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.