Highly efficient gene editing of using CRISPR/Cas9 combined with FACS.

() is a major causative agent of feline viral rhinotracheitis and ocular lesions. Due to its large DNA genome, the construction of recombinant viruses presents considerable challenges for conventional methodologies. In this study, we implemented an integrated strategy combining CRISPR/Cas9-mediated gene editing with fluorescence-activated cell sorting (FACS) to enable the rapid and efficient generation of recombinant viruses. Specifically, the thymidine kinase () gene was disrupted by inserting a monomeric Cherry (mCherry) reporter gene, and the glycoprotein E () gene was similarly interrupted through the incorporation of a green fluorescent protein (GFP) reporter. The CRISPR/Cas9 system enables precise, site-specific genomic modifications, while FACS allows for effective enrichment and isolation of the desired recombinant viral populations. This combined approach significantly reduces the time required for recombinant virus generation from weeks to days, thereby offering substantial potential to expedite vaccine development and advance functional genomics research.