96 Evaluation of CRISPR/Cas9 alternative delivery in parthenogenetic porcine embryos

Abstract

Porcine genetic editing is considered promising in biomedical research, particularly for xenotransplantation. However, in vitro porcine embryo production is less efficient than in other animal models. Thus, we aimed to perform a rapid optimization essay by producing parthenogenetic porcine embryos to evaluate transgenesis delivery and CRISPR/Cas9 editing efficiency. First, PCX-enhanced green fluorescent protein (EGFP) plasmid was microinjected (30 ng μL⁻¹) into a diploid parthenogenic zygote with (lipo+) or without lipofectamine (lipo−), since it has been shown that this transfection reagent improves transgene delivery and increases its expression in bovine embryos (Vichera et al. 2011 Reprod. Domest. Anim. 46, 214-220; https://doi.org/10.1111/j.1439-0531.2010.01642.x). Briefly, in vitro-matured oocytes were electrically activated, followed by incubation in synthetic oviductal fluid medium containing 6-dimethylaminopyridine. Embryos were in vitro cultured until Day 7, and EGFP-positive (EGFP+) embryos were corroborated at Day 5. Data were analysed using Fisher's exact test. Cleavage rates were no different among groups (EGFP/lipo−: 40%, n = 28; EGFP/lipo+: 45%, n = 41; control: 41, 3%, n = 45; P > 0.05). Also, no significant difference in the percentage of EGFP+ embryos was observed between EGFP/lipo+ (31%, n = 13) and EGFP/Lipo− (18%, n = 5) groups. Although blastocyst rate showed no statistical difference among groups, a lower blastocyst percentage tendency was observed in the EGFP/Lipo+ group compared with the control group (EGFP/Lipo+: 5%, n = 2; control: 20%, n = 9; P = 0.051), suggesting that the presence of lipofectamine and EGFP plasmid may affect embryo development. Next, two guides (single guide (sg) RNA) were designed for the internal regions of GGTA1, CMAH, and VWF target genes, involved in hyperacute rejection and coagulation in xenotransplantation. A liposome-DNA mixture was used: DNA for sgRNA and Cas9, with and without lipofectamine (10× dilution; CRISPR/lipo+, CRISPR/lipo−), diluted to half concentration with 10% polyvinylpyrrolidone, resulting in a final concentration of 20 ng ul⁻¹ for all sgRNA and 40 ng ul⁻¹ for Cas9. A total of 2 pL of the mixtures was microinjected into the diploid parthenogenetic zygotes which were in vitro cultured until Day 7. Genetic editing was corroborated by analysing the presence of a double cut directed by the two sgRNA designed for the target genes, resulting in an amplicon with lower molecular weight compared to the wild-type PCR fragment. No differences in cleavage or blastocyst rates were observed among groups (blastocyst rates: CRISPR/Lipo−: 12%, n = 13; CRISPR/Lipo+: 10%, n = 7; control: 15%, n = 18; P > 0.05). Finally, one embryo showed a single deletion for GGTA1 and another showed a double deletion in GGTA1 and VWF genes, out of seven embryos from the CRISPR/Lipo+ group. No gene deletion was confirmed in any of the embryos from the CRISPR/Lipo− group. These results are preliminary data (more experiments are currently being done) suggesting that microinjection of CRISPR/Cas9 with lipofectamine could be used as an alternative delivery system, since it seems to have no impact on porcine embryo development.