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CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model

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dc.contributor.authorChey, Y.C.J.
dc.contributor.authorCorbett, M.A.
dc.contributor.authorArudkumar, J.
dc.contributor.authorPiltz, S.G.
dc.contributor.authorThomas, P.Q.
dc.contributor.authorAdikusuma, F.
dc.date.issued2024
dc.description.abstractBackground The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. Results To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. Conclusions Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies. Keywords CRISPR-Cas9, Duchenne muscular dystrophy, Humanized mouse model, Zygote microinjection, Transgenic mouse model, Dystrophin, Megabase scale deletion, Nanopore sequencing, Illumina sequencing, Gene editing
dc.description.statementofresponsibilityYu C. J. Chey, Mark A. Corbett, Jayshen Arudkumar, Sandra G. Piltz, Paul Q. Thomas, and Fatwa Adikusuma
dc.identifier.citationBMC Biology, 2024; 22(1):214-1-214-15
dc.identifier.doi10.1186/s12915-024-02008-7
dc.identifier.issn1741-7007
dc.identifier.issn1741-7007
dc.identifier.orcidChey, Y.C.J. [0000-0001-7553-5163]
dc.identifier.orcidCorbett, M.A. [0000-0001-9298-3072]
dc.identifier.orcidArudkumar, J. [0000-0002-4856-6332]
dc.identifier.orcidAdikusuma, F. [0000-0003-2163-0514]
dc.identifier.urihttps://hdl.handle.net/2440/144889
dc.language.isoen
dc.publisherBMC
dc.rights© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
dc.source.urihttp://dx.doi.org/10.1186/s12915-024-02008-7
dc.subjectCRISPR-Cas9
dc.subjectDuchenne muscular dystrophy
dc.subjectDystrophin
dc.subjectGene editing
dc.subjectHumanized mouse model
dc.subjectIllumina sequencing
dc.subjectMegabase scale deletion
dc.subjectNanopore sequencing
dc.subjectTransgenic mouse model
dc.subjectZygote microinjection
dc.subject.meshAnimals
dc.subject.meshMice, Transgenic
dc.subject.meshHumans
dc.subject.meshMice
dc.subject.meshMuscular Dystrophy, Duchenne
dc.subject.meshDisease Models, Animal
dc.subject.meshDystrophin
dc.subject.meshGene Duplication
dc.subject.meshTransgenes
dc.subject.meshCRISPR-Cas Systems
dc.subject.meshClustered Regularly Interspaced Short Palindromic Repeats
dc.subject.meshGene Editing
dc.titleCRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model
dc.typeJournal article
pubs.publication-statusPublished

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