Precise Multi‐Gene Editing Strategies for Xenotransplantation Donor Pigs: Overcoming Immune and Coagulation Barriers

ABSTRACT Organ transplantation is the preferred treatment for end‐stage organ failure, but the severe shortage of donors severely restricts its clinical application. Xenotransplantation, especially using pigs as donors, is considered an ideal source of alternative donors due to the high similarity between their organ structures and those of humans. However, significant differences in immune recognition and coagulation regulation between species can easily induce a series of rejection reactions, including hyperacute rejection, acute humoral rejection, T‐cell‐mediated rejection, and chronic vascular complications. It also carries risks such as physiological metabolic incompatibility and potential viral transmission. In recent years, with the development of tools such as CRISPR/Cas, precise multi‐gene editing technology has become possible, enabling the simultaneous knockout of multiple xenoantigen genes (such as GGTA1, CMAH, and B4GALNT2) and the introduction of human genes regulating complement, coagulation, and immune responses (such as hCD55, hTBM (THBD), and hCD47), significantly improving the immune tolerance and physiological compatibility of donor organs. This article systematically reviews the immune and coagulation barriers in xenotransplantation, focusing on precise multi‐gene editing strategies for pigs used in xenotransplantation. It highlights editing pathways such as tandem knock‐in at the same site, simultaneous multi‐site editing, stepwise modular editing, and homology‐directed repair (HDR) enrichment. Combined with representative organ‐specific examples (heart, kidney, liver, and lung), including key non‐human primate studies and early human exploratory cases where available, it explores the application prospects of these strategies in creating safe clinical‐grade donor pigs and promoting the clinical translation of xenotransplantation.