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Gene editing and specific MicroRNA inhibition

Student thesis: Doctoral ThesisDoctor of Philosophy

Background: MicroRNAs (miRNAs) are short, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. They are important regulators of diverse physiological and pathophysiological processes. MiRNA families and clusters are two key features in miRNA biology. Previous work on miRNAs in abdominal aortic aneurysm has focused on miR-195, a member of the miR-15 family. MiR-195 exhibits high homology with the other members of the family and therefore, its manipulation, overexpression or inhibition, inevitably affects the expression of other miRNAs of the family. Objective: The current project explores the use of CRISPR/Cas9 as a gene editing tool to delineate the expression of miRNA families. Methods and Results: We employed CRISPR/Cas9, an RNA-guided system able to provide highly specific alterations of targeted sequences in the genome of eukaryotic cells. We designed RNA guides and validated their efficiency in editing the mir-195 locus in various cell types while no off-target mutagenesis was observed. Quantification of the levels of miR-195 and other members of the miR-15 family, revealed a significant decrease of miR-195 and in the expression of the other miRNA in the cluster, miR-497. Three more miRNA clusters composed of miRNA members of the same or different family were analysed. Although no gene editing was detected in the miR-497 genomic locus, computational simulation revealed alteration in the three-dimensional structure of the pri-miR-497~195 that may affect its processing. RNA pull-down experiments, using wild type and an edited pri-miRNA of shorter length, were performed and the eluted proteins were analysed by Mass Spectrometry. Conclusions: Our findings suggest that CRISPR/Cas9 is a powerful gene editing tool able to provide highly specific editing of the genomic locus of miRNAs, resulting in decrease of the levels of their mature transcripts. They also highlight different regulatory mechanisms in miRNA cluster regulation and function.
Original languageEnglish
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Award date1 Sep 2019

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