Preparation Of PRV-targeted CRISPR/Cas9-sgRNA Complex And Its Antiviral Effect Research

Pseudorabies is an infectious disease of domestic animals caused by Pseudorables virus(PRV).The disease has severely affected animal husbandry and threatened human health.PRV belongs to the genus Herpesvirus,a neurotropic virus that can establish a latent infection in the infected host.The PRV DNA in latent infection is in a transcriptional silent state,which is difficult to detect and identify by conventional serological methods,and the existing vaccines have little effect on the prevention and control of PRV in latent infection,which makes it difficult to completely eliminate PRV from pig farms.Studies have shown that CRISPR/Cas-based gene programming technology can effectively interfere with the early infection of herpes virus and prevent the latent infection of the virus.However,there is the problem of Cas9 protein stimulating immune response and off-target effects.It has been reported that the pre-assembled Cas9 and g RNA complex(ribonucleoprotein,RNP)can effectively overcome these problems due to its short half-life and other characteristics,and is expected to be used to eliminate latent infection of PRV.However,existing RNP pre-assembly methods exist Insufficiency such as high cost and long cycle.This study will explore the efficient,low-cost,and short-period preparation method of Cas9 RNP complex targeting PRV genome,and detect the effect of Cas9 RNP complex interfering with PRV replication in PK-15 cells.In the early stage of the laboratory,a new method for efficiently preparing Cas9 RNP was established,that is,the modified cold shock vector ptac-Cas9-T7sgRNA was used to co-express Cas9 protein and sgRNA in E.coli,and the co-expressed Cas9 protein and sgRNA can self-assemble into Cas9 RNP complex in bacteria.This method can prepare Cas9 RNP complex with high efficiency and low cost,which will greatly expand its application.In this study,we selected IE180,EP0,genes related to early PRV replication,and genes related to viral virulence,US8,US7,UL23,as targets,and then constructed a co-expression vector for the Cas9 RNP complex of these 5 genes.Firstly,the co-expression vectors were transferred into E.coli cells respectively,and the optimal induction conditions were obtained by screening:the final concentration of IPTG was 1 m M,the culture temperature was 18?,and the expression was induced in a shaker at 220 rpm for 16 h.Then ultrasonically break the harvested cells,and use nickel beads to purify the recombinant protein.The purity of the Cas9RNP can reach more than 90%,and about 7 mg of Cas9 RNP complex can be obtained from 1L of bacterial culture.Using laboratory efficient vector construction methods and protein purification methods,from vector construction to high-purity Cas9 RNP complex of target genes,the entire cycle can be completed in only 3 days.This experiment further tested the ability of co-expressing Cas9 RNP complex to inhibit virus replication in cells.Firstly,PK-15 cells were infected with different doses of PRV(2000×TCID₅₀,200×TCID₅₀,20×TCID₅₀),and then the Cas9 RNP complex was transferred to the cells with transfection reagent,and finally the supernatant of the infected cells was harvested.The TCID₅₀ experiment was used to detect the virus titer in the supernatant,and the effect of each anti-PRV Cas9 RNP complex on inhibiting virus proliferation was further evaluated.The test results confirmed that Cas9 RNPs targeting the EP0,IE180 and TK genes of PRV can significantly inhibit virus proliferation on cells infected with 200×TCID₅₀ and2000×TCID₅₀ doses,but cells infected with 20×TCID₅₀ doses There is no obvious inhibitory effect.And through sequence determination,we found that the Cas9 RNPs targeting the EP0,IE180 and TK genes of PRV had a significant editing effect on the newly generated virus.We further explored whether multiple Cas9 RNPs targeting different genes of PRV can jointly inhibit PRV replication.PK-15 cells were infected with PRV virus at a dose of2000×TCID₅₀ first,and then co-transformed into Cas9 RNPs targeting the EP0 and TK genes of PRV.The results showed that when the two were added together,there was a significant superimposed anti-virus proliferation effect.The experiment further confirmed that when the cell mass is 10^6 and the amount of infected virus does not exceed 200×TCID₅₀,at least 2.5?g of Cas9 RNP can effectively inhibit the proliferation of the virus.Finally,we explored whether anti-PRV Cas9 RNPs can interfere with PRV replication through fluorescence quantitative experiments.We selected PRV US6 as the target gene.Experimental results showed that the US6 mRNA level in cells transfected with Cas9RNP targeting the PRV TK gene was reduced by 20%,and the US6 mRNA level in cells transfected with Cas9 RNP targeting the PRV EP0 gene was reduced by 80%.However,the US6 mRNA level in the cells transferred by the two was reduced by 90%,and there was an obvious superimposing effect.The above results confirm that Cas9 RNP targeting the PRV genome can inhibit the replication of PRV in cells,and when the two work together,they show a significant synergistic inhibitory effect.This article lays the foundation for the study of the anti-PRV Cas9 RNP co-expressed in Escherichia coli on the elimination of PRV virus.