The potential of CRISPR/Cas9 gene editing to repress CyHV-3 was tested in vitro. By targeting two basic target genes necessary for the early transcription of CyHV-3, we show that virus transcription and particle release were significantly decreased by CRISPR/Cas9, as measured by quantitative real-time PCR and virus titration experiments, respectively. (A) The effectiveness is confirmed of the CRISPR/Cas9 system at repressing exogenous genes, including large viral genomic DNA, by introducing site-specific mutations in vitro. (B) The CyHV-3 virus replicates poorly in Cas9-positive cells. (C) The inhibition of thymidine kinase alone cannot block viral particle release.