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Gene editing technology can be used to make small-scale mutation corrections to cure genetic diseases. However, in cases where a disease can be caused by multiple mutations, it is generally more efficacious to use a strategy that permanently integrates entire healthy copies of a gene to replace mutant copies.
Here, the student will use the novel DNA editing technology “eePASSIGE” to insert healthy copies of the KRT1/10 gene in keratinocytes/fibroblasts from patients with epidermolytic ichthyosis (EI) caused by mutations in KRT1/10. The relative efficacy and optimal conditions for gene correction will be evaluated by Sanger sequencing and by assessing restoration of mRNA and protein expression.
This PhD project aims to address the hypothesis that optimizing the efficacy of gene editing will advance the development of a future therapy for EI (and other skin disorders). To prove this hypothesis, the strategy for the PhD student will be to:
I. Optimize the efficiency of KRT1/10 whole-gene replacement using the eePASSIGE system.
II. Assess gene editing safety by sequencing the inserted gene around the insertion sites as well as genome-wide off-target activity using deep sequencing analysis.
III. Explore the translational potential of gene editing in EI through functional validation of the edited cells.
Throughout the 3-year project the following aims have to be achieved by the student in the given time:
Aim1: Insert a full-length copy of the KRT1/10 gene into the genome of KRT1/10-mutant cells using eePASSIGE.
Aim2: Next generation sequencing (NGS) and extensive bioinformatics analysis to confirm on-target efficiency and evaluate potential off-target and nonspecific changes in gene expression and splicing events (year 2).
Aim3: Functional evaluation of the efficiency of edited patients’ cells (year 3).
The selected student must have a keen interest in (and ideally expertise and experience with) DNA editing technology derived from CRISPR-Cas9. Experience with mammalian cell culture and expertise in dermatology is desirable but not essential.
The student will work closely with Dr Jackow the primary supervisor of the project. The student will be hosted in St John’s Institute of Dermatology, part of the Division of Genetics and Molecular Medicine, Kings College London (KCL) which has an active clinical and scientific genetic skin research programme. For questions related to this application the interested candidate can contact Dr Jackow or Dr. Carina Graham directly: joanna.jackow@kcl.ac.uk; carina.graham@kcl.ac.uk.
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