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Influenza A virus (IAV) infection in pigs causes significant economic losses in the swine industry. More importantly, pigs serve as an intermediate host for generating reassortant viruses with pandemic potential. Previously, gene-edited pigs lacking the gene were generated and tested for susceptibility to IAV infection. Experimental infection of knockout pigs ( KO pigs) resulted in limited nasal shedding, posing the question about their ability to transmit IAV. Therefore, this study evaluated IAV transmission from principal-infected (donor) KO pigs to sentinel (recipient) KO and wild-type (WT) pigs. As expected, limited amounts of infectious virus were shed via the nasal cavity by the principal-infected KO pigs. After co-housing with principal-infected KO pigs at 2 days post-challenge, sentinel KO pigs remained uninfected until the end of observation period of 21 days as shown by the absence of infectious virus and seroconversion. In addition, there was significantly delayed transmission of IAV from principal-infected KO pigs to sentinel WT pigs when compared to WT-to-WT transmission. These results support a significant impairment of IAV transmission in KO pigs.

Based on promising results obtained in primate models, pioneers in the US have now started to explore the new frontier of genetically-edited pig-to-human transplantation. The recent transition of xenotransplantation into clinical medicine has included transplants in brain-dead subjects and the compassionate use of xenotransplants in living recipients without options for allotransplantation. While the barrier of hyperacute rejection seems to be successfully overcome by gene editing of donor pigs, the occurrence of accelerated rejection could pose significant limitations to the success of the procedure. Ultimately, the establishment of efficient and safe strategies to overcome immunologic barriers will, among other critical factors, such as potential xenozoonotic disease transmission or physiological differences, determine whether and for which indications xenotransplantation will be viable. Considering preliminary outcomes of compassionate use xenotransplantions, which may raise questions about how faithfully data from non-human primate models translate into human outcomes, further research in decedents may be necessary before proceeding with additional clinical transplants. Looking ahead, designing systematic trials in xenotransplantation, including the definition of acceptable eligibility criteria for such high-risk transplants, will be an immense challenge, especially in kidney transplantation, where dialysis provides an effective alternative to transplantation in most cases.

Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro . The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated. Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro , using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.

Xenotransplantation is considered a solution for the shortage of organs, and pigs play an indispensable role as donors in xenotransplantation. The biosecurity of pigs, especially the zoonotic viruses carried by pigs, has attracted attention. This review introduces several viruses, including porcine endogenous retroviruses that are integrated into the pig genome in a DNA form, herpesviruses that have been proven to clearly affect recipient survival time in previous xenotransplant surgeries, the zoonotic hepatitis E virus, and the widely distributed porcine circoviruses. The detail virus information, such as structure, caused diseases, transmission pathways, and epidemiology was introduced in the current review. Diagnostic and control measures for these viruses, including detection sites and methods, vaccines, RNA interference, antiviral pigs, farm biosecurity, and drugs, are discussed. The challenges faced, including those posed by other viruses and newly emerged viruses, and the challenges brought by the modes of transmission of the viruses are also summarized.

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5 + subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5 + cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5 + pig model in exploring the role of LGR5 + cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.