Genomics and Applied Biology 2024, Vol.15, No.5, 264-275 http://bioscipublisher.com/index.php/gab 266 3.2 Targeting ASFV genes with CRISPR/Cas9 In the context of African Swine Fever Virus (ASFV) research, CRISPR/Cas9 has been employed to target several key viral genes. These include genes responsible for viral replication, virulence, and immune evasion. For instance, targeting the p72 gene, which encodes a major capsid protein, has shown promise in disrupting viral assembly and replication (Lin et al., 2021). Another target is the B646L gene, which encodes the ASFV major capsid protein p72, crucial for virus infectivity (Adli, 2018). Recent studies have demonstrated the successful application of CRISPR/Cas9 in editing ASFV genes, leading to attenuated viral strains that could serve as potential vaccine candidates. For example, the deletion of the DP71L gene, which is involved in inhibiting host immune responses, resulted in a virus with reduced virulence, providing insights into ASFV pathogenesis and potential vaccine development (Li et al., 2021; Lin et al., 2021). These advancements highlight the potential of CRISPR/Cas9 not only in understanding ASFV biology but also in developing effective control measures against the virus. 3.3 Limitations and challenges Despite its potential, the application of CRISPR/Cas9 to ASFV research faces several technical challenges. One major issue is the delivery of the CRISPR/Cas9 components into ASFV-infected cells, which can be complicated by the virus's ability to evade host defenses and the lack of efficient delivery systems (Lino et al., 2018). Additionally, off-target effects, where the Cas9 enzyme cuts at unintended sites, can lead to unwanted mutations and complicate the interpretation of results (Liu et al., 2016; Manghwar et al., 2020) (Figure 1). Figure 1 Major concerns/outcomes of off-target effects (Adopted from Manghwar et al., 2020) Image caption: CRISPR/Cas systems usually offer great potential in genome editing, but off-target activity, causing unintended consequences, is limiting its applications for therapeutic and agricultural purposes. a) CRISPR/Cas9 with a specific sgRNA may sometimes bind and edit at a site other than its target sequence, known as off-target editing. This may result in unexpected serious consequences, such as the activation/inactivation of off-target genes which can result in lethal or undesired phenotypes, or the activation of oncogenes causing cancer in animals. b) CRISPR/Cas9 that accurately edits its target gene is termed on-target editing. CRISPR/Cas9 has been used in wide range of plants and animals due to its robust on-target editing efficiency. On-target editing leads to desired targeted phenotypes. Created with BioRender.com (Adopted from Manghwar et al., 2020)
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