Production of Genetically Modified Porcine Embryos via Lipofection of Zona-Pellucida-Intact Oocytes Using the CRISPR/Cas9 System.

The generation of genetically modified pigs has an important impact thanks its applications in basic research, biomedicine, and meat production. Cloning was the first technique used for this production, although easier and cheaper methods were developed, such as the microinjection, electroporation, or lipofection of oocytes and zygotes. In this study, we analyzed the production of genetically modified embryos via lipofection of zona-pellucida-intact oocytes using Lipofectamine CRISPRMAX Cas9 in comparison with the electroporation method. Two factors were evaluated: (i) the increment in the concentration of the lipofectamine-ribonucleoprotein complexes (LRNPC) (5% vs. 10%) and (ii) the concentration of ribonucleoprotein within the complexes (1xRNP vs. 2xRNP). We found that the increment in the concentration of the LRNPC had a detrimental effect on embryo development and a subsequent effect on the number of mutant embryos. The 5% group had a similar mutant blastocyst rate to the electroporation method (5.52% and 6.38%, respectively, > 0.05). The increment in the concentration of the ribonucleoprotein inside the complexes had no effect on the blastocyst rate and mutation rate, with the mutant blastocyst rate being similar in both the 1xRNP and 2xRNP lipofection groups and the electroporation group (1.75%, 3.60%, and 3.57%, respectively, > 0.05). Here, we showed that it is possible to produce knock-out embryos via lipofection of zona-pellucida-intact porcine oocytes with similar efficiencies as with electroporation, although more optimization is needed, mainly in terms of the use of more efficient vesicles for encapsulation with different compositions.