Genome manipulation is an effective method for studying the function of viral genes and can help scientists understand the biology of viruses, such as discovering virulence factors and exploring new targets for developing novel vaccines and antiviral drugs for the prevention and treatment of viral infection. Herpesviruses are a group of double-strand DNA (dsDNA) viruses which can cause lifelong persistent infections and are major pathogens in humans and a wide range of animals. The genomes of herpesviruses are between 125 and 295 kilobases in length [1], which makes it difficult for genome manipulation.

2. Genome Editing Techniques of Herpesviruses Based on BAC

2.1. RecA Recombination Technique

The RecA recombination technique is a bacterial endogenous homologous recombination system composed of RecA recombinase and related auxiliary proteins (such as RecBCD, RecFOR, RuvABC, etc.). RecBCD is a multifunctional enzyme complex consisting of RecB, RecC, and RecD. When binding to the incision on the double-stranded donor DNA, RecB and RecD exhibit 3′ to 5′ and 5′ to 3′ helicase activities, respectively, which together result in the opening of the complementary double-stranded DNA (Figure 1a) [16]. Then, RecC can mediate the recognition of chi sites in the sequence and thereby cause allosteric changes, leading to a decrease in the pace of movement (Figure 1b) [17]. At this point, RecB also has exonuclease activity, which mediates the generation of single-stranded DNA at the 3′ end (Figure 1c) ^[18][19][20][18,19,20]. The RecA recombinase binds to single-stranded DNA sites generated by the exonucleases and engages in the process of identifying homologous regions on the BAC for subsequent annealing and interaction. After successful interaction, the nucleoprotein filaments invade the dsDNA and undergo strand exchange to form heteroduplex DNA (Figure 1d). Finally, RuvABC assists in catalyzing branch migration and degradation of the heteroduplex DNA, resulting in homologous recombination (Figure 1e) [21]. Although the RecA-mediated homologous recombination method has shown improved efficiency(10⁻⁶ to 10⁻⁴) compared to traditional methods for editing the herpesvirus genomes in eukaryotic cells (Table 1) [22], it still has significant drawbacks. One major issue is that due to the presence of repeated sequences in the herpes virus genomes, the expression of RecA can lead to instability in the herpes virus’s BAC clone, which in turn can cause mutations in the viral genome (Table 1) [1]. In addition, 500 bp to 3 kb long homologous arms were typically used in RecA-mediated recombination (Table 1) ^[7][23][24][7,23,24], resulting in a cumbersome process for constructing shuttle plasmids required for recombination. These drawbacks limit the application of RecA-mediated recombination.

2.2. λ-Red Recombination Technique

Homologous recombination mediated by the λ-red recombination system is currently the most widely used recombination technique for editing herpesvirus BAC clones ^[2][25][2,25]. The λ-red recombination system is derived from the phage λ and consists of Gam, Exo, and Beta proteins. The Gam protein is an auxiliary protein of Exo protein and Beta protein. The Gam protein can inhibit the binding of Rec BCD to the ends of dsDNA and thus inhibit the function of Rec BCD exonuclease, preventing the degradation of the exogenous double-stranded DNA (Figure 2a) ^[26][27][26,27]. Exo proteins can bind to the ends of a dsDNA donor, which contains homologous fragments on both sides of the target gene. Concurrently, Exo protein possesses 5′ to 3′ exonuclease activity, producing a stretch of single-stranded DNA at the 3′ end (Figure 2b) [28]. Beta protein plays a decisive role in the process of λ-red homologous recombination. As a single-stranded DNA binding protein, the beta protein binds to the single-stranded DNA produced by the Exo protein. The binding of Beta protein enhances the annealing of the donor DNA fragment and the homologous sequence at the target site of the replicating herpes virus BAC (Figure 2c). Homologous recombination is complete with the replication of DNA (Figure 2d) ^[29][30][29,30]. Compared with the RecA recombination technique, the λ-red recombination technique avoids the risk of a partial deletion of the herpes virus genome in BAC during the recombination process as only homologous double-strand ends can be used as a substrate (Table 1). Additionally, this method typically only requires 30–50 base pairs of homologous arms for recombination (Table 1), making it easier to obtain the donor through techniques such as oligonucleotide synthesis or polymerase chain reaction (PCR), eliminating the need for shuttle plasmids as required in RecA recombination ^[31][32][31,32]. Importantly, the recombination efficiency mediated by the λ-red recombination technique has been improved to a maximum of 0.68% when the donor is double-stranded DNA (Table 1) ^[33][34][35][33,34,35].

