Engineering the Cas9/CRISPR tool-box to study the role of nuclear architecture during the regulation of DNA replication timing in mouse embryonic stem cells

This is a UK Centre for Mammalian Synthetic Biology Studentship.

Interested individuals must follow Steps 1, 2 and 3 at this link on how to apply:

http://www.ed.ac.uk/biology/prospective-students/postgraduate/pgr/how-to-apply

Supervisors: Dr Sara Buonomo ([Email Address Removed]) and Prof Susan Rosser ([Email Address Removed])

DNA replication is a fundamental step for the transmission of genetic as well as epigenetic information and is controlled at multiple levels. DNA replication starts from genomic regions called origins. The eukaryotic genome contains multiple origins that are activated throughout the S-phase of the cell cycle, in a specific temporal order. The pathway that controls this order is called the DNA replication-timing program. The biological role of this program is still unknown, although there are indications that it is fundamental. It is conserved throughout eukaryotes and dynamically regulated during embryonic development and cell fate transitions. The Buonomo group has identify the first genome-wide regulator of DNA replication timing, a protein called Rif1, describing its function during the architectural re-organization of the mammalian genome in the G1 phase of the cell cycle. The Buonomo group has discovered that Rif1 deletion induces simultaneously changes of replication timing and of spatial positioning of the late replicating genomic regions. Supported by independent data, this observation suggests the possibility that localization at the nuclear is a key determinant of late replication. Traditionally, tethering genomic regions to the nuclear periphery relies on knock-in of tet or lac operators (tetO/lacO), sequences of bacterial origin recognized and bound with very high affinity by the tet repressor (tetR) protein. Fusion of the tetR with a peripheral protein such as Lap2b induces the relocation to the nuclear periphery of the teto-tagged genomic region. However, this approach has some major drawbacks. For the system to work, it has been necessary to introduce very large arrays containing hundreds of tetOs. Such large insertions alter the epigenetic and chromatin environment of the locus, potentially interfering with the results.

The PhD project available will harness the new Cas9/CRISPR technology to tether any genomic sequence of choice to the nuclear periphery. By guiding a fusion protein Cas9-Lap2b to the desired genomic locus through the expression of multiple guide RNAs (sgRNAs), we will be able to reposition them to the nuclear periphery without altering the DNA sequence or chromatin environment. The student will be involved in designing and engineering multiple sgRNAs and generically manipulate mouse embryonic stem cells to stably express this multiple-components system. We will also employ advanced protein engineering methods to asses the contribution of protein multimerization versus DNA-binding affinity. Finally, the student will be trained in state-of-the-art confocal and super-resolution microscopy, and bioinformatics analysis of the data acquired.

This is a joined position between the Rosser and the Buonomo groups. The student will therefore be exposed and learn directly from experts in both synthetic and cell biology fields. If you are interested in synthetic biology, cell cycle, DNA replication, nuclear organization and want to work with mammalian stem cells join our groups. We are looking for enthusiastic, flexible and hard working candidates driven by curiosity and passion for science. Basic knowledge of cell/molecular biology and ability to work in an international environment are required.