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

Area of Science:

• Immunology
• Genetics
• Animal Models

Background:

• Pigs are valuable large animal models for biomedical research.
• The Major Histocompatibility Complex (MHC) is crucial for immune system function, particularly in infection and transplant rejection.
• Class I MHC-deficient pigs are needed to study viral immunity and transplant rejection.

Purpose of the Study:

• To create and characterize pigs deficient in class I MHC using CRISPR-Cas9 technology.
• To assess the viability and immunological potential of these genetically engineered pigs.

Main Methods:

• Genetic engineering of pig fetal fibroblasts using Cas9 nuclease and guide RNAs to knock out class I MHC genes.
• Somatic cell nuclear transfer using engineered fibroblasts to produce piglets.
• Characterization of the resulting piglets for the absence of cell surface class I proteins and assessment of T cell populations.

Main Results:

• Successfully produced three piglets lacking all cell surface class I proteins.
• These pigs exhibit reduced levels of CD4(-)CD8(+) T cells in peripheral blood.
• The class I MHC knockout pigs appear healthy and are developing normally.

Conclusions:

• The creation of class I MHC knockout pigs is successful using Cas9 technology.
• These pigs represent a valuable new reagent for immunological research, including viral immunity and transplantation studies.
• Further research can utilize these animals to investigate MHC-related immune responses and xenotransplantation models.