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

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.