The immune protective effects of pseudorabies virus (PRV) vaccines in the market have gradually reduced and they have failed to provide complete protection against the new PRV variant. In this study, a triple gene-deleted live PRV strain–rZDΔTK-gE-gI was successfully constructed by simultaneously knocking out the three major virulence genes (gE/gI and TK) with CRISPR/Cas9 and Cre/LoxP gene editing system and low melting point agarose purification method. After challenge with the virulent PRV

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... iant, all the 3-week-old piglets vaccinated with the rZDΔTK-gE-gI PRV vaccine candidate survived without any clinical symptoms, whereas all the unvaccinated piglets exhibited PRV respiratory and neurological signs with 100% mortality rate within 7 days post infection. High levels of anti-gB antibodies were induced in the vaccinated piglets after vaccination with the rZDΔTK-gE-gI vaccine candidate, which elicited a better immune protective effect than the classical strain Bartha-K61. Therefore, the triple gene-deleted live PRV vaccine candidate is expected to control the current outbreak of pseudorabies caused by the PRV variants.