Background: Genetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection. Materials and Methods: Multiplex genetic knockout of GGTA1, β4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats–associated protein 9 system was used to target genes relevant to xenotransplantation, and a method for highly efficient editing of multiple genes in primary porcine fibroblasts was described. Results: Editing efficiencies greater than 85% were achieved for knockout of GGTA1, β4GalNT2, and CMAH. Conclusion: The high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblast cells for in vitro studies and the production of animal models.
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