Qiao Jie, Zhang Junqi, Jiang Qingyuan, Jin Shuqi, He Ruyi, Qiao Bin, Liu Yi
Pilot Base of Food Microbial Resources Utilization of Hubei Province, School of Life Science and Technology, Wuhan Polytechnic University, Hubei 430023, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Hubei 430042, China.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf510.
The CRISPR/Cas12a [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a] system is known for its intrinsic RNA-guided trans-cleavage activity; however, its RNA detection sensitivity is limited, with conventional methods typically achieving detection limits in the nanomolar range. Here, we report the development of a "pseudo hybrid DNA-RNA" (PHD) assay that significantly enhances the RNA detection capability of Cas12a. The PHD assay achieves a striking detection limit of 7.7 pM using single CRISPR RNA (crRNA) and 33.8 fM using pooled crRNAs. Importantly, this assay exhibits ultra-high specificity, capable of distinguishing mutated RNA target sequences at the protospacer adjacent motif (PAM)-distal region. It can also detect ultrashort RNA sequences as short as 6-8 nt and long RNAs with complex secondary structures. Additionally, the PHD assay enables PAM-free attomolar-level DNA detection. We further demonstrate the practical utility of the PHD assay by successfully detecting miR-155 biomarkers and human pappilloma virus 16 DNA in clinical samples. We anticipate that the design principles established in this study can be extended to other CRISPR/Cas enzymes, thereby accelerating the development of powerful nucleic acid testing tools for various applications.
CRISPR/Cas12a（成簇规律间隔短回文重复序列/CRISPR相关蛋白12a）系统以其固有的RNA引导的反式切割活性而闻名；然而，其RNA检测灵敏度有限，传统方法的检测限通常在纳摩尔范围内。在此，我们报告了一种“假杂交DNA-RNA”（PHD）检测方法的开发，该方法显著提高了Cas12a的RNA检测能力。使用单个CRISPR RNA（crRNA）时，PHD检测方法的检测限低至7.7 pM，使用混合crRNAs时为33.8 fM。重要的是，该检测方法具有超高的特异性，能够区分原间隔序列临近基序（PAM）远端区域的突变RNA靶序列。它还能检测短至6-8个核苷酸的超短RNA序列以及具有复杂二级结构的长RNA。此外，PHD检测方法能够实现无PAM的阿托摩尔级DNA检测。我们通过成功检测临床样本中的miR-155生物标志物和人乳头瘤病毒16型DNA，进一步证明了PHD检测方法的实际应用价值。我们预计，本研究中确立的设计原则可扩展到其他CRISPR/Cas酶，从而加速开发适用于各种应用的强大核酸检测工具。
