97 Generation of GGTA1 knockout porcine blastocysts by intracytoplasmic sperm injection mediated gene edition

Pigs are considered to have great promise in biomedical research, and one option is as an organ supply for xenotransplantation. For this purpose, direct zygote gene editing is the most widely used approach to obtain edited piglets. However, IVF porcine zygote production is still inefficient due to polyspermy problems, and in vivo embryo recovery involves additional complexity and higher costs. We propose intracytoplasmic sperm injection-mediated gene edition (ICSI-MGE) as an alternative technique to produce edited porcine embryos avoiding polyspermy. The α-1,3-galactosyltransferase gene (GGTA1) is an enzyme that places galactose residues on the surface of porcine cells, causing hyperacute rejection if placed in the human body. Therefore, the aim of this work was to evaluate the efficiency of ICSI-MGE to produce GGTA1 knockout (KO) porcine embryos. Briefly, two guides (sgRNA) were designed to the internal regions of GGTA1. Ribonucleoprotein complex (RNP) was diluted to half concentration with 10% polyvinylpyrrolidone (PVP), resulting in a final concentration of 200 ng μL⁻¹ for Cas9 protein and a total of 100 ng μL⁻¹ for the two sgRNAs. Cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured in vitro for 44 h. A group of matured and denuded oocytes were subjected to electrical activation followed by incubation in PZM medium containing 6-dimethylaminopurine (6-DMAP), as a diploid parthenogenetic embryo development control (DAP). Next, a total of 2 pL of the mixtures was microinjected into the diploid parthenogenetic zygotes (DAP-ggta1) and co-microinjected with sperm in another group of matured oocytes (ICSI-ggta1). Embryos were in vitro cultured until Day 7. Genetic edition was corroborated by analysing the presence of double cut directed by the two sgRNAs designed on the target gene, resulting in an amplicon with lower molecular weight compared with the wild-type PCR fragment. Data was analysed by Fisher’s exact test using GraphPad software (GraphPad Inc.). Differences were considered significant at P < 0.05. No differences in cleavage rates were found among groups (DAP: 55.7%, n = 741; DAP-ggta1: 51.7%, n = 180; ICSI-ggta1: 50.5%, n = 105; ICSI: 54.8%, n = 73). However, blastocyst rates were significantly lower in ICSI groups (ICSI: 6.8% and ICSI-ggta1: 10.5%) compared with parthenogenetic groups (DAP: 20% and DAP-ggta1: 19.3%), suggesting a delay in development of ICSI-produced embryos compared with parthenogenic ones. One group of blastocysts obtained by ICSI-MGE and other by microinjection were analysed by PCR and sequencing. From those groups, 4/4 of ICSI-ggta1 and 11/19 of DAP-ggta1 embryos showed deletion and/or edition. These results are preliminary data and suggest that ICSI-MGE could be a powerful technique to produce gene-edited porcine embryos.