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Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple disease loci

A Murillo(1) M Alpaugh(2) M Larin(1) E L Randall(1) L Heraty(1) R R Durairaj(1) A N Aston(1) A S Taylor(1) A M Monteys(3) N Stöberl(4) A ER Heuchan(1) P Aeschlimann(1) S Bhattacharyya(1) N D Allen(4) J Puymirat(5) B L Davidson(3,6) F Cicchetti(7) M J Lelos(4) V Dion(1)

1:UK Dementia Research Institute at Cardiff University, Hadyn Ellis Building, Maindy Road, Cardiff, United Kingdom, CF24 4HQ; 2:Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; 3:Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, Philadelphia, 19104, USA; 4:School of Biosciences, Cardiff University, The Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX; 5:LOEX, CHU de Québec-Université Laval Research Center, Quebec City, QC, Canada.; 6:Department of Pathology, University of Pennsylvania, Philadelphia, 19104, USA.; 7:Centre de Recherche du CHU de Québec, Axe Neurosciences; Département de Psychiatrie et Neurosciences, Université Laval, Québec, QC, Canada.

Expanded CAG/CTG repeats cause at least 15 different neurodegenerative and neuromuscular diseases that all remain without an effective disease-modifying treatment. Because the repeat tract size accounts for most of the variation in disease severity, contracting them presents an attractive therapeutic avenue. Here, we show that the CRISPR-Cas9 nickase targeting the CAG/CTG repeat itself leads to efficient contractions in Huntington’s disease patient-derived neurons and astrocytes, as well as in myotonic dystrophy type 1 patient-derived neurons. Using single-cell DNA sequencing, PCR-free whole genome sequencing, and targeted long-read sequencing of the HTT locus, we found no off-target mutations above background in neurons and astrocytes. Furthermore, we delivered the Cas9 nickase and sgRNA stereotactically to a mouse model of Huntington’s disease using adeno-associated viruses and found contractions accumulating in over half of the infected cells over a period of 5 months. We also found that the Cas9 nickase was prone to silencing, further improving the safety of the approach. Our results provide proof of concept for using the Cas9 nickase to contract the repeat tract safely in multiple cell types and diseases.

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