| dc.description.abstract | Rare diseases, when considered as a whole, affect up to 7% of the population, which
would represent 3.5 million individuals in the United Kingdom alone. However, while
“personalised medicine” is now yielding remarkable results using recent sequencing
technologies in terms of diagnosing genetic conditions, we have made much less
headway in translating this patient information into therapies and effective treatments.
Even with recent calls for greater research into personalised treatments for those
affected by a rare disease, progress in this area is still severely lacking, in part due to
the astronomical cost and time involved in bringing treatments to the clinic.
Gene correction using the recently-described genome editing technology
CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue
of treatment, if not cure, for rare diseases; up to 80% of which have a genetic
component. This system allows the researcher to target any locus in the genome for
cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG
sequence motif. If a repair sequence is then also provided, such as a wild-type copy of
the mutated gene, it can be incorporated by homology-directed repair (HDR), leading
to gene correction. As both guide-RNA and repair template are easily generated, whilst
the machinery for editing and delivery remain the same, this system could usher in the
era of ‘personalised medicine’ and offer hope to those with rare genetic diseases.
However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene
correction, especially in vivo.
Therefore, in my PhD I have developed a novel fluorescent reporter system which
provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and
homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon
a cassette which is stably and heterozygously integrated into a ubiquitously expressed
locus in the mouse genome. This cassette contains a strong hybrid promoter driving
expression of membrane-tagged tdTomato, followed by a strong stop sequence, and
then membrane-tagged EGFP. Unedited, this system drives strong expression of
membrane-tdTomato in all cell types in the embryo and adult mouse. However,
following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato
is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to
imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear
tagged-EGFP is also supplied, the editing machinery may then use the precise HDR
pathway, which results in a rapid transition from membrane-tdTomato to nuclear-
EGFP. Thereby this system allows the kinetics of editing to be visualised in real time
and allows simple scoring of the proportion of cells which have been edited by NHEJ
or corrected by HDR. It therefore provides a simple, fast and scalable manner to
optimise reagents and protocols for gene correction by CRISPR/Cas9, especially
compared to sequencing approaches, and will prove broadly useful to many
researchers in the field.
Further to this, I have shown that methods which lead to gene correction in our reporter
system are also able to partially repair mutations found in the disease-causing gene,
Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia
(PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare
disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary
clearance leading to neonatal respiratory distress and recurrent airway infections, often
progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF,
with progressive deterioration of lung function and lower airway bacterial
colonization. However, unlike CF which is monogenic, over 40 genes are known to
cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to
such a genome editing strategy, which can be tailored for the correction of any mutated
locus. | en |
