CRISPR manipulation of gene expression in retinal organoids to model and rescue human disease

University of Manchester

Faculty of Biology, Medicine and Health

Project Description

Human pluripotent stem cells can be used in the lab to grow 3D retinal structures called retinal organoids. This allows us to investigate both the early and later events of human eye development and disease.

Abnormal eye development including microphthalmia (small eye), anophthalmia (no eye) and coloboma (abnormal eye development) (MAC) can result from a large number of genetic mutations. In these conditions the balance of stem cell proliferation and differentiation is often awry, however, the underlying mechanisms are poorly understood. Retinal dystrophies (RDs) are a range of conditions that lead to deteriorating vision but it is unclear how early the first retinal changes begin. RDs can also result from single gene mutations, including those that cause a lack of sufficient protein activity resulting from haploinsufficiency or hypomorphic alleles.

CRISPR modulation of gene expression is a useful technology to study human disorders. CRISPR activation (CRISPRa) can be used to up-regulate the expression of hypomorphic or wildtype alleles to compensate for the lack of normal protein function and has been used successfully to up-regulate protein expression to attenuate the disease phenotype in animal models of muscular dystrophy, retinitis pigmentosa, obesity and epileptic seizures.

Aims:

1. Investigate stem cell proliferation and differentiation in microphthalmia

Candidate genes for microphthalmia will be modulated by CRISPR in retinal organoids followed by immunohistochemical staining for proliferation and differentiated cell-type markers and different stages of organoid development.

2. Rescue RD gene expression in retinal organoids

We will identify a number of RD genes for CRISPRa based on their molecular pathologic mechanism. Expression of the target gene in retinal organoids will be determined by qRT-PCR and western blot. Immunohistochemical staining and qRT-PCR will determine whether the disease phenotype is rescued.

The successful applicant will also work closely with the Genome Editing Unit.

Funding Information

Applications are invited from self-funded students. This project has a Band 3 tuition fee. Details of our different fee bands can be found on our website.

Eligibility Requirements

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.

Application Process

For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website. Informal enquiries may be made directly to the primary supervisor. On the online application form select PhD Genetics.

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 www.internationalphd.manchester.ac.uk

Supplementary Information

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.

References

Liu J, Sadeh TT, Lippiat JD, Thakker RV, Black GC, Manson F. Small molecules restore the function of mutant CLC5 associated with Dent disease. J Cell Mol Med. 2021 Jan;25(2):1319-1322. doi: 10.1111/jcmm.16091.
Liu J, Taylor RL, Baines RA, Swanton L, Freeman S, Corneo B, Patel A, Marmorstein A, Knudsen T, Black GC, Manson F. Small Molecules Restore Bestrophin 1 Expression and Function of Both Dominant and Recessive Bestrophinopathies in Patient-Derived Retinal Pigment Epithelium. Invest Ophthalmol Vis Sci. 2020 May 11;61(5):28. doi: 10.1167/iovs.61.5.28.
David J. Green; Eva Lenassi; Cerys S. Manning; David McGaughey; Vinod Sharma; Graeme C. Black; Jamie M. Ellingford; Panagiotis I. Sergouniotis. North Carolina Macular Dystrophy: Phenotypic Variability and Computational Analysis of Disease-Associated Noncoding Variants. Investigative Ophthalmology & Visual Science June 2021, Vol.62, 16.
Veronica Biga, Joshua Hawley, Ximena Soto, Emma Johns, Daniel Han, Hayley Bennett, Antony D Adamson, Jochen Kursawe, Paul Glendinning, Cerys S Manning, Nancy Papalopulu. A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord. Mol Syst Biol (2021)17:e9902
Manning CS, Biga V, Boyd J, Kursawe J, Ymisson B, Spiller DG, Sanderson CM, Galla T, Rattray M, Papalopulu N. Quantitative single-cell live imaging links HES5 dynamics with cell-state and fate in murine neurogenesis. Nat Commun. 2019 Jun 27;10(1):2835.

To apply for this PhD, please use the following application link: https://www.bmh.manchester.ac.uk/study/research/apply

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