| Currently,pseudorabies virus(PRV)variant strains are outbreaking in China;these variants belong to genotype ?PRV.The traditional Bartha-K61 vaccine has failed to provide complete protection against the emergent variant strains.Therefore,the studies focusing on this variant strain is urgent.The PRV genome is large and difficult to manipulate,but is more feasible using clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology.In this study,we first report a rapid method for editing the PRV genome using the CRISPR-Cas9 system.We developed a triple gE/gI/TK gene-inactivated HeN1 PRV strain,and evaluated the attenuation of PRV in the mice and demonstrated that modified PRV was fully attenuated.Furthermore,the attenuated strain also induced immune protection in response to a parental PRV challenge.Overall,we showed that PRVs can be rapidly attenuated using CRISPR-Cas9 technology,which will be critical for PRV control,especially when new variant PRV strains emerge.Next,identification of single guide RNA(sgRNA)through screening is critical to the CRISPR/Cas9 system,and is traditionally time and labor intensive,and not suitable for rapid and high throughput screening of effective PRV sgRNAs.In this study,we developed a recombinant PRV strain expressing firefly luciferase and EGFP as a reporter virus for PRV specific sgRNA screens and rapid evaluation of antiviral compounds.Luciferase activity was apparent as soon as 4 hours after infection and was stably expressed through 10 passages.In a proof of principle screen we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods.We also attempted that large fragment gene deletions by CRISPR/Cas9 system,and the ratio of knockout nearly reaches 100%.We also demonstrated that CRISPR/Cas9 was useful tool for inhibiting PRV.Last,we screened several virulence gene by this technology.CRISPR/Cas9 will be a useful tool in PRV research. |
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