Pilot Base of Food Microbial Resources Utilization of Hubei Province, School of Life Science and Technology, Wuhan Polytechnic University, Wuhan, Hubei, 430023, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, Hubei, 430042, China.
Nat Commun. 2024 Sep 27;15(1):8342. doi: 10.1038/s41467-024-52691-x.
The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system's specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings.
CRISPR-Cas12a 系统凭借其快速、精准的特性革新了核酸检测（NAT），但其传统上需要 RNA 预扩增。在这里，我们开发了一种利用分割 Cas12a 系统（SCas12a）的快速荧光和横向流动 NAT 检测方法，该系统由 Cas12a 酶和分割 crRNA 组成。SCas12a 检测法能够灵敏、无需扩增且对 miRNA 和长 RNA 进行多重检测，即使它们具有复杂的二级结构。它可以区分成熟 miRNA 和其前体（pre-miRNA），这对于精确的生物标志物识别和癌症进展监测至关重要。该系统的特异性还体现在其能够检测 DNA 和 miRNA 点突变的能力上。值得注意的是，SCas12a 系统能够定量检测宫颈癌患者血浆中的 miR-21 生物标志物，并且与 RPA 结合使用时，能够在临床样本中检测到 HPV 的皮摩尔水平。总之，我们的工作提出了一种简单、经济高效的基于 SCas12a 的 NAT 平台，适用于各种诊断环境。
