The Study On New Methods For Nucleic Acid Isothermal Detection Based On CRISPR/Cas Protein

Nucleic acid analysis and detection are of great significance to basic research in life sciences and human health.The traditional polymerase chain reaction(PCR)relies on a thermal cycler and is not suitable for testing in remote,poor places without related instruments.Nucleic acid isothermal amplification technology has got rid of the dependence on sophisticated equipment,and has shown good application prospects in clinical and on-site rapid diagnosis.However,the current isothermal amplification technology still has some problems in terms of sensitivity,specificity,and scope of application.The CRISPR/Cas system,as a site-specific genome editing technology derived from the acquired immune system of prokaryotes,has attracted a lot of scientific attention since its discovery.In recent years,it has been widely used in clinical nucleic acid analysis and detection and biosensing technology.In this paper,we used the unique nucleic acid cleavage activity of CRISPR/Cas protein to develop new methods for rapid detection of microRNA and isothermal amplification of nucleic acid.First,we took advantage of Cas 12a trans-cleavage activity to develop a multi-enzyme cascade reaction method for microRNA(miRNA)specific detection.In the presence of the target miRNA,RNA ligase initiated the ligation reaction to generate a transcription template containing the T7 promoter.T7 RNA polymerase transcribed and synthesized a large number of RNA sequences containing crRNA and 8-17E DNAzyme substrate.8-17E DNAzyme specifically cleaved the RNA sequence to generate crRNA and RNA with the same sequence as the target miRNA.After crRNA bound to Cas12a,the free double-stranded DNA molecule in the reaction system specifically activated the trans-cleavage activity of CRISPR/Cas 12a,resulting in the single-stranded fluorescent probe being cleaved and generating fluorescence.The quantitative analysis of miRNA was realized by detecting the fluorescent signal.The detection limit of this method was 21.9 fM,and it could be applied to the quantitative analysis of miRNA in different cell lysates.Second,we prepared the Cas9n-Klenow fusion protein to improve the universal isothermal DNA amplification method(Cas9nAR)coupled by two enzymes(CRISPR Cas9n and DNA polymerase).A plasmid expressing the Cas9n-Klenow fusion protein was constructed.The fusion protein was expressed and purified in vitro,and proved to have nickase and polymerase activities.Two groups of sgRNA:Cas9n-Klenow complexes combined with the target DNA sequence adjacent to the protospacer adjacent motif(PAM),and a target ssDNA sequence was obtained by the single-stranded nicking activity and strand displacement activity of the fusion protein.Under the action of sgRNA:Cas9n-Klenow complex and primers 1 and 2 with Cas9n cleavage sequence,a large number of target dsDNA sequences were obtained via cyclic processes of primer hybridization,extension,nicking,and replacement reactions.SYBR Green I fluorescent dye combined with dsDNA products to produce fluorescence,and the fluorescence intensity was proportional to the concentration of dsDNA products.The Cas9n-Klenow fusion protein was used to successfully amplify the target sequence at a constant temperature of 37?,proving that it can be applied to the Cas9nAR method.