2.3. Base Editing Technique

CRISPR/Cas 9 is a powerful genome editing technique [36] that has been successfully applied in a wide range of eukaryotic cells, including human cell lines, embryonic stem cells, mice, Arabidopsis, and Drosophila ^{[37][38][39][40]}[37,38,39,40]. In 2013, Jiang et al. successfully edited Streptococcus pneumonia’s genome using CRISPR/Cas-9-only gene editing [41]. Since then, it has been successfully employed in a variety of prokaryotic species as well ^[42][43][42,43]. The versatility and effectiveness of CRISPR/Cas9 in modifying the genome make it a valuable tool for a wide range of scientific and medical applications. Due to the lack of the non-homologous end-joining (NHEJ) pathway ^{[44][45][46][47]}[44,45,46,47] and the low efficiency of their endogenous homologous recombination system, it is difficult to achieve stable genome editing in most bacteria using CRISPR/Cas9 gene editing technology alone ^{[41][48][49][50]}[41,48,49,50]. Until now, there was still no publication reporting CRISPR/Cas 9-based gene editing technique in the stably preserved herpesvirus BAC gene in E. coli. This highlights the need to explore alternative approaches to achieve efficient and reliable gene editing in these organisms. Recently, scientists have constructed an efficient gene editing method by combing CRISPR/Cas 9 gene editing technique with precise base editing technology. This method consists of an sgRNA and a complex that includes modified Cas9 proteins, cytosine deaminases, and an uracil glycosylase inhibitor (UGI) [51]. Unlike wild-type Cas9 proteins, the modified Cas9 is catalytically dead, lacking endonuclease activity. Therefore, it can only facilitate genome targeting via sgRNA but cannot induce a double strand break (DSB) due to the absence of cleavage activity (Figure 3a). Cytosine deaminase converts the specified cytosine (C) site to uracil (U) (Figure 3b). At this point, uracil glycosylase inhibitors can prevent the excision of intermediate product U, increasing the efficiency of converting C to T on the DNA chain, ultimately achieving single-base precise editing of C to T and G to A (Figure 3c). Base editing allows for site-directed mutagenesis of multiple prokaryotic genomes [52], including E. coli [22] and even herpesvirus genome BACs preserved in E. coli [53]. Zheng et al. ^[22][53][22,53] utilized this technology, directly converting cytidine (C) to uridine (U) at specific positions on the US8 and UL34 genes of the pseudorabies virus genome BAC, thus achieving the premature termination of the corresponding genes and approaching 100% editing efficiency (Table 1). Due to the modifications that occur in the base-editing window, many studies have demonstrated that base editing is associated with off-target effects ^[54][55][54,55], thereby limiting its practical application. To effectively utilize base editing, optimization of the cytidine deaminase and/or UGI is necessary ^[56][57][56,57]. Despite this, the technology has the advantage of directly editing the target site nucleotide without the need for donors. It provides an efficient alternative method for point mutation editing. Moreover, the novel combined editing technology that has emerged in bacterial genome editing but has not yet been applied to BACs and may also provide insights for future BAC editing. Combining CRISPR/Cas 9 with λ-red recombination could increase the efficiency of recombination in editing the genome of E. coli, with reported knockout efficiency of up to 100% for deletion lengths of up to 3.4 kb, which is higher than in previous reports ^[48][58][59][48,58,59]. The use of this system could potentially offer a new approach for efficient editing of the BAC of herpesvirus genomes that are stably stored in E. coli.