International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 155-166 http://medscipublisher.com/index.php/ijmms 160 (Liang et al., 2015). These studies underscore the potential and the current limitations of CRISPR/Cas9 technology in clinical applications. CRISPR/Cas9 technology has transformed the landscape of genome editing with its simplicity, efficiency, and versatility. Its applications in medical research are vast, ranging from cancer studies to early human embryo editing, although challenges such as off-target effects and repair mechanism fidelity remain to be addressed. 4 Applications of CRISPR/Cas9 in Xenotransplantation 4.1 Genetic modification of donor animals to reduce immunogenicity CRISPR/Cas9 technology has been instrumental in reducing the immunogenicity of donor animals, particularly pigs, which are commonly used in xenotransplantation. By targeting genes responsible for xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, researchers have successfully created genetically modified pigs with reduced levels of these antigens. This genetic modification helps to mitigate hyperacute rejection, a major barrier in xenotransplantation (Tanihara et al., 2021). The ability to perform precise gene editing with CRISPR/Cas9 has significantly improved the feasibility of using animal organs for human transplantation by reducing the immune response triggered by these foreign tissues (Ryczek et al., 2021). 4.2 Prevention of zoonotic disease transmission through genome editing One of the critical concerns in xenotransplantation is the potential transmission of zoonotic diseases from donor animals to human recipients. CRISPR/Cas9 technology offers a solution by enabling the precise editing of animal genomes to eliminate endogenous retroviruses and other pathogens that could pose a risk. This genome editing approach not only enhances the safety of xenotransplantation but also ensures that the transplanted organs are free from infectious agents that could compromise the health of the recipient (Ryczek et al., 2021; Wei et al., 2020). By targeting and inactivating specific viral sequences within the donor genome, CRISPR/Cas9 helps to create safer and more reliable sources of transplantable organs. 4.3 Enhancing organ compatibility and function Beyond reducing immunogenicity and preventing disease transmission, CRISPR/Cas9 technology is also being used to enhance the compatibility and function of transplanted organs. By editing genes that influence organ size, function, and metabolic compatibility, researchers can create donor organs that are better suited for human physiology. For example, modifications to genes involved in metabolic pathways can help ensure that the transplanted organs function more efficiently within the human body (Ryczek et al., 2021; Tanihara et al., 2021). This level of customization and optimization is crucial for improving the success rates and long-term outcomes of xenotransplantation procedures. 4.4 Case studies and successful experiments using CRISPR/Cas9 in xenotransplantation Several successful experiments have demonstrated the potential of CRISPR/Cas9 in advancing xenotransplantation. One notable study involved the one-step generation of multiple gene-edited pigs by introducing CRISPR/Cas9 into zygotes via electroporation. This approach targeted the GGTA1, CMAH, and B4GALNT2 genes simultaneously, resulting in pigs with significantly reduced xenoantigen levels (Tanihara et al., 2021). Tanihara et al. (2021) conducted a study that utilized electroporation technology to introduce the CRISPR/Cas9 system into fertilized eggs, as a method to rapidly generate multi-gene-edited pigs with reduced xenoantigen biosynthesis. The study targeted the GGTA1, CMAH, and B4GALNT2 genes to create pigs devoid of xenoantigens. Initially, the study optimized the gRNA combinations for GGTA1 and CMAH, and then introduced these gRNAs along with Cas9 into in vitro fertilized oocytes via electroporation. The electroporated embryos were subsequently transplanted into recipient sows. The results demonstrated that this method successfully generated pigs with dual gene edits for GGTA1/CMAH and triple gene edits for GGTA1/CMAH/B4GALNT2. Immunohistochemical analysis indicated a significant reduction in the expression levels of xenoantigens in these multi-gene-edited pigs, although some pigs exhibited gene mosaicism. The study suggests that, despite the issue of mosaicism that still needs to be addressed, the electroporation technique holds great potential for rapidly
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