The double-sgRNA CRISPR/Cas9 system and the protocol used to generate the recombinant virus rPRV HN1201-EGFP-Luc. A Schematic diagrams of the double -sgRNA CRISPR/Cas9 system and the CRISPR/Cas9 cleavage positions. B Diagrams showing the PRV HN1201 genome and the donor plasmid. In the donor plasmid, the left homologous recombination arm (left HR arm) and the right HR arm are located upstream and downstream of the sgRNA1 and sgRNA2 target sites, respectively. There were 1693 base pairs (bp) between the two sgRNA target sites. After the PRV genome is cut at the two sgRNA target sites and homologous recombination occurs, the PRV genome loses a 2459 bp DNA fragment, and the cassette containing the EGFP and luciferase genes is knocked in at the DNA deletion sites. C Diagram depicting the protocol used to obtain and purify recombinant PRV for the expression of EGFP and luciferase genes. A mixture of PRV HN1201 genomes, Cas9/sgRNAs and donor plasmid was cotransfected into PK-15 cells. CPEs with EGFP were observed 2–4 days after transfection. Cells and media were collected after three freeze–thaw cycles and then inoculated into cells in 96-well plates after serial dilutions to obtain single viral clones. Subcloned viruses were subjected to luciferase assays and sequence analysis.