21 GENERATION OF GGTA1 AND CMAH BIALLELIC KNOCKOUT PORCINE FIBROBLAST CELL LINES BY TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE AND CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9

Abstract

The use of porcine organs for pig-to-human transplantation is considered the most promising solution to overcome the growing shortage of human grafts for all transplantation. Unfortunately, human antibodies recognise Gala-1, 3-gal (Gal), and N-glycolylneuraminic acid (Neu5Gc) epitopes on the surface of pig cells, causing hyperacute rejection. Transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are 2 powerful tools for producing gene knockouts with high efficiency. Here we generate α-1, 3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene knockout porcine fibroblasts cell lines with TALEN and CRISPR/Cas9. Porcine fibroblasts were treated with TALEN and CRISPR/Cas9 designed against the region coding for the catalytic core of GGTA1 and CMAH. A magnetic separation of these cells was used in somatic cell NT. The cloned fetuses and piglets were characterised by T7E1, fluorescent PCR, and DNA sequencing. Nineteen fetuses were harvested using TALEN; 4 of them were GGTA1 gene mutated, and 11 were CMAH gene mutated. The DNA sequence analysis confirmed 5 of 11 are biallelic disruptions of the CMAH gene. For the CRISPR/Cas9, 6 piglets were obtained, and DNA sequence analysis confirmed 1 of 6 is biallelic disruptions of the GGTA1 and 2 of 6 are mono-allelic disruptions of the CMAH gene. We produced several fetuses or piglets via gene editing technology (TALEN and CRISPR/Cas9) and somatic cell NT technology. Furthermore, GGTA1 and CMAH gene knockout porcine fetal fibroblast cell lines derived from the fetus individuals were successfully established. However, expression of α-Gal and Neu5Gc epitopes is needed to be further examined by flow cytometry and confocal microscopy. Our study will provide the possibility of making GGTA1 and CMAH knockout pigs in the future.