Application Of CRISPR/Cas9 Technology Construction Of Recombinant Pseudorabies Virus And Biological Analysis

Pseudorabies virus(PRV),belonging to herpesviruses,has a double-stranded DNA genome.The virus gene has a strong foreign gene capacity,which can be used as a viral gene carrier.PRV infects a broad range of wild and domestic animals,including cattles,sheeps,mouse and fox.PRV-infected animals exhibit neurological signs and abortions.Pseudorabies virus has caused enormourous economic losses to the breeding industry in the whole world.In our country,PR is usually controled by prevention.However,in recent years,porcines immunized gE gene-deleted vaccine showed severe,stillbirth and neurological symptoms.And the research of pseudorabies virus on porcines relatively deeper than wild animals.At present,recombinant virus is usually obteined by the method of homologous recombination with a low efficiency.So,it is urgent to develop a simple and rapid method to construct PRV vacinne.In this study,genome PRV-HB1 from fox was edited by CRISPR/Cas9 system rapidly.Firstly,the Cas9-GFP gene was amplified by PCR and cloned into transfer vector.The recombinant plasmid was transfected into HEK293T cells with adenovirus backbone plasmid to produce recombinant adenovirus.The recombinant virus was amplified and purified,and its titer was measured.The recombinant adenovirus was rescued and identified by Western blot,and the results showed that the recombinant adenovirus Ad-Cas9-GFP expressing Cas9-GFP protein was constructed successfully.TK and gE genes are two of PRV genes which are closely related to PRV virulence.It has been reported that PRV strains with TK or gE gene-deleted can weaken its virulence obviously without affecting its replication ability.In this study,sgRNAs targeting TK and gE open reading frames were designed and shuttle plasmids were constructed.Subsequently,the shuttle plasmids were cotransfected with PRV-HB1 genome DNA into HEK293T cells which were infected by recombinant adenovirus later.The gene-deleted strain rPRV-TK~-was purified by T7E1 enzyme,plaque purification and PCR amplication.On the basis of this,the gE gene was disrupted and the gene-deleted strain rPRV-TK~-/gE~-was obtained.Meanwhile the transfer vector carrying EGFP gene was constructed.EGFP cassette was inserted into viral genome through the error-prone non-homologous end joining(NHEJ)mediated DNA repair mechanism,and recombinant strain rPRV-TK~-/EGFP~+was obtained.One-step curves of the viruses rPRV-TK~-and rPRV-TK~-/gE~-were similar but weaker than parent virus.Animals tests showed that these viruses were safe to mouse with reduced virulence,with the possibility of being developed as a vaccine.In this study,recombinant pseudorabies virus was constructed by CRISPR/Cas9 system rapidly,which established an efficient technical platform for the development of genetic engineering vaccines to wild animals.