The swine industry suffers significant financial losses worldwide due to the presence of pseudorabies virus (PRV), which is a crucial pathogen for pigs (1, 2). PRV is a highly contagious virus that affects the nervous system of pigs, leading to neurological symptoms, such as paralysis and death (1, 2). The virus is highly contagious and can spread rapidly among pigs through direct contact or through contact with contaminated objects. Moreover, there has been a concerning trend regarding the increased ability of PRV to transmit across different species, as evidenced by outbreaks of PRV variants. This poses a significant risk to humans, a fact that has been well documented in several recent reviews (3–5). PRV belongs to the alphaherpesvirus group, which is closely related to herpes simplex virus-1 (HSV-1) and varicella-zoster virus (VZV). These viruses are known for their neurotropism and ability to establish lifelong latency in their natural hosts (6, 7). Consequently, PRV is frequently utilized as a model to understand the molecular details of alphaherpesviruses and examine the functions of the nervous system in mammals (8, 9).

Due to its large genome, which contains more than 70 genes with varying functions, the genomic manipulation of PRV was a major challenge in herpesvirus research prior to the emergence of CRISPR technology (10). In early studies, the genome of herpesviruses was manipulated by subjecting the infected cells to a range of physical, chemical, or biological conditions (11, 12), but the resulting mutation is not site-specific and is often randomly scattered throughout the whole genome. Furthermore, the mutation frequency is extremely low, and acquiring interesting mutations is always time-consuming and labor-intensive (11, 12). When the recombination strategy was introduced to manipulate the herpesvirus genome, precise manipulation of the desired specific gene became possible. By transfecting an interesting DNA fragment with homologous arms into infected cells or cotransfecting it with viral genomic DNA, the homologous DNA fragment recombined with the target herpesvirus genome. However, the efficiency of homologous recombination using these methods was extremely low (ranging from 1 in 10⁶ to 1 in 10⁷). The development of bacterial artificial chromosome (BAC) technology has revolutionized the genetics of herpesviruses (11–15). Advances in BAC-based genome editing have been instrumental in helping us gain insight into herpesvirus gene function and vaccine development, thus furthering our knowledge of herpesviruses and paving the way for more effective vaccines and treatments (this is well reviewed by Xia and coworkers (16)). Despite its advantages, BAC-based mutagenesis has some drawbacks. For instance, it can only be used after an infectious BAC has been created, and in some cases, the BAC vector must be removed from herpesviruses (10, 17, 18).

The emergence of genome editing technologies, specifically programmable nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system, has opened up a wide range of possibilities for their use in various life science applications (19–21). In regard to editing at specific sites, ZFNs and TALENs use DNA-binding proteins and the FokI nuclease domain, whereas CRISPR/Cas9 utilizes guide RNAs and the Cas9 protein. The fundamental concept behind these technologies is to cut DNA in a site-specific manner, which generates double-strand breaks (DSBs) at targeted sites. DSBs then stimulate the activation of endogenous DNA repair systems, which can lead to targeted genome modification through either homology-directed repair (HDR) or error-prone nonhomologous end joining (NHEJ). In this mini-review, we focus on the application of CRISPR/Cas9 technology in studies of PRV and mainly concentrate on strategies to efficiently edit the PRV genome.

As a versatile genetic modification tool, CRISPR/Cas9 has emerged as a valuable tool for genetic engineering in a variety of organisms (22, 23). CRISPR–Cas systems have been discovered in numerous bacterial and archaeal organisms, which use these systems as a means of protecting themselves against mobile genetic elements, which utilize the RNA-guided Cas9 nuclease to selectively target and cleave specific foreign DNA sequences (24, 25). The CRISPR–Cas9 system offers a simple and efficient method for manipulating cells in diverse organisms, including those relevant to medicine, agriculture, and scientific investigation. This approach is applicable to virtually all cell types, which makes it a versatile tool for researchers in various fields (24, 25). The mechanism of DNA editing by CRISPR/Cas9 involves creating DSBs in the targeted DNA, which then triggers the activation of cellular repair pathways such as NHEJ and HDR. Knock-out and knock-in of genes of interest can be achieved by utilizing both repair pathways. In comparison to BAC and homologous recombination (HR) methods, CRISPR/Cas9 presents more advantages for the editing of DNA viruses because it only requires the design of effective single-guide RNA (sgRNA) (26).

Large-genome DNA viruses, including adenovirus (27), herpes simplex virus 1 (17, 27–29), and Epstein–Barr virus (30–32), have been manipulated using the CRISPR/Cas9 system. In fact, CRISPR/Cas9 for PRV editing was first conducted by Xu et al. (33). The application of CRISPR/Cas9 in PRV includes the construction of reporter viruses (34–38), vaccine development (39–47), the exploration of virulence genes (48, 49), the studying of viral protein function (49–56), single virus tracking (57), and the development of CRISPR/Cas9-based antiviral strategies (58, 59).

This mini-review describes how PRV may be edited efficiently by CRISPR/Cas9 and provides some insights for PRV researchers. In summary, we recommend using a genomic DNA cotransfection-based method and optimizing the use of two sgRNAs for knock out or knock in. However, CRISPR/Cas9 also has limitations, such as difficulty in single-base editing, whereas the BAC system can efficiently achieve single-base editing (61, 62). In future PRV research, a variety of genome editing tools should be employed; for example, the combination of single-base editing and CRISPR mediates knockdown (63).

Y-DT and H-MW conceptualized the study and generated the figures. All authors contributed to the article and approved the submitted version.

This study was supported by the grants from Jiangsu Agri-animal Husbandry Vocational College (NSF2023CB17).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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