Abstract:In this study,CRISPR/cas9mediated homologous recombination technology was used to genetically edit the pseudorabies virus (PRV) HB2017 strain isolated from a pig farm in Hubei Province,and a PRV with deletion of TK, gE and gI genes was constructed. Afterwards,the genedeleted PRV strain was obtained by techniques such as plaque purification. Subsequently,characteristics of the attenuated PRV were preliminarily studied by PCR,gene sequencing,indirect immunofluorescence assay,growth curve determination,vaccine safety and efficacy tests. The results showed that the TK,gEand gI genes of the PRV HB2017 strain had 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 were not significantly different and had high virus titer. After the PRV HB2017ΔTKΔgE/gI strain was transmitted to the 30th generation,the deletion sequences of the TK and gE/gI genes were stable and could not be recovered. The PRV HB2017ΔTKΔgE/gI strain is safe for piglets. Vaccinating piglets with 106.0 TCID50 or 107.0 TCID50 of the PRV HB2017ΔTKΔgE/gI strain could protect them from the attack of 108.0 TCID50 of the PRV virulent strains.