Journal Article

C07 A CRISPRI platform to assess the role of HD risk modifiers in CAG repeat expansion in iPSC derived striatal neurons

Ferguson, Ross, Flower, Michael, Tabrizi, Sarah J

2022

Overview

BackgroundThe pathological HTT CAG repeat continues to expand throughout life. As CAG repeat length predicts rate of expansion and age of HD onset, slowing expansion is an attractive therapeutic approach. The GeM-HD GWASs associated changes in age of onset with many loci, including DNA damage repair genes. Understanding if/how these risk modifiers affect repeat expansion rate, and their interplay in vulnerable neurons, will contribute to understanding the underlying expansion mechanism.Aims1) Generate a human HD iPSC-based model capable of rapidly assaying risk associated genes for effects on somatic expansion of the HTT CAG repeat in striatal neuron cultures. 2) Target expression of components of the mismatch repair MutL complex (MLH1, PMS1, MLH3 & PMS2) as validation.MethodsdCas9 fused to a transcriptional repressor was knocked-in to the CLYBL safe harbour locus of the 125Q iPSC juvenile HD line. Guides targeting individual MutL components were introduced by lentiviral transduction. Knock-down pools for each gene were differentiated to post-mitotic MSNs and assayed for CAG expansion.ResultsKnock-in clones retained parental CAG expansion rates and differentiation potential. CRISPRi achieved 70-90% reduction in target expression. Knock-down of the individual MutL proteins suppressed expansion in striatal neuron cultures.ConclusionsThis model successfully recapitulates previously reported effects of MutL components on somatic expansion of the HTT CAG repeat. This indicates our model will be a useful platform for rapidly dissecting the effects of other risk modifier gene expression on CAG instability and pathophysiology in a human model of HD.

• Publisher: BMJ Publishing Group Ltd,BMJ Publishing Group LTD
• Subject: C: Genetic modifiers / CRISPRi / Gene expression / iPSC / MMR / Repeat expansion