Bioscience Methods 2024, Vol.15, No.4, 162-172 http://bioscipublisher.com/index.php/bm 1 65 Zheng et al. (2022) investigated the efficiency of base editing in the PERV (porcine endogenous retrovirus) genome using the MAIO-epiCBE system. Their experimental approach included transfecting ST cells with the editing system, followed by selection and Sanger sequencing to identify genomic modifications. The study demonstrated that the MAIO-epiCBE system effectively induced base substitutions at multiple target sites within the PERV genome. This was confirmed by sequencing data, which revealed significant base conversion rates, marked by red arrows, across various sgRNA target sites. The results indicated successful base editing at high efficiency, producing amino acid changes and introducing stop codons at specific positions. These findings underscore the potential of MAIO-epiCBE for precise genome editing in PERV, offering a promising tool for future biotechnological applications in the pig-to-human xenotransplantation field. 3 CRISPR/Cas9 in Eliminating Porcine Endogenous Retroviruses (PERVs) 3.1 Mechanism of CRISPR/Cas9 and its application in targeting PERVs The CRISPR/Cas9 system is a revolutionary genome-editing tool that allows for precise modifications of DNA sequences. It consists of two key components: the Cas9 enzyme, which acts as molecular scissors to cut DNA, and a guide RNA (gRNA) that directs Cas9 to the specific DNA sequence to be edited. In the context of eliminating PERVs, CRISPR/Cas9 can be designed to target and disrupt the PERV pol genes, which are essential for viral replication and integration into the host genome. This approach has been successfully applied to create PERV-inactivated pig cell lines, such as the PK15 clone 15, which showed impaired viral particle production and non-infectious virions (Godehardt et al., 2019). 3.2 Experimental evidence and outcomes of perv inactivation Several studies have demonstrated the effectiveness of CRISPR/Cas9 in inactivating PERVs. For instance, the PK15 clone 15 cell line, which underwent CRISPR/Cas9-mediated inactivation of PERVs, exhibited a significant reduction in viral protein production and the assembly of non-infectious viral particles (Figure 2) (Godehardt et al.,2019). Another study highlighted the successful disruption of PERV pol genes in porcine embryos using electroporation of the Cas9 protein (GEEP) system, which resulted in high-frequency indel mutations and affected embryonic development7. Additionally, the evaluation of CRISPR/Cas9 constructs for targeting porcine genes relevant to xenotransplantation showed efficient disruption with minimal off-target effects, further supporting the potential of this technology in creating PERV-free pigs (Ryczek et al., 2022). Godehardt et al. (2019) explored the presence and localization of PERV nucleocapsid protein p10 in various cell types using immunohistological techniques. Their study revealed that PERV nucleocapsid p10 was detected in PERV-positive PK15 cells and PERV-B positive 293T/B (33) cells, indicated by prominent green fluorescence, demonstrating active viral protein expression. In contrast, no significant fluorescence was observed in PERV-inactivated PK15 clone 15 cells and PERV-negative 293T cells, suggesting the absence or significant reduction of viral protein expression in these lines. The study used an anti-PERV nucleocapsid p10 antibody to specifically target and visualize the viral protein, validating the antibody's specificity and the robustness of the immunohistological method. These findings highlight the differential expression of PERV proteins in infected versus non-infected cell lines, providing crucial insights into PERV biology and the potential for developing targeted antiviral strategies. 3.3 Potential challenges and limitations in perv eradication Despite the promising results, several challenges and limitations remain in the eradication of PERVs using CRISPR/Cas9. One major concern is the potential for off-target effects, where the Cas9 enzyme may introduce unintended mutations in the genome, which could have deleterious consequences (Ryczek et al., 2022). Additionally, the efficiency of PERV inactivation can vary depending on the specific gRNAs used, as seen in the differential impact on embryonic development when targeting the PERV pol gene (Hirata et al., 2019). Another challenge is the possibility of incomplete inactivation, where residual viral particles or defective proviruses may still be present, posing a risk of reactivation or recombination with other viral elements (Godehardt et al., 2019). Furthermore, the long-term stability and safety of CRISPR/Cas9-modified pigs need to be thoroughly evaluated before clinical applications in xenotransplantation can be realized. In conclusion, while CRISPR/Cas9 offers a
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