Genome-wide Genetic Mutations Accumulated in Pigs Genome-edited for Xenotransplantation and Their Filial Generation

Although xenotransplantation has been revolutionized by the development of genome-edited pigs, it is still unknown whether these pigs and their offspring remain genomically stable. Here, we showed that GGTA1-knockout (GTKO) pigs accumulated an average of 1205 genome-wide genetic mutations, and their filial 1 (F1) offspring contained an average of 18 de novo mutations compared with wild-type controls and their parents. The majority of mutations were in regions annotated as intergenic without altering protein functions, and none were located at predicted off-target sites. RNA sequencing analysis and phenotypic observations indicated that the accumulated mutations may have only a limited influence on GTKO pigs, and most of the mutations in the GTKO pigs could be attributed to the electrotransfection of plasmids into cells. This is the first report demonstrating that genetic mutations in genome-edited pigs are inherited stably by the next generation, providing a reference for the safe application and a standard approach to breed genome-edited pigs for xenotransplantation.