Abstract：Abstract N-acetylneuraminic acid (Neu5Gc) is an important non-galactose antigen which can cause xenograft rejection, and it is catalyzed synthesis by cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In this study, the CMAH gene knockout Bama mini pigs (CMAH-/-) were generated by CRISPR/Cas9 based on the alpha-1, 3-galactosyltransferase (GGTA1) gene knockout pigs. The health and reproductive condition of the CMAH-/- pigs were evaluated. Single guide RNA (sgRNAs) were designed to target the porcine CMAH gene and the efficiency of the sgRNAs was verified. High-efficiency CMAH-gRNA1 expression vector was transfected into the ear fibroblasts of Bama mini pigs (BMEF). Cells identified as positive were used as nuclear donors, and somatic cell nuclear transfer were used to prepare CMAH-/- pigs. CMAH gene mutations in piglets were identified by PCR and sequencing; Immunofluorescence detection of CMAH-/-porcine organs with anti-Neu5Gc antibody to determine the expression of Neu5Gc in piglets; The 4-month-old CMAH-/- pigs' blood was collected for the detection of its physiological indexes, the 6 months old CMAH-/-boars mating with wide type sows, the litter size of sows was computed to evaluate the CMAH-/- pigs'breeding capacity. The CMAH-/- piglets had two homozygous genotypes: -2 bp/-2 bp and -1bp/-1bp. Immunofluorescence detection results showed that the Neu5Gc was not detected on the surface of CMAH-/-pigs'organs, indicating that the CMAH gene was knockout. Compared with the wild type pigs (WT), there was no significant difference in blood routine and blood biochemical parameters, and the number of sows mating with CMAH-/- boars was normal. In this study, CRISPR/Cas9 technology was used to generate GGTA1/CMAH knockout pigs, the health and reproductive condition of CMAH-/- pigs were normal. The pigs could provide a low immune antigen donor for further reducing the acute immune rejection of xenotransplantation.
