Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease

• Published in: The Journal of immunology (1950) Vol. 193; no. 11; pp. 5751 - 5757
• Main Authors: Reyes, Luz M, Estrada, Jose L, Wang, Zheng Yu, Blosser, Rachel J, Smith, Rashod F, Sidner, Richard A, Paris, Leela L, Blankenship, Ross L, Ray, Caitlin N, Miner, Aaron C, Tector, Matthew, Tector, A Joseph
• Format: Journal Article
• Language: English
• Published: United States 01.12.2014
• Subjects: Animals; Cells, Cultured; Endonucleases - metabolism; Fibroblasts - physiology; Gene Knockdown Techniques - methods; Genetic Engineering; Graft Rejection; Histocompatibility Antigens Class I - genetics; Models, Animal; Nuclear Transfer Techniques; RNA, Guide, CRISPR-Cas Systems; Swine - genetics; Swine - immunology; T-Lymphocytes - immunology; Transplantation, Heterologous; Virus Diseases - immunology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.1402059

Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.