Efficient production of GGTA1-/-/Fah+/- knockout pigs by CRISPR/Cas9 and somatic cell nuclear transfer
Xenotransplantation is promising to combat the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of multi-transgenic pigs to overcome the various rejection responses after pig-to-non-human primate xenotransplantation, the possibility to grow human organs in pigs by producing chimeric pigs is an attractive alternative. Pig embryos with impaired liver generation (fumaryacetoacetate hydrolase knockout) could form a niche which subsequently could be colonized by microinjected pluripotent human cells that form a humanized liver. Vascularization of the organ would still be of porcine origin albeit liver parenchyma is formed by human cells. To avoid the hyperacute rejection of the porcine organ after transplantation, a complete knockout of the major antigen producing gene, the alpha1,3-galactosyltransferase (GGTA1) is necessary. Fah- knockout pigs could also serve as a large animal model for studies on metabolic liver disease. Here, we describe the efficient production of GGTA1-/-/ Fah+/- knockout pigs by CRISPR/Cas9 and somatic cell nuclear transfer. In total, 3x106 fetal fibroblasts were co-transfected with two CRISPR/CAS9 vectors (px330, Addgene 42230). One codes for a single guide RNA targeting exon 9 of the GGTA1 locus, the other targets exon 5 of the FAH locus. Cells were counter-selected for the absence of Gal epitopes by magnetic bead selection. Gal-negative cells were used as donor cells for somatic cell nuclear transfer. Two hormonally synchronized recipients received 90 cloned embryos each. Both sows remained pregnant and one was sacrificed and two healthy fetuses were recovered (#465/1,#465/2). The remaining pregnancy was allowed to go to term and delivered 5 live female offspring. The 5 piglets (#471/1-5) and the two fetuses carried a bi-allelic knockout of the GGTA1 gene as determined by flow cytometry after staining with GS-IB4. A heterozygous Fah knockout was confirmed by Cel-I assay in three of the five piglets and in one of the two fetuses. Sequencing of the GGTA1 and Fah locus in fetuses and piglets revealed indels at the GGTA1 ranging between +1 to -78bp. Indel formation at the Fah locus was +1bp in piglets #471/2 and #471/5. Piglet 471/3 and fetus 465/1 showed a loss of 4bp and an insertion of 6 bp on one of the Fah alleles. The next step is to produce GGTA1-/-/Fah-/-pigs by conventional breeding. In a first proof-of-concept attempt, GGTA1-/-/Fah-/- blastocysts could be injected with brain-incompetent nonhuman primate iPS cells for blastocyst complementation. The growth of humanized organs in pigs could then be determined This project was funded by DFG TR CRC-127.
Petersen, Björn* / Petersen, Björn / Frenzel, Antje / et al: Efficient production of GGTA1-/-/Fah+/- knockout pigs by CRISPR/Cas9 and somatic cell nuclear transfer. 2015.
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