Elimination of GGTA1, CMAH, β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

Background Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles. Methods Thus, we generate a 4‐gene (GGTA1, CMAH, β4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously. Results We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole‐genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3 months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment. Conclusions 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion. The GGTA1, CMAH, β4GalNT2 and CIITA knockout Bama pigs were successfully generated using CRISPR/Cas9, and it was confirmed that the deletion of four genes occurred at the gene, cell and tissue levels. Knockout of CIITA induced significant changes in the immune system of piglets. The low immunogenicity of 4KO piglets was verified by mixed lymphocyte reaction, serum‐mediated antibody binding reaction and complement‐dependent cytotoxicity assay.