α-Gal antigen-deficient rabbits with GGTA1 gene disruption via CRISPR/Cas9

Background: Previous studies have identified the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope), known as the a-Gal antigen as the primary xenoantigen recognized by the human immune system. The alpha-Gal antigen is regulated by galactosyltransferase (GGTA1), and alpha-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop alpha-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system. Results: The mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the alpha-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits. Conclusions: alpha-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these alpha-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.