Introduction: Xenotransplantation is considered a promising solution to overcome the continuous organ shortage in allotransplantation. Besides, significant improvements in the past, long-term xenograft survival is still challenging and needs extensive genetic modifications of the pig genome. The knockout of specific genes encoding for xenoantigens requires a selection process of the transfected cells which is often not compatible with cell survival. Besides, using the CRISPR/Cas9 system guided by a single guide RNA leads to unpredictable modifications at the targeted location. The use of two guide RNAs separated several base pairs downstream from each other results in predictable editing and enables the simple selection of transfected cell clones by PCR.

Methods: We used the CRISPR/Cas9 system to target the CMAH gene in GGTA1-KO/5tg porcine adult fibroblasts. Two guide RNAs were designed to target exon 10 of the CMAH gene 64 bp from each other. After transfection, cells were seeded out in a 25 cm2 flask and allowed to grow until they reach 80% confluency. Cells were then transferred to 96-well plates at a density of 7 cells/well. Individual wells were analyzed by PCR. Cell clones showing two bands (289 bp=wild-type, 225 bp= targeted allele) were further diluted to enrich cell clones carrying a homozygous 64 bp deletion. Cell clones with a homozygous biallelic modification of the CMAH gene were tested by flow cytometry using a Neu5GC-specific antibody (poly21469, Biolegend, chicken IgG, donkey anti-chicken, Jackson lab). Two cell clones (C9: faint wild type band and D5: pure deletion band) were pooled and chosen as donor cells for somatic cell nuclear transfer (SCNT) to generate living offspring.

Results: Several cell clones with a double band were detected in the PCR. After further dilution and culture of the cells, two promising cell clones (C9, D5) were selected as donor cells for SCNT. In total, 74 cloned embryos were transferred to a hormonally synchronized recipient, which became pregnant and gave birth to two offspring (52-1, 52-2). One piglet was liveborn (52-1) and one was stillborn (52-2). PCR analysis and Sanger sequencing revealed that 52-1 carried a biallelic 64 bp deletion in the CMAH gene. In contrast, the stillborn piglet carried a wild-type allele and a deleted allele (64 bp deletion, Fig. 1). The remaining piglet developed normally and healthy and will be used as a founder for our edited pig lines.

Discussion/Conclusion: Employing two guide RNAs to induce a defined deletion at a specific locus in the pig genome is an efficient and predictable approach to knock out genes encoding for xenoantigens and allows for a simple selection of transfected cells by PCR. Double KO/5tg cells are currently used to add more genetic modifications to reduce the immunogenicity of pig organs to bring xenotransplantation closer to the clinics.