Advances in Amplification-Free Nucleic Acid Detection Technologies Based on CRISPR/Cas System

doi:10.12141/j.issn.1000-565X.230705

Abstract

Abstract:

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.

Key words: CRISPR/Cas, nucleic acid detection, amplification-free, signal amplification

CLC Number:

TS207.3

SHEN Xing, LI Yan, ZHANG Xu, GAI Zuoqi, LIU Yan, HUANG Yingyin, LEI Hongtao, CHEN Jiahong. Advances in Amplification-Free Nucleic Acid Detection Technologies Based on CRISPR/Cas System[J]. Journal of South China University of Technology(Natural Science Edition), 2025, 53(1): 136-146.

Figures/Tables 6

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免核酸扩增策略	优点	缺点
Cas介导的级联或生化回路	检测方便，灵敏度高	探针设计复杂
电化学传感	灵敏度较高	稳定性差，电极片可重复利用性低，部分芯片制造昂贵
微体积CRISPR/Cas系统	样品需求量少，可拓展性强	灵敏度较低，微流控芯片制造复杂
优化信号报告物	设计简单，可拓展性强	灵敏度低，有时需要进行额外的修饰或偶联