CRISPR/Cas13a-based Genome Editing For Establishing The Detection Method Of H9N2 Subtype Avian Influenza Virus

H9N2 subtype Avian influenza virus（AIV）is an influenza A virus that is transmitted between different bird species.In recent years,H9N2 AIV has significantly threat the poultry business by having became the predominant subtype in flocks of chickens,ducks,and pigeons.In addition to,the six internal genes of H9N2 AIV can frequently recombine with other influenza viruses to generate new influenza viruses that infect people,generating a significant public health threat to people.For pandemic prevention and control,early and quick diagnosis of the pathogen is crucial.However,existing diagnostic techniques for H9N2 AIV suffer from low sensitivity,low specificity,time-consuming and require specialized equipments and skilled personnel.Therefore,an accurate,quick and effective approach for the diagnosis of H9N2 AIV is essential.Almost all archaea and about half of bacteria have clustered regularly interspaced short palindromic repeats system-related proteins（CRISPR/Cas）adaptive immune systems.The CRISPR/Lw Cas13a in the CRISPR system has RNA cleavage activity to cleave single-stranded RNA in trans,and Cas13a also causes a trans cleavage effect on bystander RNA molecules.Using this cleavage activity in combination with recombinase-based isothermal amplification,Zhang Feng’s team created a nucleic acid detection method named SHERLOCK（Specific high-sensitivity enzymatic reporter unlocking）.Pathogen detection methods based on CRISPR/Cas13a technology are more suitable for practical applications because of their high specificity,sensitivity and ease of detection.The purpose of this research was to establish a new detection method for H9N2 AIV using CRISPR/Cas13a technology.The major studies and outcomes were as follows:1.Expression purification and activity identification of Lw Cas13a proteinThe expression of soluble Lw Cas13a protein was induced using E.coli.The soluble Lw Cas13a protein was purified using a His Trap^TM Ni Sepharose excel column,and a negative control was set up to detect the cleavage activity of Lw Cas13a protein.The results showed that the soluble Lw Cas13a protein could be successfully expressed in E.coli using a final concentration of 0.5 m M IPTG at 21°C.Relatively high purity Lw Cas13a protein was obtained after purification using His Trap^TM Ni Sepharose excel column.When the activity of Lw Cas13a protein was identified,The fluorescence signal of the experimental group was significantly different from that of the negative control group,which indicated that the purified Lw Cas13a protein had good RNAase cleavage activity.2.Establishment,optimization and evaluation of the H9N2 detection system based on CRISPR/Cas13a technologyThe 30 H9N2 AIV HA and NA genes published by NCBI were compared with the HA and NA genes of the H9N2 AIV strain used in this experiment,the conserved sequences were selected as templates,three RPA primer pairs were designed according to the RPA primer design principles,the target genes were amplified using RT-RPA technology,and the RPA primer pairs with the highest amplification efficiency were selected.Three cr RNAs were designed based on the cr RNA design principle,and the best cr RNA was selected by fluorescence intensity using CRISPR/Cas13a technology for nucleic acid detection of H9N2 subtype avian influenza virus.The best Cas13a protein concentration and cr RNA concentration were determined by fluorescence intensity.The results showed that the optimal concentration of Lw Cas13a protein was12.5 ng/μL,the optimal concentration of cr RNA was 0.9375 ng/μL.In order to evaluate the CRISPR/Cas13a-based H9N2 assay,a 10-fold dilution of treated H9N2 genomes was tested using a pre-optimised developed assay to determine the minimum detection limit of the assay based on fluorescence intensity.The specificity of the test was determined by whether generated a fluorescent signal using a pre-optimised test system for the detection of different viruses to which the birds were susceptible.The results showed that the minimum detection limit of the CRISPR-based H9N2 assay was 10⁰ copies/μL and the sensitivity was high.The H9N2detection system based on CRISPR/Cas13a technology showed no fluorescent signals when detecting different viruses susceptible to avian species except H9N2,confirming the high specificity of the detection system.In conclusion,this experiment has constructed a method CRISPR/Cas13a-based genome editing technology for detection of H9N2 subtype avian influenza virus.It can provides a new method and idea for the diagnosis of H9N2 subtype avian influenza virus,and also lays the base for the On-site detection of H9N2 subtype avian influenza virus.