International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 155-166 http://medscipublisher.com/index.php/ijmms 157 2) Zoonotic Diseases: The risk of transmitting zoonotic diseases, particularly porcine endogenous retroviruses (PERVs), from donor pigs to human recipients is a significant concern. These viruses, embedded in the pig genome, have the potential to infect human cells and could lead to unforeseen diseases (Cowan et al., 2019). Recent studies have demonstrated the potential of CRISPR/Cas9 to inactivate PERVs in pigs, thereby reducing the risk of zoonotic infections and making xenotransplantation safer (Ross et al., 2018). 3) Genetic Modifications and Mosaicism: While CRISPR/Cas9 allows for precise genetic modifications, the generation of genetically uniform animals remains a challenge. Mosaicism, where not all cells in the organism carry the desired genetic modifications, can limit the effectiveness of xenotransplantation. Techniques such as electroporation have been developed to improve the efficiency of gene editing, but further optimization is required (Tanihara et al., 2021). 4) Ethical and Regulatory Issues: The use of animals for organ harvesting raises ethical concerns regarding animal welfare and the moral implications of genetic modifications. Additionally, stringent regulatory frameworks are necessary to ensure the safety and efficacy of xenotransplantation procedures (Naert and Vleminckx, 2018). 2.3 Overview of donor species and recipient compatibility Pigs are the primary donor species for xenotransplantation due to their physiological similarities to humans and the feasibility of genetic modifications. They are physiologically and anatomically similar to humans, including comparable organ size and function. Moreover, pigs have a relatively short gestation period and large litters, making them a practical choice for genetic modification and breeding programs (Tanihara et al., 2021).The compatibility between donor pigs and human recipients is enhanced through the targeted editing of specific genes to reduce immunogenicity and improve graft survival. Key genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are commonly targeted to create genetically modified pigs with reduced antigenicity (Kararoudi et al., Tanihara et al., 2021; Cowan et al., 2019). Additionally, the use of CRISPR/Cas9 has enabled the development of pigs with multiple genetic modifications, including the knockout of glycosyltransferases and the inactivation of PERVs, which are crucial for improving the safety and efficacy of xenotransplantation (Ryczek et al., Ross et al., 2018; Cowan et al., 2019). These advancements have paved the way for more successful preclinical trials and hold promise for future clinical applications. While xenotransplantation has made significant strides with the help of CRISPR/Cas9 technology, ongoing research is essential to overcome the remaining challenges and ensure the safety and efficacy of this promising therapeutic approach. 3 CRISPR/Cas9 Technology 3.1 Principles and mechanisms of CRISPR/Cas9 The CRISPR/Cas9 system, derived from the adaptive immune system of bacteria, has revolutionized genome editing. The technology relies on two key components: the Cas9 nuclease and a guide RNA (gRNA). The gRNA directs Cas9 to a specific DNA sequence through complementary base pairing, where Cas9 introduces a double-strand break. This break can then be repaired by the cell's endogenous repair mechanisms, primarily non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Figure 2) (Wang et al., 2017; Liang et al., 2015). The precision of CRISPR/Cas9 is largely determined by the design of the gRNA, which can be optimized using various bioinformatics tools (Wang et al., 2017). 3.2 Advantages of CRISPR/Cas9 Over traditional gene editing techniques CRISPR/Cas9 offers several advantages over traditional gene-editing methods, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). These include: 1) Simplicity and Efficiency: CRISPR/Cas9 is easier to design and implement compared to ZFNs and TALENs, which require complex protein engineering. The design of guide RNA sequences for CRISPR/Cas9 is straightforward and can be rapidly synthesized (Blitz and Nakayama, 2021).
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