PhD in Dementia Research - Molecular mechanisms of CRISPR Cas9 nickase-induced CAG/CTG repeat contraction: implications for gene editing in expanded repeat disorders

Background:
Expanded CAG/CTG repeats cause 14 clinically different neurological, neuromuscular, and neurodegenerative diseases, including Huntington’s disease and myotonic dystrophy. There is currently no way of curing these diseases or even of slowing their progression. The repeats are highly unstable, with changes in size occurring in every cell for some organs. The instability phenomenon alters the phenotypic outcome, with longer repeats generally leading to more severe phenotypes. Thus, an attractive therapeutic possibility is to shrink repeats down to a normal size.
We have recently developed the first way to contract repeat tracts at high efficiencies (Cinesi et al, Nat Comms 2016). The method relies on using the CRISPR/Cas9 nickase and the results have important implications in designing a treatment for these devastating diseases. Indeed, targeting the Cas9 nickase to expanded CAG/CTG repeats leads to efficient contractions in a reporter system as well as in cells derived from patients with myotonic dystrophy, effectively correcting the mutation. Importantly, we could not detect off-target mutations, making this approach especially attractive.
Gap in knowledge:
How the CRISPR/Cas9 nickase provokes CAG/CTG repeat contractions is unknown.
Goal of the project:
Here we aim to understand how contractions occur at the molecular level such that we can make a potential gene editing-based treatment safer and more efficient.
Methods:
The project will use a combination of molecular biology, genome engineering, genetics, and high throughput sequencing to uncover genetic interactors and map the network leading to nickase-induced contractions. For this, we will use our reporter system, which is ideal for screening. Next, we will apply the knowledge gained to improve the efficiency of contractions using Huntington’s disease patient-derived induced pluripotent stem cells as a model of the disease. We will further estimate the frequencies of the off-target mutations induced by the CRISPR/Cas9 nickase using next-generation sequencing. Finally, we will assay whether the cellular phenotypes of the disease are affected by the presence of contractions.
Significance:
The CRISPR/Cas9 technology is already used in about 25 different clinical trials. It therefore holds great promise for the treatment of a wide variety of diseases. Defining the mechanism of contractions induced by the Cas9 nickase will bring us a step closer to the clinic by potentially making the treatment safer, more specific, and more efficient. The knowledge uncovered here could also help identify patients who would respond either poorly or particularly well to a Cas9 nickase-based treatment.
Supervision:
Both Vincent Dion and Nick Allen have a track record of graduating students on time and of supporting their intellectual, technical, and scientific growth.
Reference:
Cinzia Cinesi, Lorène Aeschbach, Bin Yang & Vincent Dion. Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase. Nature Communications 7 ; 13272 (2016).