Year: 2024
Type: conference-abstract
Abstract: Background The Tabrizi lab primarily utilises stem cells to investigate genetic modifiers of Huntington’s disease (HD) and CAG repeat instability. These stem cells were generated from the blood of a juvenile HD patient in 2017, and the CAG repeat was measured to be 125 CAGs in length. While this model has been essential to our lab, to assess new areas of interest, (i.e. DNA damage response pathways) it was decided that an isogenic allelic series should be generated and CRISPR technology would be used to accomplish the task. Aims Reduce the CAG repeat tract of the mutant HTT allele to a non-pathological length, creating isogenic control lines which will be used for future work investigating DNA damage response in HD. Methods/Techniques CRISPR technology created and gifted by Benjamin Kleinstiver was used to target and shorten the CAG repeat tract of the HTT gene. Nanopore sequencing was used to verify the mutations were in-frame, and western blotting confirmed that the edited allele could be used to synthesise HTT protein. Fragment analysis was used to assess the instability of the edited HTT CAG tract. Results/Outcome An isogenic allelic series of the 125Q line was generated with varying CAG repeat sizes and changes to CAG instability were measured. Initial experiments characterising the DNA damage response in cells with different CAG lengths were performed. Conclusions This work will provide invaluable insights to the role of expanded CAG repeats on DNA damage response pathways in HD.
Authors: Freja Sadler, Michael Flower, Lucy Coupland, Benjamin Kleinstiver, Sarah Tabrizi
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4402373745