Abstract
Dyslexia is a disorder that impairs reading skills and is strongly influenced by genes. One of the genes more robustly associated with dyslexia is KIAA0319. This gene is highly expressed in brain during embryonic life and some studies suggest that it plays a role in neuronal migration. KIAA0319 is a transmembrane protein, the structure of which is consistent with a role in mediating interactions between cells or between cells and the extracellular matrix. Traditional molecular methods and animal models have been used to investigate its function but have failed to unveil its molecular mechanism.The recent development of CRISPR-Cas9 has opened possibilities for the study of genes associated with disorders by simplifying the editing of DNA and the modulation of gene expression. The work described in this thesis applied these new methods to the study of KIAA0319 and led to the development of new tools to investigate genes implicated in complex disorders.
I used CRISPR-Cas9 to eliminate the start codon and to generate mutations that cause premature stop codons in KIAA0319 in RPE1 cell line. These modified cell lines present differences in attachment, migration and cilia length compared to the wild type. I have also used CRISPR-Cas9 to attach a GFP tag to the endogenous KIAA0319 in HEK293 cells to study the dynamics of the protein. The observation of this tagged cell line suggests that the expression of KIAA0319 in HEK293 is very low and restricted to a small percentage of the cells.
In parallel I developed light sensitive gene upregulation systems based on the nuclease-null dCas9, that combine dCas9 proteins originated in different species of microorganisms with a strong transcriptional activator. These systems respond to blue light and are able to upregulate an inducible GFP reporter.
| Date of Award | 29 Jun 2018 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Silvia Paracchini (Supervisor) |
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