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,de-pendence 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 ar-ticle reviewed recent advancements in CRISPR-based amplification-free nucleic acid detection technologies.Ac-cording 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 ar-ticle 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 sys-tem,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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