收稿日期: 2023-11-13
网络出版日期: 2024-02-27
基金资助
国家自然科学基金项目(32072316)
Advances in Amplification-Free Nucleic Acid Detection Technologies Based on CRISPR/Cas System
Received date: 2023-11-13
Online published: 2024-02-27
Supported by
the National Natural Science Foundation of China(32072316)
成簇规则间隔短回文重复序列（CRISPR）是来源于古细菌的一种免疫系统，可在特定向导RNA的引领下对外来核酸进行识别与切割。由于具有高特异性靶标识别能力以及靶激活的核酸酶活性，近年来该系统被广泛应用于核酸检测领域。为实现高灵敏检测，成簇规则间隔短回文重复序列及相关基因编码的系统（CRISPR/Cas）通常需与预扩增技术相结合，但这同时带来了扩增子气溶胶污染、依赖专有设备、检测时间延长等问题。因此，许多研究人员致力于开发免扩增CRISPR核酸检测技术以解决上述限制。CRISPR/Cas靶向激活的高周转率非特异性切割活性为这种技术的开发提供了可能性，利用各种提高系统反式切割效率或者增强信号的策略，许多免扩增CRISPR核酸检测技术被成功开发出来。该文综述了近年开发的基于CRISPR的免扩增核酸检测技术，依据策略不同将这些技术分为Cas效应器的联用或构筑生化回路、电化学传感、微体积CRISPR/Cas系统、优化信号报告物等4个方面，并从策略的角度分析了这些技术实现免扩增核酸检测的原理，即通过累积多个蛋白复合物传导的信号、增强信号传感能力、提高反应体系浓度、放大报告底物信号等原理实现灵敏度提升。基于已有的技术原理，该文对几种策略的优缺点进行讨论。此外，该文进一步展望免扩增CRISPR核酸检测技术的发展趋势并提出未来可能的研究方向，为开发更快速、灵敏、简便的分子检测技术以促进其更深入的应用提供参考。
关键词： CRISPR/Cas; 核酸检测; 免扩增; 信号放大
沈兴 , 李妍 , 张旭 , 盖作启 , 刘艳 , 黄颖茵 , 雷红涛 , 陈佳虹 . 基于CRISPR/Cas系统的免扩增核酸检测技术研究进展[J]. 华南理工大学学报(自然科学版), 2025 , 53(1) : 136 -146 . DOI: 10.12141/j.issn.1000-565X.230705
Clustered regularly interspaced short palindromic repeats (CRISPR) are an immune system derived from archaea that recognizes and cleaves alien nucleic acids, with the assistance of specific guide RNAs. Due to the highly specific target recognition ability and target-activated nuclease activity, the system has been widely used in nucleic acid detection in recent years. To achieve highly sensitive detection, CRISPR / Cas systems usually need to be combined with pre-amplification technology, but this also brings problems with amplicon aerosol pollution, dependence on proprietary equipment, and prolonged detection time. Thus, many researchers have focused on deve-loping amplification-free CRISPR nucleic acid assays to deal with the above limitations. The high turnover non-specific cleavage activity of CRISPR / Cas targeted activation provides the possibility of developing this technology, and a number of amplification-free CRISPR nucleic acid detection technologies have been successfully developed using various strategies to improve the efficiency of trans-cleavage of the system or to enhance the signal. This article reviewed recent advancements in CRISPR-based amplification-free nucleic acid detection technologies. According to different strategies, these technologies are divided into four aspects: the combination or construction of Cas effectors, electrochemical sensing, microvolume CRISPR / Cas system, and signal reporter optimization. The article also analysed the principles of these technologies to achieve amplification-free nucleic acid detection from the perspective of strategies, that is, to achieve sensitivity enhancement through the accumulation of multiple protein complex-conducted signals, enhancement of signal sensing ability, increasing the concentration of the reaction system, and amplification of the signal. Based on the existing technical principles, the article discussed the advantages and disadvantages of several strategies. In addition, the article further outlooked the development trend of amplification-free CRISPR nucleic acid detection technology and proposes possible future research directions, which will provide a reference for the development of more rapid, sensitive, and simple molecular detection techno-logy to promote its deeper application.
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