Evaluation of CRISPR/Cas9 alternative delivery in parthenogenetic porcine embryos

BRISKI O; FERNANDEZ Y MARTIN R; LA MOTTA GE; RATNER LD; SALAMONE DF

Nueva York

Congreso; International Embryo Technology Society?s 46th Annual Meeting; 2020

International Embryo Technology Society

Porcine genetic edition is considered a promise in biomedical research, particularly for xenotransplantation. However, in vitro embryo production is less efficient than in other animal models. So, we aim to perform a rapid optimization essay by producing parthenogenetic porcine embryos to evaluate transgenesis delivery and CRISPR/Cas9 edition efficiency. First, pcx-egfp plasmid was microinjected (30 ng/ul) into a diploid parthenogenic zygote with (lipo+) or without lipofectamine (lipo-), since it has been showed that this transfection reagent improves transgene delivery and increases its expression in bovine embryos (Vichera et al. 2011). Briefly, in vitro matured oocytes were electrically activated, followed by incubation in SOF medium containing 6-DMAP. Embryos were in vitro cultured until day 7 and egfp positive (EGFP+) embryos were corroborated at day 5. Data were analyzed by 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 egfp/lipo+ group respect to control (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 (sgRNA) were designed to 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 times dilution) (crispr/lipo+, crispr/lipo-) diluted to half concentration with 10% PVP resulting in a final concentration of 20 ng/ul for all sgRNAs and 40 ng/ul for Cas9. A total of 2 pl of the mixtures, was microinjected into the diploid parthenogenetic zygotes and were in vitro cultured until day 7. Genetic edition was corroborated by analyzing the presence of a double cut directed by the 2 sgRNAs designed on the target genes; resulting in an amplicon with lower molecular weight compared to WT PCR fragment. No differences in cleavage and blastocysts 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 7 embryos from 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) that suggest 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.