PAM-free activation of CRISPR/Cas12a via semi-nested asymmetric RPA: highly specific detection of HPV16 dsDNA

Jul 25, 2025The Analyst

Highly specific detection of HPV16 DNA using CRISPR/Cas12a activated without PAM by semi-nested asymmetric DNA amplification

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Abstract

A limit of detection as low as 18 aM was achieved for HPV16 dsDNA using a novel detection method.

The semi-nested asymmetric recombinase polymerase amplification (SNA-RPA) method enhances specificity for target sequences.
Asymmetric primer ratios facilitate the efficient production of single-stranded DNA (ssDNA) that activates Cas12a without requiring a protospacer adjacent motif (PAM) site.
This approach offers rapid and highly specific detection of HPV16 dsDNA.
Improved amplification fidelity is associated with the new method, potentially reducing the risk of false-positive results.

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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 remains 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 and highly specific 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.

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PAM-free activation of CRISPR/Cas12a via semi-nested asymmetric RPA: highly specific detection of HPV16 dsDNA

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