Friedreich's ataxia (AF) is caused by an expansion of the GAA triplet in the first intron of the FXN gene, encoding the frataxin protein, whose expression is decreased. The work analyzed in this thesis aims to study the effect of the insertion, via CRISPR / Cas9, of about 200 GAA into the intron of the fh gene of Drosophila melanogaster (homolog of FXN). Deficiency in protein expression is associated with developmental delay and high lethality. To overcome the early lethality of the mutation, genetic tools were used that allowed the protein to be overexpressed during development. RNA Seq has shown alterations in amino acid metabolism and oxidative stress markers. In particular, a general increase in Tspo expression was noted, proposed as a biomarker for the disease, totally restored to normal by the expression of frataxin in the adult. Finally, it was possible to evaluate the positive effect of N-acetyl cysteine which increases the survival rate and improves locomotor skills, resistance to oxidative stress and aconitase activity. This study also highlights the effectiveness of CRISPR / Cas9 genome editing technology for introducing human mutations in Drosophila’s orthologous genes.
L’atassia di Friedreich (FA) è causata da un’espansione della tripletta GAA nel primo introne del gene FXN, codificante la proteina fratassina, la cui espressione risulta diminuita. Il lavoro analizzato in questo elaborato di laurea ha lo scopo di studiare l’effetto dell’inserzione, mediante CRISPR/Cas9, di circa 200 GAA nell’introne del gene fh di Drosophila melanogaster (omologo di FXN). Al deficit d’espressione della proteina si associano ritardo nello sviluppo e elevata letalità. Per ovviare alla precoce letalità della mutazione, sono stati utilizzati tools genetici che hanno consentito di sovraesprimere la proteina durante lo sviluppo. L’RNA Seq ha evidenziato alterazioni nel metabolismo degli amminoacidi e marcatori di stress ossidativo. In particolare, si è notato un generale aumento dell’espressione di Tspo, proposto come biomarker per la malattia, totalmente riportata alla normalità dall’espressione di fratassina nell’adulto. Infine, è stato possibile valutare l’effetto positivo dell’N-acetil cisteina che aumenta il tasso di sopravvivenza e migliora le capacità locomotorie, la resistenza allo stress ossidativo e l’attività aconitasica. Questo studio evidenzia anche l’efficacia della tecnologia di genome editing CRISPR/Cas9 per introdurre mutazioni umane in geni ortologhi di Drosophila.
L’inserzione di ripetizioni GAA nel gene di fratassina di Drosophila con CRISPR/Cas9 fornisce un modello per lo studio dell’atassia di Friedreich e dell'effetto di protezione in vivo con N-acetil cisteina
FAVERO, GIACOMO
2021/2022
Abstract
Friedreich's ataxia (AF) is caused by an expansion of the GAA triplet in the first intron of the FXN gene, encoding the frataxin protein, whose expression is decreased. The work analyzed in this thesis aims to study the effect of the insertion, via CRISPR / Cas9, of about 200 GAA into the intron of the fh gene of Drosophila melanogaster (homolog of FXN). Deficiency in protein expression is associated with developmental delay and high lethality. To overcome the early lethality of the mutation, genetic tools were used that allowed the protein to be overexpressed during development. RNA Seq has shown alterations in amino acid metabolism and oxidative stress markers. In particular, a general increase in Tspo expression was noted, proposed as a biomarker for the disease, totally restored to normal by the expression of frataxin in the adult. Finally, it was possible to evaluate the positive effect of N-acetyl cysteine which increases the survival rate and improves locomotor skills, resistance to oxidative stress and aconitase activity. This study also highlights the effectiveness of CRISPR / Cas9 genome editing technology for introducing human mutations in Drosophila’s orthologous genes.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/11152