Genomics and Applied Biology 2024, Vol.15, No.5, 264-275 http://bioscipublisher.com/index.php/gab 267 The use of CRISPR/Cas9 in ASFV research also raises ethical and biosafety concerns. The potential for creating genetically modified viruses necessitates stringent biosafety protocols to prevent accidental release and ensure that modified viruses do not pose a greater threat than the wild-type strains (Sharma et al., 2020). Moreover, ethical considerations regarding the use of gene editing in animals and the potential long-term impacts on ecosystems and biodiversity must be carefully evaluated (Lino et al., 2018; Li et al., 2021). 4 Use of TALENs and Other Gene Editing Tools 4.1 TALENs (transcription activator-like effector nucleases) 4.1.1 How TALENS differ from CRISPR/Cas9 in ASFV research TALENs and CRISPR/Cas9 are both powerful tools for genome editing, but they have distinct mechanisms and applications. TALENs are composed of a DNA-binding domain derived from transcription activator-like effectors and a nuclease domain, typically FokI, which induces double-strand breaks at specific DNA sequences. In contrast, CRISPR/Cas9 uses a guide RNA to direct the Cas9 nuclease to the target DNA sequence, where it creates double-strand breaks (Demirci et al., 2018; Gupta et al., 2019; Janik et al., 2020). One of the key differences in ASFV research is the specificity and off-target effects. TALENs are known for their high specificity due to the modular nature of their DNA-binding domains, which can be customized to recognize long DNA sequences, reducing off-target effects. CRISPR/Cas9, while easier to design and implement, can have higher off-target effects, although various strategies have been developed to mitigate these (Manghwar et al., 2020; Naeem et al., 2020). Additionally, TALENs can target mitochondrial DNA, which is challenging for CRISPR/Cas9 due to difficulties in importing guide RNA into mitochondria (Bhardwaj and Nain, 2021). 4.1.2 Case studies of TALENs in ASFV gene editing TALENs have been successfully used in various studies to edit genes in ASFV, demonstrating their utility in understanding the virus's pathogenesis. For instance, TALENs have been employed to knock out specific genes in ASFV to study their roles in viral replication and virulence. These studies have provided insights into the genetic factors that contribute to the virus's ability to infect and cause disease in swine (Bhardwaj and Nain, 2021). Moreover, TALENs have been used to create ASFV-resistant pig models by targeting and modifying genes associated with susceptibility to the virus, showcasing their potential in developing disease-resistant livestock (Li et al., 2020). 4.2 Other emerging gene editing technologies 4.2.1 Overview of other gene editing tools like ZFNs and base editors In addition to TALENs and CRISPR/Cas9, other gene editing technologies such as Zinc Finger Nucleases (ZFNs) and base editors have emerged. ZFNs are engineered proteins that combine a zinc finger DNA-binding domain with a FokI nuclease domain, similar to TALENs. They are highly specific but more challenging to design and construct compared to CRISPR/Cas9 (Eş et al., 2019; Gupta et al., 2019). Base editors, on the other hand, are a newer class of gene editing tools that enable precise nucleotide changes without inducing double-strand breaks. They use a modified Cas protein fused to a deaminase enzyme to convert specific bases (e.g., cytosine to thymine) at targeted sites. This technology offers a high degree of precision and is particularly useful for correcting point mutations (Naeem et al., 2020). 4.2.2 Potential applications in ASFV research The potential applications of these emerging gene editing technologies in ASFV research are vast. ZFNs can be used to create specific gene knockouts or insertions in the ASFV genome, aiding in the identification of viral genes critical for infection and replication. This can lead to the development of targeted antiviral therapies (Gupta et al., 2019; Li et al., 2020). Base editors offer the possibility of correcting point mutations in the ASFV genome, which could be used to study the effects of specific genetic changes on the virus's behavior and pathogenicity. This precise editing capability can also be applied to develop ASFV-resistant pig models by correcting susceptibility-related mutations in the host genome (Naeem et al., 2020).
RkJQdWJsaXNoZXIy MjQ4ODYzMg==