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

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

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of <i>Bbs1</i>

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate