo and C. Hou, Analyst, 2025, Accepted Manuscript , DOI: 10.1039/D5AN00491H

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