Precision Medicine DTP - Synthetic chromosomes to understand genome instability

Background

Genomic instability in cancer has been known for many years; the advent of genomic analyses in the late 1990’s enabled these changes to be characterized in detail. Despite these breakthrough studies the molecular mechanisms for genomic instability are poorly understood and even genome scale sequencing of tumours has not shed much light on initiating tumour events.

During every cell cycle, chromosomes are replicated and segregated to daughter cells. In normal cells this process happens with high fidelity ensuring the persistence of a stable genotype. However, in cancer it is beneficial for cell populations to acquire genetic diversity that can be selected for to promote tumour evolution, promoting the growth of selected clones. One of the main determinants of a stable karyotype is the formation of the centromere; aberrant centromere formation leads to inefficient segregation and lagging chromosomes giving rise to aneuploidy whilst deletion of an endogenous centromere drives selection to form a new or “neo” centromere that can maintain genomically unstable karyotypes.

Recent high throughput sequencing studies have shown that many chromatin or chromosome proteins are mutated in cancer. However, this information alone is insufficient to determine whether these are primary “driver” mutations or secondary “passenger” mutations, making it very difficult to determine molecular mechanisms from cancer sequencing data.

Aims

To understand the mechanistic basis for genome stability and how this can be a cancer driver we have developed a synthetic chromosome (called Neo3) with a unique “neocentromere”. This chromosome is stable and has been propagated through many generations, but is located on a chromosome arm within non-repetitive DNA sequences. The Neo3 chromosome will be engineered using CRISPR/Cas9 to insert selectable and fluorescent markers into the chromosome arms to provide an amenable platform to address significant research questions. In particular this project will draw on the recent identification of genetic mutations in the cancer genome atlas (TCGA) to identify candidate genes important for genome stability. This unique resource will enable us to undertake experiments to address our overarching aim to understand the molecular basis for genome instability in disease. In turn this will enable us to identify novel targets for future drug development. To achieve this ambitious aim we have a series of distinct objectives

Centromere transcription: Studies from model organisms indicate that the stability of centromeres is dependent on transcription. We will use synthetic transactivators to probe the role of transcription in regulating chromosome stability on the non-repetitive Neo3 centromere.
Neocentromere formation: Genetic selection of re-arranged chromosomes necessitates the formation of new centromeres. The genomic locations at which centromeres can form are not known. Using CRISPR/Cas9 and Cre/LoxP we will delete the functioning centromere and put the cells under strong selection to form a library of new neocentromeres that will be characterized by deep sequencing.
Identify “driver” mutations for genome instability: We will develop a CRISPRi library based on candidate gene mutations observed in TCGA. Neo3 cells will be transfected with the library, the centromere will be deleted as in aim 2 and cells will be scored for loss or maintenance of the synthetic chromosome.
Training Outcomes

The applicant will learn mammalian chromosome biology, genetic screens and genomic engineering. In addition, the applicant will learn data mining of complex whole genome sequencing data sets and will analyse libraries of clones using next generation sequencing. This combination of computational and laboratory training is crucial for training the next generation of molecular scientist

This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.

All applications should be made via the University of Edinburgh, irrespective of project location. For those applying to a University of Glasgow project, your application along with any supporting documents will be shared with University of Glasgow.

http://www.ed.ac.uk/studying/postgraduate/degrees/index.php?r=site/view&id=919

Please note, you must apply to one of the projects and you must contact the primary supervisor prior to making your application. Additional information on the application process is available from the link above.

For more information about Precision Medicine visit:
http://www.ed.ac.uk/usher/precision-medicine