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

Genetically modified pigs are very useful thanks to their applications in basic research, biomedicine, and meat production. There are different methods for producing them, including cloning and the microinjection or electroporation of oocytes and zygotes. Easier techniques are being developed, such as lipofection, which involves the encapsulation of the CRISPR/Cas9 system into vesicles that are introduced into cells. We compared the embryo development and mutation rates associated with different conditions of lipofection treatment with the electroporation technique in zona-pellucida-intact porcine oocytes. We found that the lipofection treatment, once optimized, was as effective as the electroporation technique in terms of the embryo development and mutation rates. In addition, an increment in the concentration in the media of the liposomes-CRISPR/Cas9 system complexes had a detrimental effect on the embryo development parameters, which could indicate a possible toxic effect. The achievement of generating mutant embryos via lipofection without removing the zona pellucida could open up a new, easy, and cheap way of producing genetically modified pigs. 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 (TM) CRISPRMAX (TM) 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, p > 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, p > 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.