The shortage of donor resources has greatly limited the application of clinical xenotransplantation. As such, genetically engineered pigs are expected to be an ideal organ source for xenotransplantation. Most current studies mainly focus on genetically modifying organs or tissues from donor pigs to reduce or prevent attack by the human immune system. Another potential organ source is interspecies chimeras. In this paper, we reviewed the progress of the genetically engineered pigs from the view of immunologic barriers and strategies, and discussed the possibility and challenges of the interspecies chimeras.

Gene-modified miniature pigs serve as alternative tissue and organ donors for xenotransplantation to alleviate the shortage of human allogenic organs. However, the high copy number of porcine endogenous retrovirus (PERV) genomes integrates with the porcine genome, which has a potential risk of cross-species transmission and hinders the clinical practice of xenotransplantation. Recently, CRISPR/Cas9 has been used to inactivate PERVs. However, Cas9 also triggers severe DNA damage at multiple integrated PERV sites in the porcine genome, which induces senescence and apoptosis of porcine cells. In this study, the cytosine base editor (CBE), an efficient and safe editor that does not cause DNA double strand breaks (DSBs), was used for PERV editing to reduce cytotoxic effects. Seven sgRNAs were set to target gag and pol loci of PERVs to induce premature stop codons. We found that approximately 10% of cell clones were completely inactivated for PERVs in pig ST cells, and the plasmid that was used for editing the PERVs did not integrate into host genome and influence the karyotype of the modified cells. Our studies offer a powerful and safe strategy for further generating PERV-knockout pigs using base editors.