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A CRISPR/Cas9-Based Approach For Editing Immortalised Human Myoblasts To Model Duchenne Muscular Dystrophy In Vitro

View ORCID ProfileP. Soblechero-Martín, E. Albiasu-Arteta, View ORCID ProfileA. Anton-Martinez, View ORCID ProfileI. Garcia-Jimenez, View ORCID ProfileG. González-Iglesias, View ORCID ProfileI. Larrañaga-Aiestaran, View ORCID ProfileA. López-Martinez, View ORCID ProfileJ. Poyatos-García, E. Ruiz-Del-Yerro, F. Gonzalez, View ORCID ProfileV. Arechavala-Gomeza
doi: https://doi.org/10.1101/2020.02.24.962316
P. Soblechero-Martín
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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  • ORCID record for P. Soblechero-Martín
E. Albiasu-Arteta
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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A. Anton-Martinez
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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I. Garcia-Jimenez
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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G. González-Iglesias
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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I. Larrañaga-Aiestaran
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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A. López-Martinez
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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J. Poyatos-García
2La Fe Health Research Institute, Hospital La Fe, Valencia, Spain
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E. Ruiz-Del-Yerro
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
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F. Gonzalez
3Pluripotent Stem Cells and Activation of Endogenous Tissue Programs for Organ Regeneration (PR Lab), Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain
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V. Arechavala-Gomeza
1Neuromuscular Disorders, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain
4Ikerbasque, Basque Foundation for Science, Bilbao, Spain
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  • For correspondence: virginia.arechavalagomeza@osakidetza.eus
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Abstract

CRISPR/Cas9-mediated gene editing may allow treating and studying rare genetic disorders by respectively, correcting disease mutations in patients, or introducing them in cell cultures. Both applications are highly dependent on Cas9 and sgRNA delivery efficiency. While gene editing methods are usually efficiently applied to cell lines such as HEK293 or hiPSCs, CRISPR/Cas9 editing in vivo or in cultured myoblasts prove to be much less efficient, limiting its use. After a careful optimisation of different steps of the editing protocol, we established a consistent approach to generate human immortalised myoblasts disease models through CRISPR/Cas9 editing. Using this protocol we successfully created a coding deletion of exon 52 of the DYSTROPHIN (DMD) gene in wild type immortalised myoblasts modelling Duchenne muscular dystrophy (DMD), and a microRNA binding sites deletion in the regulatory region of the UTROPHIN (UTRN) gene leading to utrophin upregulation in in Duchenne muscular dystrophy patient immortalised cultures. Sanger sequencing confirmed the presence of the corresponding genomic alterations and protein expression was characterised using myoblots. To show the utility of these cultures as platforms for assessing the efficiency of DMD treatments, we used them to evaluate the impact of exon skipping therapy and ezutromid treatment. Our editing protocol may be useful to others interested in genetically manipulating myoblasts and the resulting edited cultures for studying DMD disease mechanisms and assessing therapeutic approaches.

Summary We report two novel immortalised myoblast culture models for studying Duchenne muscular dystrophy (DMD), generated through CRISPR/Cas9 gene editing: one recapitulates a common DYSTROPHIN (DMD) deletion and the other a regulatory mutation leading to UTROPHIN (UTRN) ectopic upregulation.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted February 25, 2020.
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A CRISPR/Cas9-Based Approach For Editing Immortalised Human Myoblasts To Model Duchenne Muscular Dystrophy In Vitro
P. Soblechero-Martín, E. Albiasu-Arteta, A. Anton-Martinez, I. Garcia-Jimenez, G. González-Iglesias, I. Larrañaga-Aiestaran, A. López-Martinez, J. Poyatos-García, E. Ruiz-Del-Yerro, F. Gonzalez, V. Arechavala-Gomeza
bioRxiv 2020.02.24.962316; doi: https://doi.org/10.1101/2020.02.24.962316
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A CRISPR/Cas9-Based Approach For Editing Immortalised Human Myoblasts To Model Duchenne Muscular Dystrophy In Vitro
P. Soblechero-Martín, E. Albiasu-Arteta, A. Anton-Martinez, I. Garcia-Jimenez, G. González-Iglesias, I. Larrañaga-Aiestaran, A. López-Martinez, J. Poyatos-García, E. Ruiz-Del-Yerro, F. Gonzalez, V. Arechavala-Gomeza
bioRxiv 2020.02.24.962316; doi: https://doi.org/10.1101/2020.02.24.962316

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