Produção e avaliação de modelos de cultivo celular para teste de RNAs guia para o sistema CRISPR-Cas9

Abstract

Background: The CRISPR-Cas9 gene editing system has emerged as a powerful tool for targeted genetic modifications. This doctoral thesis aimed to optimize the CRISPR-Cas9 system for use in bovine mammary epithelial cells (MDBK) and embryos. Methods: Specific sgRNAs were designed and produced to target loci of interest for gene editing in MDBK cells. Additionally, stable Cas9- expressing MDBK cells were generated. In vitro evaluation of sgRNA activity was conducted, and mRNA reporters were utilized for transfection efficiency confirmation. Antibiotic selection was employed to promote stable Cas9 expression in human and bovine cell lines. The efficiency of gene editing in embryos was also evaluated through PCR and sequencing. Results: The in vitro evaluation of sgRNA activity in MDBK cells showed variations in editing efficiency, emphasizing the need for cell lines expressing Cas9 with relevant genomes. The mRNA reporter method demonstrated its usefulness for confirming transfection efficiency. Antibiotic selection successfully promoted stable Cas9 expression in human and bovine cell lines. Gene editing in embryos resulted in a total editing proportion of 48%, with the majority of mutant cells (35.5%) exhibiting a single-nucleotide deletion. Conclusion: The designed sgRNAs enabled gene editing in MDBK bovine cell lines, and the optimized CRISPR-Cas9 model was effective for editing the target loci in embryos. These findings contribute to the advancement of bovine genome engineering, offering potential applications in future studies involving the CRISPR-Cas9 system for bovine genome editing.