Bioscience Methods 2024, Vol.15, No.4, 162-172 http://bioscipublisher.com/index.php/bm 1 64 transmission (Tanihara et al., 2019). Another critical target is the porcine cytomegalovirus (PCMV), which can adversely affect the survival of transplanted organs. Genetic modifications have been applied to enhance the pig's antiviral response, reducing the replication and transmission of PCMV (Perleberg et al., 2018). Additionally, pathways involved in immune evasion and inflammation have been modified to improve the compatibility of pig organs with human recipients. For instance, genes responsible for the expression of alpha-gal, a sugar molecule that triggers hyperacute rejection in humans, have been knocked out to prevent immune responses against pig organs. 2.3 Case studies of successful genetic modifications reducing pathogen loads Several case studies highlight the success of genetic modifications in reducing pathogen loads and enhancing the biosafety of porcine organs. One study demonstrated the use of CRISPR/Cas9 to produce triple gene-knockout (TKO) pigs, targeting GGTA1, CMAH, and β4GalNT2. These modifications resulted in the elimination of major xenoantigens, significantly reducing human IgG/IgM binding and immunogenicity without affecting the physical properties of the porcine pericardium (Zheng et al., 2018). Another study utilized cytosine base editors to inactivate PERVs in pig cells, achieving approximately 10% complete inactivation of PERVs without causing DNA double-strand breaks or cytotoxic effects, thus offering a safer strategy for generating PERV-knockout pigs (Figure 1) (Zheng et al., 2022). These advancements underscore the potential of genetic engineering in creating safer and more compatible pig organs for xenotransplantation. Figure 1 Efficient base editing of PERV genome by MAIO-epiCBE system (Adopted from Zheng et al., 2022) Image caption: (A) Schematic of selection for PERV genome-edited clones. (B) Base conversion efficiency of clone #1 and #2 detected by Sanger sequencing. All seven target sites were detected and compared to cells transfected with null group. The positions of base substitutions are marked with red arrows. The amino acid sequence and base conversion efficiency are listed under sequence map. *: stop codon site (Adopted from Zheng et al., 2022)
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