Jiang Qingyuan, Jin Shuqi, Qin Zhichao, Zhang Junqi, He Ruyi, Chen Zhuo, Qiao Bin, Qiao Jie, Liu Yi
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Hubei 430042, China.
Pilot Base of Food Microbial Resources Utilization of Hubei Province, School of Life Science and Technology, Wuhan Polytechnic University, Hubei 430023, China.
Nucleic Acids Res. 2025 Sep 5;53(17). doi: 10.1093/nar/gkaf893.
The CRISPR/Cas12a technology has revolutionized molecular diagnostics. However, existing Cas12a systems depend on continuous target DNA activation, which limits them to single-target detection. In this study, we developed a novel topology-guided Cas12a system, the double-target responsive (DTR) system, capable of being activated by noncontiguous dual RNA/DNA targets. The DTR system employs two split CRISPR RNA (crRNA) fragments and two Cas12a proteins that cooperatively reconstitute upon recognizing two nucleic acid activators. We demonstrated the DTR system's ability to specifically detect dual nucleic acid substrates in a single readout, achieving a detection limit of 78 fM for RNA and exceptional specificity for single-nucleotide variations. Additionally, we successfully applied the DTR system to clinical samples, enabling simultaneous detection of two oral squamous cell carcinoma-related microRNAs (miR-155 and miR-let-7a), thereby distinguishing healthy individuals from patients. This work establishes an efficient Cas12a-based platform for sensitive, simultaneous, and discriminative detection of RNA and DNA targets, enhancing the versatility of Cas12a in analytical detection and clinical diagnosis.
CRISPR/Cas12a技术彻底改变了分子诊断。然而，现有的Cas12a系统依赖于持续的靶DNA激活，这限制了它们只能进行单靶标检测。在本研究中，我们开发了一种新型的拓扑结构引导的Cas12a系统——双靶标响应（DTR）系统，它能够被不连续的双RNA/DNA靶标激活。DTR系统采用两个分开的CRISPR RNA（crRNA）片段和两个Cas12a蛋白，它们在识别两个核酸激活剂时协同重组。我们证明了DTR系统能够在一次读数中特异性地检测双核酸底物，实现了对RNA 78 fM的检测限以及对单核苷酸变异的卓越特异性。此外，我们成功地将DTR系统应用于临床样本，能够同时检测两种口腔鳞状细胞癌相关的微小RNA（miR-155和miR-let-7a），从而区分健康个体和患者。这项工作建立了一个基于Cas12a的高效平台，用于灵敏、同时且有区分性地检测RNA和DNA靶标，增强了Casl2a在分析检测和临床诊断中的多功能性。
