Abstract
Gene edition methods are an attractive putative therapeutic option for Duchenne muscular dystrophy and they have an immediate application in the generation of research models. To generate two new edited myoblast cultures that could be useful in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene: DMDΔ52-Model, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures and, to show their use in the assessment of DMD treatments, we have performed exon skipping in the DMDΔ52-Model and have used the unedited cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene edition protocol, DMD-UTRN-Model offers a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
After participating in the preprint review option, we realised that the focus of the manuscript should be in the new cell culture models generated by CRISPR/Cas gene edition rather than in the methodology used to create them, so we have included new results from further characterization of these cells and changed the text and the title to reflect these changes from -A CRISPR/Cas9-Based Approach For Editing Immortalised Human Myoblasts To Model Duchenne Muscular Dystrophy In Vitro- to -Duchenne Muscular Dystrophy Cell Culture Models Created By CRISPR/Cas 9 Gene Editing And Their Application To Drug Screening.- The main changes in the results section are in the characterization of both cell culture models by the evaluation of fusion indexes, the quantification of dystrophin, utrophin and several differentiation markers expression by droplet digital PCR, and new myoblot experiments to quantify dystrophin, utrophin and MF20 expression at different culture times. The structure has also been changed: the editing process of both cultures has been included in figure 1, cell models characterisation has been included in figures 2 and 3 and the editing workflow and the new myoblot experiments results have been moved to supplementary figures.
