Highly efficient multiplex genetic engineering of porcine primary fetal fibroblasts

Abstract 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 (KO) of GGTA1, β4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the CRISPR/Cas9 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 KO 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.