Rapid Virulent Genes Knockout Of Pseudorabies Virus Using The CRISPR/Cas9 System

Pseudorabies(PR),is also known as Aujeszky's disease,is caused by porcine herpes simplex virus type I(Suid heipes virus I,Su HV-I),also known as pseudorabies virus(PR virus,PRV)that infected pigs,cattle and sheep.PRV can infect a variety of livestock and wild animals,its main features were itching,fever,encephalomyelitis,respiratory system and nervous system disorders.Vaccination of classic attenuated PRV Bartha-K61 vaccine is one of the effective ways to prevent pseudorabies.However,in recent years,many regions of China outbreak of a new pseudo-rabies which vaccinated Bartha-K61 vaccine.Studies have shown that the current prevalence of PRV was a variant which belong to the PRV gene type II.The traditional Bartha-K61 vaccine failed to provide complete protection against the currently variants.Therefore,a quick method to attenuated current epidemic strain is nesscery for effective prevention and control of PRV.Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)RNA system is an RNA-mediated acquired immune system that is widely found in bacteria and archaebacteria.The immune system provides host cells with a foreign gene(Such as phage plasmid)immune function.In type II CRISPR systems CRISPR-Derived RNA(cr RNA)and Trans actating cr RNA(tracr RNA)forms a complex by reverse-translating with a base pair that specifically recognizes the genomic sequence and directs the Cas9 endonuclease in the desired fragment cut the DNA double strand breaks(DSBs).And through the artificial design of these two RNA,the formation of a guide role of sg RNA(single guide RNA).By expressing the original expression of single guide RNA(sg RNA)and expressing the original of cas9 to obtain a plasmid capable of expressing both at the same time.After transfecting with CRISPR-Cas9 plasmid,certain virus was used to infect when the cas9 endonuclease was expressed which is enough to edit PRV genome and produce gene-knockcout virus.CRISPR-Cas9,an efficient genomic editing function system,has been used to edit large viral genomes,including HIV-I,Adenoviruses(ADV)etc.In this study,the PRV genome was quickly edited by CRISPR/Cas9 technique.To determine whether viral DNA cleavage by designed sg RNA,we screened sg RNA by viral plaque formation.We first transfected sg RNA and then infected with He N1,and viruses were purified by plaque assay.The gene deletion verified at protein level.The study successfully constructed a g E gene-deficient strain g E-PRV.The replication ability of g E-PRV was assessed by Vero cells.The results showed that the pathogenicity in susceptible animal mice was nosignificant difference compared to parental strain.The g E-/g I-PRV gene was successfully constructed and evaluated in mice.The results showed that the g E-/g I-PRV gene-deficient strain had no pathogenicity to mice.The g E/g I and TK tri-gene deletion strains were successfully constructed on the basis of g E-/g I-PRV gene and were evaluated in mice.The results showed that the pathogenicity of g E-/g I-/TK-PRV strain was significantly decreased compared with the parent strain He N1.At the same time g E-/g I-/TK-PRV strain can also induce robust immune response that protecte mice from PRV He N1 infection.This study demonstrates that CRISPR-Cas9 technology can rapidly attenuate PRV virus,which is critical for the current control of PRV,especially when the current variant PRV strain occurs.