Feline herpesvirus type 1 (FHV-1), a member of the Alphaherpesvirinae subfamily, is responsible for approximately 50% of diagnosed viral upper respiratory tract diseases and represents a major cause of ocular disease in domestic cats globally (Nasisse, 1990). FHV-1 possesses a double-stranded DNA genome of approximately 134 kb, which is organized into unique long (UL) and unique short (US) regions flanked by inverted repeats (IR/TR) (Grail et al., 1991; Tai et al., 2010). Due to its large genome size and high GC content (~60%), targeted genetic modification presents considerable challenges (Tai et al., 2016). Traditional methods for generating recombinant FHV-1 rely primarily on homologous recombination in mammalian cells. However, this approach is inefficient due to low recombination frequencies, requiring multiple rounds of plaque purification-a labor-intensive process that typically spans 3–4 weeks. An alternative strategy for manipulating the genomes of large DNA viruses involves the use of bacterial artificial chromosome (BAC)-based systems. Although BAC technology has enhanced genome editing capabilities for large DNA viruses, it still faces several limitations: (1) BAC cloning requires extensive restriction enzyme mapping and in vitro recombination; (2) maintaining large viral genomes in E. coli often leads to instability; and (3) viral reactivation following BAC transfection into mammalian cells remains inefficient (4). These constraints significantly hinder functional genomics research and the rapid development of vaccines against FHV-1.

The CRISPR/Cas9 system has revolutionized the field of genetic engineering, particularly in the context of large DNA viruses such as herpesviruses, whose complex genomes pose significant challenges to conventional homologous recombination techniques (Suenaga et al., 2014). By employing a programmable single-guide RNA (sgRNA) in combination with the Cas9 endonuclease, the CRISPR/Cas9 system enables precise double-strand breaks (DSBs) at specific genomic loci, thereby facilitating targeted insertions, deletions, or substitutions through either homology-directed repair (HDR) or non-homologous end joining (NHEJ). This method has demonstrated high efficiency in modifying herpes simplex virus (HSV) (Lin et al., 2016), and similar success has been reported for cytomegalovirus (CMV) (King and Munger, 2019), Epstein-Barr virus (EBV) (Yuen et al., 2015), pseudorabies virus (PRV) (Tang et al., 2016b; Tang et al., 2016a; Wang et al., 2023), and vaccinia virus (Yuan et al., 2015). These advancements have enabled the rapid generation of gene-deletion mutants for vaccine development and functional genomics studies. Recent studies have also shown that FHV-1 can be genetically modified using CRISPR/Cas9, allowing for the integration of fluorescent reporter genes and other genetic elements (Tang et al., 2023; Yang et al., 2023; Yang et al., 2024). However, the current protocol requires three to seven rounds of plaque purification, which is both time-consuming and labor-intensive. This underscores the need for further improvements in homologous recombination efficiency when applying CRISPR/Cas9 to FHV-1. Fluorescence-activated cell sorting (FACS) offers a promising solution by enabling the high-throughput isolation of recombinant viruses expressing fluorescent markers such as GFP or mCherry. In this study, we combined CRISPR/Cas9 with FACS technology to streamline the generation and isolation of desired recombinants, reducing the experimental timeline from weeks to days.

