6 Comparison of three methodologies for producing gene-edited pigs for xenotransplantation

Pigs are considered excellent candidates for xenotransplantation. However, multiple genetic modifications are necessary to overcome immunological rejection and physiological incompatibilities. In this study, we evaluated the knockout (KO) of the GGTA1, CMAH, β4GalNT2, and growth hormone receptor (GHR) genes. We compared pregnancy and birth rates of edited piglets using three different CRISPR-Cas9-based methodologies: intracytoplasmic sperm injection-mediated gene edition (ICSI-MGE), oocyte microinjection followed by IVF, and microinjection of in vivo-fertilized zygotes. First, oocytes were collected from slaughterhouse ovaries and in vitro matured for 44 h. Zygotes obtained through in vivo fertilization were recovered by flushing the oviducts of superovulated and inseminated sows. Single guide RNAs (sgRNAs) were designed using the Breaking-Cas program and then incubated with the Cas9 endonuclease (New England Biolabs, M0646T) to form ribonucleoprotein (RNP) CRISPR complexes. They were evaluated by in vitro digestion of DNA fragments and in parthenogenetic microinjected embryos. After confirming that sgRNAs successfully edited all genes, RNP complexes were microinjected into the three experimental groups. Twenty-four hours later, 80 to 100 ICSI-MGE embryos, 100 to 150 IVF embryos, and 50 to 70 in vivo embryos were surgically transferred into synchronized nulliparous recipient oviducts. Fragment analysis was performed by capillary electrophoresis from DNA extracted from tissue of expelled embryos and newborn piglet skin cells. Phenotype analysis of the GGTA1 gene KO was performed by detecting the presence of α-1,3-galactose (α-Gal) in piglet skin cells, using a specific FITC-conjugated lectin. Phenotype analysis of the GHR gene KO was evaluated by comparing weight and length measurements weekly from birth to 109 days, among wild-type, monoallelic and biallelic GHR-edited piglets. Bonferroni least squares means were adjusted, and differences between groups were statistically analysed (P < 0.05) using SAS 9.4 software. Out of the six sows that received ICSI-MGE embryos, all pregnancies were lost, one at 28 days, and the rest presented embryo resorption between 40 and 90 days. Out of the six sows that received IVF embryos, all pregnancies were lost at 28 days with the presence of expelled embryos. Three of them were recovered and showed mutations in GGTA1. Among the five sows that received in vivo embryos, one gave birth to five piglets with GGTA1 biallelic mutations. Three of these piglets showed GHR mutations, with only one having a biallelic mutation. No mutations were observed for CMAH and β4GalNT2 genes. The absence of α-Gal was observed on the surface of GGTA1 KO piglet cells. The GHR biallelic mutated piglet was significantly shorter compared to the others, and no differences were found in weight among them. In conclusion, the microinjection of in vivo zygotes was the only technique that proved to be efficient for the birth of five edited pigs, and probably due to a lack of CMAH and β4GalNT2 sgRNAs edition efficiency, only two of the four genes proposed were successfully edited.