Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nucle...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2024-05, Vol.121 (19), p.e2321438121
Main Authors: Poukalov, Kiril K, Valero, M Carmen, Muscato, Derek R, Adams, Leanne M, Chun, Heejae, Lee, Young Il, Andrade, Nadja S, Zeier, Zane, Sweeney, H Lee, Wang, Eric T
Format: Article
Language:English
Subjects:
Animal models
Animals
Biological Sciences
Cell Nucleus - metabolism
CRISPR
CRISPR-Associated Protein 9 - genetics
CRISPR-Associated Protein 9 - metabolism
CRISPR-Cas Systems
Disease Models, Animal
Duchenne's muscular dystrophy
Dystrophy
Editing
Gene Editing - methods
Gene therapy
Genetic modification
Genetic Therapy - methods
Humans
Localization
Mice
Muscle, Skeletal - metabolism
Muscles
Muscular dystrophy
Muscular Dystrophy, Duchenne - genetics
Muscular Dystrophy, Duchenne - therapy
Myoblasts
Myoblasts - metabolism
Myotonic dystrophy
Nuclear Localization Signals - genetics
Nuclear transport
Skeletal muscle
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Summary:Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.2321438121