Our previous studies have demonstrated that for large DNA virus manipulation, extracting the genome and employing a dual-sgRNA strategy can significantly enhance PRV genomic editing efficiency (Tang et al., 2018). Based on this approach, we extracted the FHV-1 genome using the same PRV genome extraction protocol (Tang et al., 2018). However, transfection of the FHV-1 genome resulted in very low virus rescue efficiency (data not shown), suggesting that the PRV genome extraction method may not be directly applicable to FHV-1. Subsequently, we adopted a combination of transfection and viral infection strategies to construct recombinant viruses (Tang et al., 2016b; Tang et al., 2016a; Tang et al., 2017; Wang et al., 2023; Bai et al., 2025). To improve recombination efficiency, we integrated CRISPR/Cas9 technology with fluorescence-activated cell sorting (FACS), as illustrated in Figure 1 . Initially, CRFK cells were co-transfected with CRISPR/Cas9 plasmids and homology arm plasmids. After a 24-hour transfection period, the cells were infected with FHV-1 at an MOI of 0.01. The virus was harvested after 48 hours and used to infect fresh CRFK cells. Twelve hours post-infection, the cells were collected and sorted by FACS. Finally, the recombination efficiency of the sorted virus was evaluated. Accordingly, we designed a knockout of the FHV-1 tk gene using the mCherry reporter gene, as shown in Figure 2A . Following FACS sorting, we successfully obtained the FHV-1-ΔTK-mCherry recombinant virus ( Figure 2B ). This recombinant virus was validated through plaque assays and fluorescence microscopy, revealing a recombination efficiency of 40.27% ± 1.253 ( Figure 2C ). These results indicate that the integration of CRISPR/Cas9 with FACS enables efficient construction of FHV-1 recombinant viruses. To assess the general applicability of this method, we designed sgRNAs targeting the gE/gl region ( Figure 3A ) and employed the GFP reporter gene to generate recombinant viruses targeting this locus. The results confirmed successful generation of GFP-expressing recombinant viruses ( Figure 3B ), with a recombination efficiency of 52.43% ± 2.430 ( Figure 3C ).

Vaccine development plays a critical role in the prevention and control of viral diseases (Tang et al., 2024a). Several advanced technologies have contributed to the design of effective antiviral vaccines (Wang et al., 2022; Wang et al., 2023; Tang et al., 2024b). Efficient manipulation of viral genomes is essential for such vaccine development strategies. This is particularly important for large-genome viruses like FHV-1, which is the focus of this study. Through precise genetic modification techniques, key virulence genes can be targeted for modification, facilitating the development of more effective and safer vaccines (Tang et al., 2017). Such vaccines not only inhibit viral replication but also elicit robust immune responses. The CRISPR/Cas9 system has revolutionized genome engineering by enabling programmable, site-specific double-strand breaks via RNA-guided Cas9 nucleases. Key advantages of this system include: (1) Precision: Single-guide RNAs (sgRNAs) direct Cas9 to specific genomic loci, such as virulence genes (e.g., tk or gE), thereby promoting homology-directed repair (HDR) for the insertion of exogenous sequences. (2) Selective Enrichment: Cas9-mediated cleavage selectively suppresses wild-type virus replication, thereby enriching the population of recombinant viruses. Nevertheless, HDR rate in viral genomes remains relatively low, necessitating the implementation of effective enrichment strategies. FACS addresses this limitation by enabling high-throughput isolation of recombinant virions based on fluorescent reporters (e.g., GFP or mCherry). Specifically, FACS offers the following benefits: (1) It achieves high purity of fluorescent populations within hours, eliminating the need for time-consuming plaque assays that typically require weeks. (2) Recombinant frequencies are significantly increased compared to conventional methods. Together, the CRISPR/Cas9 system generates genetic diversity, while FACS physically isolates desired recombinants, thereby reducing the experimental timeline from weeks to days.

In our previous studies, we found that extracting the viral genome and co-transfecting it with two sgRNAs could achieve nearly complete gene knockout (Tang et al., 2018). However, when applying this strategy to FHV-1 gene editing, the outcomes were suboptimal (data not shown). This discrepancy requires further exploration in the future. For future FHV-1 genome manipulation, if target genes without fluorescent reporters are to be edited, they can be fused with a fluorescent reporter gene to facilitate screening during viral recombination. Once the recombinant virus is obtained, a secondary reverse selection step can be applied to remove the fluorescent reporter gene, thereby enabling rapid and efficient editing of FHV-1.

Overall, this platform enables the repurposing of FHV-1 for various critical applications. Firstly, it facilitates the rapid attenuation of FHV-1, reducing its pathogenicity in vivo, making it ideal for developing live-attenuated FHV-1 vectored vaccines. Additionally, the platform supports high-throughput analyses of viral gene functions, particularly those involved in latency. Lastly, this methodology can be applied to other large DNA viruses possessing complex genomes, such as African swine fever virus (Wang et al., 2019; Zhao et al., 2023). By addressing inefficiencies in herpesvirus engineering, the CRISPR/Cas9/FACS synergy provides a transformative toolkit that advances both fundamental virology research and translational vaccinology.