Efficient generation of a triple knockout (GGTA1/CMAH/ASGR1) of xenorelevant genes in pig fibroblasts

Porcine xenografts from pigs lacking a functional α1,3-galactosyltransferase gene (GGTA1-KO) prevent the hyperacute rejection associated with pig-to-nonhuman primate xenotransplantation. In pig-to-human xenotransplantation, the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene encoding the Neu5Gc epitopes, which are absent in humans, also play an important antigenic role. Another potential target is the porcine asialoglycoprotein receptor (ASGR1) which is involved in the onset of lethal thrombocytopenia after liver xenotransplantation. With the aid of CRISPR/Cas9, it is now feasible to target several endogenous genes in a single approach. Here we designed three independent CRISPR/Cas vectors carrying guide RNAs to target GGTA1, CMAH and ASGR1. About 3 million fetal fibroblasts from day 25 of gestation were simultaneously transfected with three different CRISPR/Cas9 constructs (px330, Addgene plasmid 42230). The first one encoded for a single guide RNA targeting exon 9 of GGTA1 (5´- CTGACGAGTTCACCTACGAG-3´), the second one targeted exon 10 of CMAH (5´- CCTGAACTACAAGGCTCGGC-3´) and the third one targeted exon 3 of ASGR1 (5´- CGCCGAAAGAGTGACTGTTG-3´). Transfected cells were cultured for 5-7 days until they reached confluency in a 75cm2 culture flask. Subsequently, cells were counter-selected for Gal-epitopes by using biotin-conjugated Griffonia simplicifolia IB4 (GS-IB4) lectin and Streptavidin-coated magnetic beads. Cells not bound by the magnetic field, remained in the supernatant and were seeded onto 96-wells. Cells were further analyzed by FACS-staining for Gal-epitopes and surveyor nuclease assay (Cel-I) to determine mutations at the GGTA1-,CMAH- and ASGR1 loci. All cells did not express Gal epitopes on their surface and showed indel formation at all three loci. Mutations at the GGTA1 locus led to a bi-allelic knockout of the gene proven by the complete absence of Gal epitopes measured by flowcytometry. We only observed heterozygous mutations at the CMAH (5bp deletion) and ASGR1 locus (13 bp deletion). Currently, triple genetically modified cells serve as donor cells for somatic cell nuclear transfer to generate offspring for further analyses. Results show that CRISPR/Cas9 is a very efficient tool to generate multiple genetic modifications in the porcine genome, which will facilitate the production of pigs suitable for xenotransplantation. This project was funded by DFG TR CRC-127.