Description
Genome editing encompasses the targeted modification of DNA, enabling insertions, deletions, or substitutions at defined genomic loci. Conventional approaches, however, rely on the generation of a double-stranded break (DSB), which limits both their precision and efficiency.
Prime editing, characterised as a "search-and-replace" genome editing technology, builds upon earlier CRISPR-based systems to enable all 12 possible transition and transversion mutations, as well as small insertions and deletions, without the requirement for a double-stranded break. This research focuses on the IFT140-associated inherited retinal ciliopathy, specifically targeting the T484M point mutation located on chromosome 16.
Using established in silico design tools, PEgIT and pegFinder, multiple prime editing components have been designed spanning successive prime editor generations, in order to identify the most effective combination for targeting this disorder.
Twelve pegRNAs have been designed, incorporating two secondary nicking sites. Designs include the use of canonical and non-canonical PAM sites, tiling strategies, PE3 secondary nicking guides, and a comparison of tevopreQ1 epegRNAs versus standard pegRNAs. Transfection experiments to assess the efficiency of introducing the T484M point mutation are currently ongoing in HEK293T cells.
This work will provide an optimisation framework of pegRNA designs capable of targeting this mutation within the genome and serve as a proof of concept for the application of prime editing to this disorder. Following HEK293T transfections to establish a cellular disease model, the optimised guides will subsequently be applied to correct the causative mutations in patient-derived iPSCs.
Lay Abstract
Our DNA contains the instructions needed to keep every cell in the body functioning correctly. A single inherited error in one of these instructions can be enough to cause progressive vision loss over time. This project focuses on one such error — a mutation in a gene called IFT140, which plays a key role in maintaining the light-detecting cells at the back of the eye.
Prime editing is a gene editing technology that can locate a specific mutation in DNA and correct it, without cutting or damaging the surrounding genetic code. Using specialist computer software, 12 different versions of this prime editing tool have been designed, each taking a slightly different approach to targeting and correcting the IFT140 mutation.
Laboratory testing will identify which version performs most accurately, correcting the intended mutation with the least risk of causing unintended changes elsewhere in the DNA.
The best-performing design will then be applied to stem cells donated by patients with this condition. These stem cells can be developed into the type of cells affected in the eye, allowing us to test whether the correction works in a clinically relevant setting and bringing us closer to a potential treatment for this inherited condition.
| Lay Title | Using Prime Editing to Target the Genetic Root of Progressive Vision Loss |
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| Role | Master Student |