Subculturing cells have no effect on CRISPR/Cas9‐mediated cleavage of UL30 gene in pseudorabies virus

CRISPR/Cas9‐mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus (PRV) could escape from CRISPR/Cas9‐mediated inhibition. In order to elucidate whether the escape of PRV from Cas9‐mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA‐expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9‐mediated cleavage of PRV. Different passages of PX459‐PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.