Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation

The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig (Sus scrofa domestica) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine GGTA1, CMAH, β4GalNT2, vWF, ASGR1 genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies

Application of Genetically Engineered Pigs in Biomedical Research

Progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. The development of transgenesis has created new directions in research and possibilities for its practical application. Generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. Animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. Another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. The further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation. Numerous studies have shown that the pig (Sus scrofa domestica) is the most suitable species both as a research model for human diseases and as an optimal organ donor for xenotransplantation. Short pregnancy, short generation interval, and high litter size make the production of transgenic pigs less time-consuming in comparison with other livestock species This review describes genetically modified pigs used for biomedical research and the future challenges and perspectives for the use of the swine animal models