About the Project
An alternative therapeutic approach is to up-regulate the expression of wildtype, haploinsufficient or hypomorphic alleles to compensate for the lack of normal protein function. This can be achieved using CRISPR-mediated activation (CRISPRa) that uses a ‘dead’ Cas9 protein (dCas9) that binds normally to its target sequence but lacks endonuclease activity. dCas9 is fused to a transcriptional activator that recruits transcription factors to the targeted promoter or enhancer. CRISPRa has been successfully used to up-regulate gene expression and correct the associated mutant phenotype for a number of human conditions.
We will select a number of IRD genes for CRISPRa based on their importance as a disease gene and disease mechanism. We will test whether CRISPRa can increase the expression of each gene in a relevant cell line. Once optimised the system will be tested in differentiated patient induced pluripotent stem cells (iPSC) with haploinsufficient or hypomorphic alleles. CRISPRa-iPSCs will then be differentiated and the expression and function of the target protein assessed.
Our groups have extensive experience in IRD research including restoration of function to mutant proteins and the generation and application of iPSC-derived tissue. The successful applicant will also work closely with the University of Manchester’s Genome Editing Unit that is expert in the application of CRISPRa and has developed a number of reagents and tools that will be employed in this project.
The PhD researcher will gain experience in a number of techniques including cell culture, iPSC culture and differentiation, CRISPRa design and optimisation, expression analysis by qRT-PCR and western blot, promoter identification and cloning, and functional analysis of the target proteins.
Candidates are expected to hold (or be about to obtain) a minimum upper second class honours degree (or equivalent) in a related area / subject. Candidates with previous laboratory experience, particularly in cell culture and molecular biology, are particularly encouraged to apply. For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website (https://www.bmh.manchester.ac.uk/study/research/apply/). Informal enquiries may be made directly to the primary supervisor.
For international students we also offer a unique 4 year PhD programme that gives you the opportunity to undertake an accredited Teaching Certificate whilst carrying out an independent research project across a range of biological, medical and health sciences. For more information please visit http://www.internationalphd.manchester.ac.uk
As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.
Kelleher J et al, Patient-Specific iPSC Model of a Genetic Vascular Dementia Syndrome Reveals Failure of Mural Cells to Stabilize Capillary Structures. 2019. Stem Cell Reports. 13:817.
Ray-Jones H et al, Mapping DNA interaction landscapes in psoriasis susceptibility loci highlights KLF4 as a target gene in 9q31. 2020. BMC Biol. 18:47.
Matharu N et al, CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. 2019. Science. 363: eaau0629
Moreno AM et al, In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation. 2018. Mol Ther. 26:1818.
Why not add a message here
Based on your current searches we recommend the following search filters.
Based on your current search criteria we thought you might be interested in these.