The PRV HN1201 genome was extracted as previously described [15, 36]. A total of 2 μg of donor plasmid pUC57-US6/7-EGFP-Luc-US8/9 was cotransfected into PK-15 cells together with 1 μg of two-sgRNA CRISPR/Cas9 plasmid pX459M-sgRNA1-sgRNA2 and 1 μg of the PRV HN1201 genome by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. EGFP was detected by using a standard FITC filter-equipped fluorescence microscope (Nikon Eclipse TS100) at 2–3 days after cotransfection. When cytopathic effects (CPEs) appeared, the cells were collected. The cell lysate was serially diluted 10–1–10–8 fold and used to infect PK-15 cell monolayers in a 96-well cell culture plate. The recombinant virus was purified from the 96-well cell culture plate with an endpoint dilution assay under an inverted fluorescence microscope. After three virus purification steps and further luciferase assays, the recombinant virus was obtained and named rPRV HN1201-EGFP-Luc. The genomic DNA of rPRV HN1201-EGFP-Luc was extracted from cell lysates to amplify the expected fragment (including the left HR arm, the EGFP expression cassette, the firefly luciferase expression cassette and the right HR arm) with the primers US 6/7-F and US 8/9-R (Table (Table1),1), and PCR products were sequenced for identification of the recombinant virus.

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