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Endogenous Reporter Systems for High-Throughput Functional Screening in Clear Cell Renal Cell Carcinoma



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Zaini, Muhammad Nazhif Bin 


Tissue-specific transcriptional programs define cancer phenotypes. However, the molecular mechanism behind the transcriptional specificity is mostly unknown, hindering the development of therapeutic approaches. In clear cell renal cell carcinoma (ccRCC), loss of von Hippel-Lindau tumour suppressor (VHL), seen in 90% of ccRCCs, leads to the stabilization of hypoxia inducible factor HIF2A and aberrant expression of its downstream targets such as the chemokine receptor CXCR4. Activation of HIF2A has been shown to be important for ccRCC tumour initiation and early progression and the expression of CXCR4 promotes metastatic progression in ccRCC. It is unclear, however, how VHL loss results in ccRCC specific tumorigenesis, one possibility being that HIF2A mRNA is expressed at a high enough level only in the cell lineage that eventually forms ccRCCs. To investigate this hypothesis, novel experimental reporter systems were developed to dissect the mechanisms regulating HIF2A mRNA expression and activity. These endogenous HIF2A and CXCR4 reporter systems express an mCherry fluorescent marker at the 3’ end of their respective locus. Homology-directed repair (HDR)- and non-homologous end joining (NHEJ)-based CRISPR-Cas9 knock-in strategies were utilized for the development of these systems that involve homology-dependent and minicircle-aided integration, respectively. These reporters were integrated precisely in the genome as evidenced by sequencing. The efficiency of these systems at detecting fluctuations in the mRNA expression of the associated endogenous gene was validated by analysing mCherry fluorescence in cells with either CRISPRi-based HIF2A inactivation or VHL-reintroduction. Moreover, these reporters also proved feasible for genetic screens as shown from an sgRNA enrichment experiment that highlighted sgRNAs targeting regulatory factors within a specific population of cells. These reporter systems were applied with focused CRISPR-Cas9 libraries targeting transcription factors (TF) and chromatin regulators for high-throughput screenings. Our screening data was analysed through a permutation analysis to remove false positive hits. In the HIF2A reporter screen with the TF library and the chromatin library, HIF2A was the sole significant hit, suggesting redundancy within the HIF2A gene regulatory machinery. In sum, I have developed and technically validated novel reporter systems that could aid in the systematic interrogation of cancer transcriptional dependencies, hence paving the way for novel therapeutic approaches.





Vanharanta, Sakari


Genetic Screens, ccRCC, Endogenous Reporters, HIF2A


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge