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Title: Analysis of the Impact of Site-specific Demethylation of N6-methyladenosine on Interferon Beta
Authors: Beard, Daniel
Advisors: Ploss, Alexander
Department: Molecular Biology
Class Year: 2022
Abstract: Epitranscriptomics is a growing field of study that refers to the factors outside of genes that affect expression via direct regulation of RNA molecules, by chemical modification. The most abundant and well-studied RNA modification is the methylation of the N6 position on adenosine, or N6-methyladenosine (m6A). The placement of this modification is mediated by the methyltransferase complex, METTL3 and METTL14. Our lab has amassed data demonstrated that the targeted disruption of METTL14 in mouse hepatocytes in vivo causes liver damage, including fibrosis and cirrhosis. It is presently unknown which specific transcript(s) – being subject to epitranscriptomic regulation – are responsible for the observed phenotype. To enable demethylation of specific transcripts, we constructed a CRISPR dCas13-FTO construct that can be directed to specific sites using a guide-RNA. As a proof-of-concept, we target interferon- (IFNB), which has been well studied in its relationship with METTL14 depletion in the liver, to demethylate three m6A modified sites on its RNA transcript. Ultimately, vector-mediated delivery of CRISPR dCas13-FTO can be used to specifically remove m6A modifications from other RNA transcripts in the liver that are up- or downregulated in response to METTL14 depletion.
Type of Material: Princeton University Senior Theses
Language: en
Appears in Collections:Molecular Biology, 1954-2024

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