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Gene Editing with Helper-Dependent Adenovirus Can Efficiently Introduce Multiple Changes Simultaneously over a Large
Donna J Palmer1, Nathan C Grove1, Dustin L Turner1
1Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Molecular Therapy. Nucleic Acids
|September 18, 2017
Summary
Helper-dependent adenoviral vectors (HDAds) can introduce multiple genetic modifications simultaneously across large genomic regions. This gene editing tool shows high efficiency for markers near the selectable marker, but caution is needed due to potential repair mismatches.
Area of Science:
- Gene therapy and editing
- Adenoviral vector technology
- Human induced pluripotent stem cells (hiPSCs)
Background:
- Helper-dependent adenoviral vectors (HDAds) feature long homology arms enabling efficient gene editing.
- These vectors hold potential for introducing multiple genetic modifications or correcting widespread mutations within a single application.
Purpose of the Study:
- To investigate the capability of HDAds to simultaneously introduce multiple genetic modifications across large genomic distances.
- To assess the efficiency of gene editing with HDAds at varying distances from the selectable marker within the homology region.
Main Methods:
- Utilized an HDAd carrying 13 genetic markers targeting the CFTR locus in human iPSCs.
- Analyzed the simultaneous introduction efficiency of these markers, with the farthest being 22.2 kb apart.
- Quantified heteroduplex DNA formation and unrepaired mismatches post-editing.
Main Results:
- All 13 genetic markers were simultaneously introduced into the target locus, spanning up to 22.2 kb.
- Editing efficiency decreased with increasing distance from the HDAd's selectable marker, with 100% efficiency at 208 bp and 21.7% at 11 kb.
- Observed extensive heteroduplex DNA formation (up to 10 kb) with up to 15% of mismatches escaping repair.
Conclusions:
- HDAds effectively facilitate simultaneous, large-scale genomic modifications in hiPSCs.
- Editing efficiency is distance-dependent relative to the selectable marker, and unrepaired mismatches necessitate careful sequence homology assessment.
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