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Probing the Role(s) of Bbs1 with CRISPR/Cas9 Gene Editing

Freke, Grace Mercedes; (2020) Probing the Role(s) of Bbs1 with CRISPR/Cas9 Gene Editing. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

BBS1 is the most commonly mutated of 21 genes which cause Bardet-Biedl syndrome, a rare, autosomal recessive “ciliopathy”. It results from dysfunction of an antenna-like organelle called the primary cilium, which participates in signalling events during development and homeostasis. Bardet-Biedl syndrome is considered a model ciliopathy in that it exhibits cardinal, multi-systemic features of ciliopathies, including kidney disease, retinal degeneration, polydactyly and obesity. There is, however, a disconnect in our comprehension of how these phenotypes manifest from mutations in ciliopathy genes. The ways in which BBS1 functions within the cell remain unclear and may extend beyond the cilium. One obstacle to understanding how mutations in BBS genes lead to disease is the great phenotypic variability that exists between patients, even siblings with the same causative variants. This project probed the cellular roles of Bbs1 in mouse kidney epithelial cells using the ground-breaking gene editing technique, CRISPR/Cas9. Clonal cell lines carrying biallelic indel mutations in Bbs1 were developed and their genomic DNA, transcripts and protein characterised. Although clones revealed an inconsistent ability to form cilia, an entirely unexpected, novel cell-cell adhesion phenotype was identified in Bbs1-/-. Consistent with recent discoveries of genetic compensation following gene knockout, the severity of this phenotype correlated inversely with Bbs1 transcript instability. Transcriptomics suggested Bbs1 knockout caused a loss of epithelial identity associated with an upregulation of mesenchymal biomarkers and, in the absence of genetic compensation, the downregulation of epithelial markers, culminating in disrupted apical-basal polarity, defective cell-cell adhesion and loss of epithelial barrier integrity. Whether genetic compensation contributes to the phenotypic variability seen in Bardet-Biedl syndrome patients remains to be seen and might provide valuable opportunities for novel therapeutics. This work suggests Bbs1 may have roles beyond the cilium and the Bbs1-/- cell models generated herein will provide a useful tool for future investigations.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Probing the Role(s) of Bbs1 with CRISPR/Cas9 Gene Editing
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Genetics and Genomic Medicine Dept
URI: https://discovery.ucl.ac.uk/id/eprint/10093086
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