Abstract:In this study, CRISPR/Cas9-mediated homologous recombination technology was used to perform gene editing on the isolated PRV HB2017 strain to construct a pseudorabies virus (PRV) with deletion of TK, gE and gI genes. After techniques such as plaque purification, we have obtained the gene-deleted PRV strain. Subsequently, the characteristics of attenuated PRV were preliminarily studied by PCR, gene sequencing, indirect immunofluorescence assay, growth curve determination and animal experiments. The results showed that the TK, gE and gI genes of the PRV HB2017 strain have been deleted, and the growth curves of the attenuated PRV strain PRV HB2017ΔTKΔgE/gI and the parent strain PRV HB2017 strain in PK-15 cells are not significantly different and have high virus titer. PRV HB2017ΔTKΔgE/gI strain has the advantages of high safety, genetic stability and superior immunogenicity.