I am developing a novel system to examine the organisation and function of the ribosomal RNA gene repeats (rDNA) in mammalian cells. rDNA forms large arrays of hundreds of tandemly repeated genes that encode the major RNA components of the ribosome. Its repetitive nature makes it notoriously difficult to study, and this is particularly true in mammalian cells, where very few genetic tools to investigate the rDNA have been developed. Moreover, the high level of sequence conservation between each rDNA repeat makes it impossible to distinguish the behaviour of individual rDNA repeats using existing approaches. To overcome this problem, I am tagging every one of the rDNA repeats with a unique “barcode” sequence in mammalian cells, using the revolutionary genome editing tool, CRISPR-Cas9.

Once this tool has been established, I will use it to help us understand the drivers of genome instability in cancer. Genome instability is a hallmark of cancer, however the link between genome instability and rDNA instability in cancer has never been addressed, despite the rDNA being the most unstable site in the genome. By barcoding each rDNA copy in a cell culture model of malignant transformation, such as the primary human fibroblasts of the BJ strain, and using deep sequencing techniques, I will be able to test whether there is a change in the organisation of the rDNA repeats as cells become malignant, which will help us understand the dynamics of instability in the rDNA in tumorigenesis. Moreover, using this system I will test whether the epigenetic profile of each rDNA repeat changes during malignancy, using ChIP-seq and/or transcriptome sequencing. Therefore, this tool will enable us to investigate the role of individual rDNA repeats in tumorigenesis for the first time.