About the Project
Mutations causing a lack of sufficient protein activity resulting from haploinsufficiency or hypomorphic alleles are the cause of most inherited retinal dystrophies (IRDs). Gene augmentation is possible for a very limited number of conditions and genome engineering using CRISPR is another treatment option but has the disadvantages of permanently altering the genome and possible off-target mutagenesis.
An alternative therapeutic approach is to use CRISPR activation to up-regulate the expression of hypomorphic or wildtype alleles to compensate for the lack of normal protein function. CRISPRa uses a ‘dead’ Cas9 protein (dCas9) that lacks endonuclease activity and is fused to a transcriptional activator (e.g. VPR). Targeting of the dCas9-VPR to the promoter or enhancer sequence of each gene under investigation is via a short guide RNA (sgRNA). CRISPRa has been used successfully to up-regulate endogenous or homologous protein expression to attenuate the disease phenotype in animal models of muscular dystrophy, retinitis pigmentosa, obesity and epileptic seizures.
We will identify a number of IRD genes for CRISPRa based on their molecular pathologic mechanism. The efficacy of the top 4 sgRNAs designed to each promoter will be determined in a relevant retinal cell line engineered to constitutively express dCas9-VPR. Expression of the target gene will be determined by qRT-PCR and western blot.
For a sub-set of genes the functional rescue will be determined in tissue derived from patient induced pluripotent stem cells (iPSCs). The assay is dependent upon the protein function. The efficacy of CRISPRa will also be quantified in retinal cell types isolated from retinal organoids grown from iPSC. The optimised sgRNAs and dCas9-VPR will also be tested as a split dCas9-VPR system. This system uses adeno-associated virus (AAV), which is commonly used in therapeutic applications, to deliver dCas9-VPR in two halves which later recombine in vivo.
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 Genome Editing Unit.
The PhD researcher will gain experience in a number of techniques including cell culture, iPSC and organoid culture and differentiation, CRISPRa, cloning, qRT-PCR, western blot and FACS.
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
Equality, diversity and inclusion is fundamental to the success of The University of Manchester, and is at the heart of all of our activities. The full Equality, diversity and inclusion statement can be found on the website View Website
2. 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.
3. 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.
4. Böhm S et al, A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation. 2020. Sci Adv. 6:eaba5614.
5. Zerti D, et al, Developing a simple method to enhance the generation of cone and rod photoreceptors in pluripotent stem cell-derived retinal organoids. 2020. Stem Cells.38:45.
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