GFP Reporters to Monitor Instability and Expression of Expanded CAG/CTG Repeats
Expanded CAG/CTG repeats are genetically unstable and, upon expression, cause neurological and neuromuscular diseases. The molecular mechanisms of repeat instability and expression remain poorly understood despite their importance for the pathogenesis of a family of 14 devastating human diseases. This is in part because conventional assays are tedious and time-consuming. Recently, however, GFP-based reporters have been designed to provide a rapid and reliable means of assessing these parameters. Here we provide protocols for quantifying repeat instability and expression using a GFP-based chromosomal reporter and the newly developed ParB/ANCHOR-mediated Inducible Targeting (PInT) and how to validate the results.
KeywordsExpanded CAG/CTG repeat diseases GFP reporters Gene expression Genome stability DNA repair
We thank members of Joachim Lingner’s lab for help setting up the ChIP protocol and Dinis Barros for help with the figures. This work is supported by Swiss National Foundation professorships (#172936) to V.D. and by the UK Dementia Research Institute, which receives its funding from DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK.
- 18.Tomé S, Nicole A, Gomes-Pereira M, Gourdon G (2014) Non-Radioactive Detection of Trinucleotide Repeat Size Variability. PLOS Currents Muscular Dystrophy. 2014 Mar 6. Edition 1. https://doi.org/10.1371/currents.md.ad50113b899fa1352ce70c087eead706
- 38.Cinesi C, Aeschbach L, Yang B, Dion V (2016) Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase. Nat Commun 7:13272Google Scholar
- 47.Yang B, Borgeaud A, Aeschbach L, Dion V (2018) Uncovering the interplay between Epigenome editing efficiency and sequence context using a novel inducible targeting system. bioRxiv. https://doi.org/10.1101/368480