Abstract:
Retinitis Pigmentosa (RP) is one of the most common genetically inherited diseases that is known to cause
retinopathy, with a global prevalence of approximately 1 in 4000. The disease affects the cells of the retina,
gradually reducing the visual field until complete blindness occurs around middle-age. Autosomal dominant
forms of the disease pose a particular challenge, with multiple members of the same family being afflicted
by the disease. With no current effective treatment, investigations into generating RP models via gene
editing are of particular interest for researchers who work in retinopathies.
We set out to create a cell model of a common mutation in the RP1 gene called R677X that is known to
cause Autosomal Dominant Retinitis Pigmentosa. This occurs through the introduction of a premature stop
codon that causes the protein coded by RP1 to be significantly shorter than a healthy copy and thereby
impede its native function. Editing experiments using CRISPR/Cas9 were undertaken in sheep fibroblasts
in order to lay the groundwork for the development of a large animal model in sheep. Using CRISPR/Cas9
guides 55 bases downstream from the mutation site, we successfully induced deletions in sheep fibroblasts
using nucleofection with an adjusted editing frequency of 47%. The mRNA from the cells carrying these
deletions indicates that the frameshifts mutations that were generated in this region would code for a
premature stop codon and subsequently delete the majority of the protein coded for by this gene, as is seen
in known pathogenic genetic variants of the disease.
Future directions include the generation of an isogenic cell line by replicating the results of this experiment
in immortalised fibroblasts. This could then be used to test the ability of CRISPR/Cas9 to knock down the
edited copy of the gene and revert the cell to using the healthy copy of RP1, creating cells that are
hemizygous for the RP1 gene. This research is incredibly encouraging for the development of a sheep model
to not only develop and test potential treatments, but to learn more about the pathology of Retinitis
Pigmentosa and how the disease progresses.