State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), Collaborative Innovation Center for Guangxi Ethnic Medicine, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin, 541004, China.
Mikrochim Acta. 2024 Jun 7;191(7):376. doi: 10.1007/s00604-024-06452-8.
CRISPR/Cas system has been widely applied in the assay of disease-related nucleic acids. However, it is still challenging to use CRISPR/Cas system to detect multiple nucleic acids at the same time. Herein, we combined the preponderance of DNA logic circuit, label-free, and CRISPR/Cas technology to construct a label-free "AND" logical gate for multiple microRNAs detection with high specificity and sensitivity. With the simultaneous input of miRNA-155 and miRNA-141, the logic gate starts, and the activation chain of Cas12a is destroyed; thus, the activity is inhibited and the fluorescence of the signal probe ssDNA-AgNCs is turned on. The detection limit of this method for simultaneous quantitative detection of double target is 84 fmol/L (S/N = 3). In this "AND" logic gate, it is only necessary for the design of a simple DNA hairpin probe, which is inexpensive and easy, and since this method involves only one signal output, the data processing is very simple. What is more important, in this strategy two types of microRNAs can be monitored simultaneously by only using CRISPR/Cas12a and a type of crRNA, which offers a new design concept for the exploitation of single CRISPR/Cas system for multiple nucleic acid assays.
CRISPR/Cas 系统已广泛应用于疾病相关核酸的检测。然而，利用 CRISPR/Cas 系统同时检测多种核酸仍然具有挑战性。在此，我们结合 DNA 逻辑电路、无标记和 CRISPR/Cas 技术的优势，构建了一种无标记的“与”逻辑门，用于高特异性和高灵敏度的多种 microRNAs 检测。当同时输入 miRNA-155 和 miRNA-141 时，逻辑门开始工作，Cas12a 的激活链被破坏，从而抑制了活性，信号探针 ssDNA-AgNCs 的荧光被打开。该方法同时定量检测双靶标的检测限为 84 fmol/L（S/N = 3）。在这个“与”逻辑门中，只需要设计一个简单的 DNA 发夹探针，既经济又简单，而且由于该方法只涉及一个信号输出，数据处理非常简单。更重要的是，在这种策略中，只需使用 CRISPR/Cas12a 和一种 crRNA 就可以同时监测两种类型的 microRNAs，为单个 CRISPR/Cas 系统用于多种核酸检测提供了新的设计理念。
