Study Of CRISPR-Cas13a-Based Technology For Detection And Clearance Of Influenza A Virus

Objective:In this study,we designed targets for the nucleoprotein(NP)and neuraminidase(NA)fragments of the influenza A virus(IAV)gene,and constructed a general and specific target database based on the CRISPR-Cas13a system.The CRISPR-Cas13a technology was combined with Reverse transcription recombinase aided amplification(RT-RAA)and colloidal gold immunochromatography strips to achieve accurate,efficient and rapid detection of IAV in the field.The aim is to rapidly identify IAV and its drug-resistant mutants in the field;to perform IAV clearance experiments with universal detection targets and to screen for efficient delivery vectors to achieve efficient clearance of multiple IAVs with high prevalence in society at the nucleic acid level,thereby solving the public health problems caused by IAV.Methods:1.The designed and synthesized crRNA was transcribed and purified in vitro,and the RT-RAA primers were designed and synthesized for amplification of IAV nucleic acid samples.The experimental group used the amplification product as a sample,and the control group used the enzyme-free water as a sample for the test.The amplification product combined with the purified crRNA was used to identify,activate CRISPR-Cas13a and cut the report RNA to capture the fluorescence emitted by the report RNA.The detection of IAV and its drug resistance sites was detected by comparing the fluorescence intensity of the experimental group and the control group.At the same time,the colloidal gold immunochromatographic test strip is used to carry out the"color development method"detection of the above detection targets.Add the amplified nucleic acid sample to CRISPR-Cas13a detection system for incubation for 30 minutes,and then insert the end of the test strip into it.Observe whether the test strip detection line develops color.Fluorescence detection and immunochromatographic strip method were used to detect samples with different copy numbers to determine the lower detection limit of the two detection methods;and use two methods to detect IAV and other respiratory transmitted viruses,and judge the specificity of the two detection methods based on the detection results.2.The two synthesized crRNA single strands were annealed to form a double strand,and the crRNA double strand was inserted into Cas13a backbone by enzyme digestion and enzyme linkage,and then the digested enzyme linkage product was introduced into DH5a,and the successfully linked plasmid was selected for plasmid extraction,and the extracted plasmid was delivered into MDCK cells using a delivery vector.After 24h,the nucleic acid extracts from the cell supernatants were quantified by RT-q PCR,and the CT values between the experimental and control groups were compared to calculate the clearance efficiency of the corresponding target sites for IAV.3.The same CRISPR-crRNA system was delivered using lipofectamine 2000 and jet OPTIMUS respectively.The cytotoxicity and delivery efficiency of the two different delivery media were compared from the aspects of cell morphology,viability,fluorescent transfection effect diagram and IAV clearance efficiency after delivery.Results:1.Seven IAV universal and specific crRNAs were screened from the conserved region of nucleoprotein(NP)gene fragment.The detection limit of this method based on CRISPR-Cas13a for IAV can reach 1×10~0 copies/μL;It can be specifically distinguished from other 6 respiratory viruses;H274Y mutation site of H1N1 virus and H274Y and R292K mutation site of H3N2 virus were detected.2.Compared with lipofectamine 2000,the cell morphology of the new nanomaterial jet OPTIMUS did not change significantly after transfection,and the relative luminescence values of the lipofectamine 2000 and jet OPTIMUS groups were 79,400 and 86,400,respectively,in the cell viability assessment assay;when transfected with equal amounts of CRISPR-Cas13a clearance The average clearance efficiency of jet OPTIMUS versus lipofectamine 2000 was increased by 21.97%±13.76%when transfected with the same clearance target.13.76%.3.The universal clearance target has a maximum clearance rate of 91.4%for H1N1 and96.1%for H3N2.Conclusion:1.The in situ IAV detection technique created in this study is comparable in sensitivity to the RT-q PCR method,and it does not rely on professional operators or expensive instrumentation;it allows for rapid and instant portable detection of multiple types of IAV in situ with a single target,with a lower limit of detection of 1×10~0 copies/μL and high stability of results.2.On-site instant detection of common IAV resistance loci based on the CRISPR-Cas13a system allows for the first identification of drug-resistant mutant strains and guidance of drug administration based on their mutation information.3.The use of generic IAV clearance targets can achieve the clearance of multiple types of IAV and expand the therapeutic range of IAV,and the generic clearance targets for H1N1and H3N2 have been initially obtained and are being repeatedly validated and optimised.4.JetOPTIMUS is a new delivery vector that is safe,cost-effective and highly efficient in delivery.The use of its delivery CRISPR-Cas13a clearance system has increased clearance efficiency by approximately 22%compared to the lipo2000 delivery vector group,and both have clearance efficiencies above 90%for IAV.