A PAM-Free One-Step Asymmetric RPA and CRISPR/Cas12b Combined Assay (OAR-CRISPR) for Rapid and Ultrasensitive DNA Detection

文献类型： 外文期刊

第一作者： Yang, Lei

作者： Yang, Lei;Chen, Guanwei;Wei, Wei;Peng, Cheng;Ding, Lin;Chen, Xiaoyun;Xu, Xiaoli;Wang, Xiaofu;Xu, Junfeng;Chen, Guanwei;Wu, Jian

作者机构：

期刊名称：ANALYTICAL CHEMISTRY （ 影响因子：7.4； 五年影响因子：7.0 ）

ISSN： 0003-2700

年卷期： 2024 年 96 卷 14 期

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收录情况： SCI

摘要： Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/mu L DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.

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