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

Yu C.J. Chey, Mark A. Corbett, Jayshen Arudkumar, Sandra G. Piltz, Paul Q. Thomas, Fatwa Adikusuma

Research output: Contribution to journalArticlepeer-review

Abstract

BACKGROUND: 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.

Original languageEnglish
Article number214
JournalBMC Biology
Volume22
Issue number1
DOIs
Publication statusPublished or Issued - 27 Sept 2024

Keywords

  • CRISPR-Cas9
  • Duchenne muscular dystrophy
  • Dystrophin
  • Gene editing
  • Humanized mouse model
  • Illumina sequencing
  • Megabase scale deletion
  • Nanopore sequencing
  • Transgenic mouse model
  • Zygote microinjection

ASJC Scopus subject areas

  • Biotechnology
  • Structural Biology
  • Ecology, Evolution, Behavior and Systematics
  • Physiology
  • General Biochemistry,Genetics and Molecular Biology
  • General Agricultural and Biological Sciences
  • Plant Science
  • Developmental Biology
  • Cell Biology

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