Development of an electroporation procedure for the generation of genetically modified mice using CRISPR/Cas9

Trabajo de fin de Grado. Grado en Biotecnología. Curso académico 2019-2020The house mouse (Mus musculus) has become the main animal model used in biomedical research, because of its physiological and genetical similarity with the human being. Over the last years, the development of the genomic editing tools has led to the generation of genetically modified (GM) mice, entailing great progress in research work. Within these tools, the discovery and improvement of the CRISPR/Cas9 system has been particularly revolutionary due to its design´s simplicity, efficiency and versatility. Originally described as an adaptive immunity mechanism in bacteria and archaea, it is composed of two elements: the single-read ribonucleic acid (sgRNA) or RNA guide and the endonuclease Cas9. The first one is made up of a 20 nucleotides sequence called CRISPR ribonucleic acid (crRNA), which defines the system’s specificity and is linked to a structural sequence named trans-activating crRNA (tracrRNA). When this hybridizes with the target sequences, the nuclease produces a double-strand break (DSB), activating endogenous mechanisms of DNA repair. This involves the gene disruption, leading to knock-out models; or the insertion of homologous molecules, producing knock-in models. The current strategy to produce GM mice is based on the microinjection of the CRISPR/Cas9 system components into one of the zygote’s pronuclei and its subsequent transfer to a female´s oviduct. The pronuclear microinjection is a quite slow process that requires expensive and sophisticated equipment and experienced personnel. As an alternative, we propose the use of electroporation to introduce the CRISPR/Cas9 system reagents in embryos and overcome the raised limitations. Thus, we have checked that the application of high voltage short electrical pulses allows the preservation of zygote’s viability. In addition, these create transient cell membrane pores which sgRNA and Cas9 protein access across. Through the in vitro genotyping process it has been confirmed that these are capable of performing genomic editing on specific locus. Finally, a specific sgRNA against tyrosinase gene (Tyr), involved in the mouse´s coat color, was designed. Therefore, the generation of albino and/or mosaic mice has allowed us to easily determine the editing and knock-out alleles’ generation efficiency with both techniques. It has been concluded that electroporation is a method as effective as pronuclear microinjection, so it could become its real substitute