摘要Background:Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic,while immunogenic genes need to be eliminated to im-prove the immune compatibility between humans and pigs.Current knockout strat-egies 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.