Genomics and Applied Biology 2024, Vol.15, No.5, 264-275 http://bioscipublisher.com/index.php/gab 265 This research aims to explore the potential of gene editing tools to enhance our understanding of ASFV pathogenesis and aid in the development of new treatments. By utilizing CRISPR/Cas9 and other gene editing technologies, we seek to identify key viral genes involved in virulence and immune evasion, and to develop attenuated virus strains that could serve as vaccine candidates. Additionally, we aim to investigate the molecular interactions between ASFV and host cells to uncover novel therapeutic targets. Ultimately, this research will contribute to the development of effective strategies to control and prevent ASFV outbreaks, thereby safeguarding global swine populations and the swine industry. 2 ASFV Genomic Structure and Key Genes 2.1 Overview of ASFV genome African swine fever virus (ASFV) is a large, double-stranded DNA virus with a genome size ranging from approximately 170 to 193 kilobase pairs (kbp), depending on the isolate (Dixon et al., 2013; Wang et al., 2021). The ASFV genome is characterized by its complex structure, which includes covalently closed termini with imperfectly base-paired hairpin loops and inverted arrays of tandem repeats adjacent to these termini. The genome contains between 150 and 167 open reading frames (ORFs) that are closely spaced and read from both DNA strands (Dixon et al., 2013). ASFV's genome encodes a variety of enzymes necessary for transcription and replication, as well as structural proteins and factors involved in evading host defense mechanisms (Dixon et al., 2013; Wang et al., 2021). The virus replicates predominantly in the cytoplasm of infected cells, forming perinuclear factory areas where DNA replication begins approximately 6 hours post-infection (Dixon et al., 2013). 2.2 Identification of key genes involved in pathogenesis ASFV encodes several key genes that play crucial roles in its replication and pathogenesis. Notable among these are genes involved in immune evasion, such as A238L, which regulates NF-κB and NFAT pathways, and A224L, an apoptosis inhibitor. The EP153R gene modulates MHC-I antigen presentation, while A276R is involved in modulating type I interferon (IFN) responses (Correia et al., 2013; Gallardo et al., 2018). Additionally, genes from multigene families (MGFs) such as MGF360 and MGF530 have been identified as important for the virus's ability to evade the host's innate immune response. The A276R gene from MGF360, for example, inhibits the induction of IFN-β by targeting IRF3 (Correia et al., 2013). These genes collectively contribute to the virus's ability to replicate efficiently and evade host immune defenses, thereby enhancing its virulence. 2.3 Challenges in targeting ASFV genes Editing the ASFV genome presents significant challenges due to its large size and complex structure. The virus's genome contains numerous closely spaced ORFs and repetitive sequences, which complicate the precise targeting of specific genes (Dixon et al., 2013; Wang et al., 2021). Additionally, the use of genome editing tools such as CRISPR-Cas9 in ASFV is hindered by potential off-target effects, which can lead to unintended mutations and affect the virus's overall fitness and pathogenicity (Chen and Gonçalves, 2015). The development of effective genome editing strategies for ASFV requires careful consideration of these factors to minimize off-target effects and ensure the accurate modification of target genes. Furthermore, the high genetic diversity and variability among different ASFV isolates add another layer of complexity to genome editing efforts (Malogolovkin and Kolbasov, 2019; Wang et al., 2020). 3 Application of CRISPR/Cas9 in ASFV Research 3.1 Mechanism of CRISPR/Cas9 The CRISPR/Cas9 system, originally derived from the adaptive immune system of bacteria, has revolutionized genome editing due to its simplicity, efficiency, and precision. The mechanism involves two key components: the Cas9 nuclease and a single-guide RNA (sgRNA). The sgRNA is designed to match a specific DNA sequence in the target genome. When introduced into a cell, the sgRNA guides the Cas9 enzyme to the complementary DNA sequence, where Cas9 induces a double-strand break (DSB) (Manghwar et al., 2019; Rodriguez-Rodriguez et al., 2019). The cell's natural repair mechanisms then attempt to repair the DSB, often resulting in insertions or deletions (indels) that can disrupt the gene, or precise modifications if a repair template is provided (Bortesi and Fischer, 2015; Manghwar et al., 2020).
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