Adenoviral vector DNA for accurate genome editing with engineered nucleases
Abstract
Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.
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CRISPR-Cas9 and DNA donor delivered via protein-capped adenoviral vector resulted in higher HDR rate than via integrase-defective lentiviral vector or non-viral vector templates.
“In a study by Holkers et al, 64 CRISPR-Cas9 and DNA donor delivered via protein-capped adenoviral vector resulted in higher HDR rate versus via integrase-defective lentiviral vector or non-viral vector templates.”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [64]
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- Authors
- Holkers, M., Maggio, I., Henriques, S. F. D., Janssen, J. M., Cathomen, T., & Gonçalves, M. A. F. V.
- Affiliations
- Leiden University Medical Center (NL)
- University of Lisbon (PT)
- University Medical Center Freiburg (DE)
- Corresponding author
- Gonçalves, Manuel A F V
- Venue
- Nature Methods
- Published
- 2014-08-24
- Volume
- 11
- Issue
- 10
- Pages
- 1051-1057
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- OpenAlex
- W2034020839
- Cited by
- 138
- References
- 57
- Citations by year
- 2026: 2 · 2025: 5 · 2024: 2 · 2023: 5 · 2022: 6 · 2021: 12 · 2020: 17 · 2019: 13 · 2018: 12 · 2017: 17 · 2016: 25 · 2015: 19 · 2014: 2
- FWCI
- 8.60
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- Leiden Repository (Leiden University) (submittedVersion)
- Funders
- Università Degli Studi di Modena e Reggio Emila
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- genome editing, homology-directed repair, adenoviral vector, TALEN, CRISPR/Cas9, engineered nucleases, illegitimate recombination, integrase-defective lentiviral vectors, human cells
- MeSH terms
- Adenoviridae, Cell Line, Cell Separation, Deoxyribonucleases, DNA, DNA, Viral, Genetic Engineering, HeLa Cells, Humans, Recombination, Genetic, Repetitive Sequences, Nucleic Acid, Reproducibility of Results, Polymerase Chain Reaction, Genome, Gene Targeting, HEK293 Cells
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