Precision genome editing using combinatorial viral vector delivery of CRISPR-Cas9 nucleases and donor DNA constructs
Nucleic Acids Research, vol. 53
Abstract
Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles.
Authors 7
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Department of Cell and Chemical Biology , Einthovenweg 20, 2333 ZC Leiden ,
Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Department of Cell and Chemical Biology , Einthovenweg 20, 2333 ZC Leiden ,
Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Department of Cell and Chemical Biology , Einthovenweg 20, 2333 ZC Leiden ,
Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Department of Cell and Chemical Biology , Einthovenweg 20, 2333 ZC Leiden ,
Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Department of Cell and Chemical Biology , Einthovenweg 20, 2333 ZC Leiden ,
Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Department of Cell and Chemical Biology , Einthovenweg 20, 2333 ZC Leiden ,
Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
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Leiden University Medical Center
Affiliation as printed
Leiden University Medical Center, Department of Cell and Chemical Biology , Einthovenweg 20, 2333 ZC Leiden ,
Leiden University Medical Center, Department of Cell and Chemical Biology, Einthovenweg 20, 2333 ZC Leiden, the Netherlands
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Cited by patents worldwide 1 (Lens.org)
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Controllable gene expression system based on homologous directional repair and application thereofCN122124288A 2026-06-02 Pending