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

Area of Science:

• Genetics
• Molecular Biology
• Animal Models

Background:

• CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) require accurate humanized animal models.
• Existing hDMDTg mouse models possess duplicated DMD transgenes, complicating CRISPR therapy outcome analysis.
• The duplicated transgene copy number does not reflect the single copy of the human DMD gene.

Purpose of the Study:

• To develop a single-copy, full-length, humanized DMD transgenic mouse model.
• To address limitations of existing duplicated transgene models for CRISPR therapy development.
• To create a more accurate preclinical model for Duchenne muscular dystrophy.

Main Methods:

• Long read nanopore sequencing to analyze transgene structure in hDMDTg mice.
• CRISPR zygotic microinjection to delete one of the duplicated transgenes.
• Generation of a single-copy transgenic mouse model (hDMDTgSc).

Main Results:

• Characterization of duplicated transgene structure using nanopore sequencing.
• Successful generation of the hDMDTgSc single-copy transgenic mouse model.
• Demonstration that the single human DMD transgene is functional and rescues the dystrophic phenotype.

Conclusions:

• The novel hDMDTgSc mouse model accurately represents the human DMD gene copy number.
• This model is suitable for preclinical assessment of sequence-specific CRISPR therapies for DMD.
• It facilitates the development of improved DMD disease models for therapeutic research.