The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficient biallelic knockout of the-1,3-galactosyltransferase gene in porcine embryonic fibroblasts

Background The recent development of the type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled genome editing of mammalian genomes including those of mice and human; however, its applicability and efficiency in the pig have not been studied in depth. Here, using the CRISPR/Cas9 system, we aimed to destroy the function of the porcine -1,3-galactosyltransferase (-GalT) gene (GGTA1) whose product is responsible for the synthesis of the -Gal epitope, a causative agent for hyperacute rejection upon pig-to-human xenotransplantation. Methods Porcine embryonic fibroblasts were transfected with a Cas9 expression vector and guide RNA specifically designed to target GGTA1. At 4days after transfection, the cells were incubated with IB4 conjugated with saporin (IB4SAP), which eliminates -Gal epitope-expressing cells. Therefore, the cells surviving after IB4SAP treatment would be those negative for -Gal epitope expression, which in turn indicates the generation of GGTA1 biallelic knockout (KO) cells. Results Of the 1.0x106 cells transfected, 10-33 colonies survived after IB4SAP treatment, and almost all colonies (approximately 90%) were negative for staining with red fluorescence-labeled IB4. Sequencing of the mutated portion of GGTA1 revealed a frameshift of the -GalT protein. Porcine blastocysts derived from the somatic cell nuclear transfer of these -Gal epitope-negative cells also lacked the -Gal epitope on their surface. Conclusions These results demonstrated that the CRISPR/Cas9 system can efficiently induce the biallelic conversion of GGTA1 in the resulting somatic cells and is thus a promising tool for the creation of KO cloned piglets.