Abstract：Bovine (Bos taurus) herpes virus type I (BHV-1) could cause infectious bovine rhinotracheitis (IBR) and result in economic losing in cattle raising industry. Extensive application of gene-deleted vaccine could significantly decrease BHV-1 infection. In order to construct gene deleted vaccines more efficiently, the present study combined CRISPR/Cas9 gene editing and homologous recombination technique to exchange the glycoprotein E (gE) gene of BHV-1 with green fluorescent protein gene (EGFP), named as BHV-1 gE-/EGFP+. Subsequently, the EGFP gene in BHV-1 gE-/EGFP+ was directly removed by specific small guide RNA (sgRNA), and the gE deleted virus was named as BHV-1 ΔgE. Notably, in vitro biological characteristics analysis showed that the BHV-1 ΔgE had similar growth characteristics and plaque morphology when compared to the parental BHV-1. In vivo animal immunization experiments showed that the CRISPR/Cas9 modifed gE deletion virus did not change the immunogenicity of BHV-1. Thus BHV-1 ΔgE could be used as a candidate for novel recombinant BHV-1 vaccine. The present study first explored the BHV-1 virus genome editing with CRISPR/Cas9 system which could provide an efficient method for constructing BHV-1 gene deleted strains and developing IBR gene deleted vaccine.
