Generation of genetically modified pigs by CRISPR-Cas9 for experimental models

Resum

Gene editing consists of the modification of the genomic sequence. The development of programmable endonucleases supposed an important advance in the efficiency of gene editing. The use of this technology is widely developed and has different applications such as basic science, improving agricultural production, or biomedicine applications. The pig (Sus scrofa domesticus) is one of the most important animals in the meat industry worldwide. The in vitro embryo production in this species is still inefficient due to the high incidence of polyspermic and suboptimal embryo development, being this an important limitation. For this reason, one of the main objectives of this Thesis was to optimize the conditions for the generation of knock-out (KO) pig embryos by different methodologies to achieve the higher efficiency of the system, in terms of blastocyst formation and mutation rates. On the other hand, the production of gene-edited embryos has an important limitation, the incidence of mosaicism, thus being another central objective of this study to produce embryos with the lowest mosaicism incidence to later obtain pigs with the desired mutation and maximum efficiency. All the studies shown in this Doctoral Thesis were based on the use of a conventional system of in vitro embryo production (IVP) involving the use of immature cumulus-oocyte complexes (COCs) and the in vitro fertilization (IVF), followed by an embryo culture procedure. In this experimental work, frozen-thawed boar semen is routinely used for IVF after being selected by a swim-up procedure that had been previously optimized too. Mutation detection was performed using the fluorescent PCR-capillary gel electrophoresis technique. Samples were considered wild type when the peak obtained by capillary electrophoresis was the same size as control peak. Other peaks were considered to be KO, and when more than two peaks were detected, this sample was evaluated as a mosaic. In chapter 2 the time of microinjection and the use of Cas9 as RNA or ribonucleoprotein (RNP) were evaluated. When Cas9 was delivered as mRNA, mutation rate was similar in all groups, being around 30-42%, whereas the mosaicism rate was between 0-21%. On the other hand, when RNP was delivered, mutation rates did not differ between groups but were higher compared with those microinjected with Cas9-mRNA. Mosaicism rate was lower in the group microinjected before insemination (15%) compared with those injected afterwards (33-57%). Therefore, in vitro-produced embryos microinjected with RNP before insemination followed by embryo transfer allowed the production of two females and one male with frame shift mutations in both alleles of the TPC2 gene. In chapter 3, after the optimization of electroporation conditions and changes to the ratio of sgRNA:Cas9, the efficiency of electroporation was tested in comparison to microinjection to analyse different strategies to generate CAPN3 KO pig embryo that could be models of LGMDR1 human disease. Regarding mutation parameters and blastocyst rate, no differences were found between either the methods (electroporation vs. microinjection) or the combination of guides. In the last chapter, the objective was to evaluate whether the application of aphidicolin makes it possible to improve the gene-editing system by reducing the mosaicism without affecting the quality and quantity of genetically modified embryos obtained. For this, sgRNAs against TPCN1 were used. As a result, a positive effect in avoiding the mosaicism and a negative effect of aphidicolin were observed in blastocyst development was observed. Therefore, under the tested conditions in these experiments, the use of aphidicolin did not show any advantage. In conclusion, after the studies carried out in the different chapters of this Thesis, an efficient and optimized system has been established to generate gene-edited embryos and pigs that could be used for human disease models.