In Vitro Effect Of CRISPR/Cas9 System On Pseudorabies Virus Proliferation

Pseudorabies virus(Suid herpesvirus 1 or PRV)is a member of the genus Varicellovirus,family Herpesviridae.PRV is not only an important pathogen of swine,resulting in devastating disease and economic losses worldwide,but also infects nearly all other mammals.Although this disease has successfully been controlled by different measures in several countries,PRV remains one of the most important pathogens worldwide.In recent years,the outbreak of a PRV variant in China resulted in a serious burden to the pig industry.Moreover,mounting evidence shows that PRV infections are more widespread in wild swine across the world.Therefore,it is necessary to develop a method for rapid detection and a strategy to effectively control PRV infection.In present study,we aimed to develop a rapid,sensitive and specific SYBR Green-based real time PCR assay for the molecular diagnosis of PRV.A pair of primers was designed according to highly conserved g D gene of PRV,yielding a 195 bp product.Sensitivity,reproducibility and specificity analysis of the SYBR Green real time PCR was evaluated.The results showed that specific PRV amplicons melted at 92 °C and there were no primer-dimers and non-specific products in negative control and other viruses.The SYBR Green real time PCR is sensitive to detect as low as 100 copies/?L standard DNA of PRV,which can be used for rapid and accurate detection of PRV.The CRISPR-Cas9 system provides an invaluable tool to target and modify the genomic sequence with high levels of efficacy and specificity.The system,consisting an RNA-guided nuclease(Cas9)and guide RNA(g RNA)complementaryto a target sequence,enables the sequence-specific cleavage of a target locus of the genome.A recent study reported the use of the CRISPR-Cas9 system to inhibit virus infection by targeting conserved regions of the viral genomic DNA.In the present study,we constructed a cell line stably expressing Cas9 endonuclease and sg RNA targeting the conserved UL30 gene of pseudorabies virus(PRV).During the PRV infection,the CRISPR-Cas9 system was efficient in cleaving the UL30 gene in each passage.However,deletions and insertions occurred at lower passages,while substitutions were frequently observed at higher passages.Furthermore,copy numbers and virus titers of PRV were significantly increased in a passage-dependent manner,indicating that viral genomic replication and assembly were more effective at the higher passages than at lower passages.These results demonstrated that PRV could escape from CRISPR-Cas9-mediated inhibition.Therefore,whether the CRISPR-Cas9 system is suitable for antiviral application should be considered and carefully verified.