title: 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.
authors.affiliation: 1] Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands. [2].
authors.forename: Maarten
authors.initials: M
authors.lastname: Holkers
authors.affiliation: 1] Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands. [2].
authors.forename: Ignazio
authors.initials: I
authors.lastname: Maggio
authors.affiliation: 1] Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands. [2] Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Sara F D
authors.initials: SF
authors.lastname: Henriques
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Josephine M
authors.initials: JM
authors.lastname: Janssen
authors.affiliation: 1] Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany. [2] Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Manuel A F V
authors.initials: MA
authors.lastname: Gonçalves
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D002469:Cell Separation; D004247:DNA / Q000737:chemistry*; D004279:DNA, Viral / Q000235:genetics*; D003851:Deoxyribonucleases / Q000737:chemistry*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results
chemical_list: D004279:DNA, Viral; D004247:DNA; D003851:Deoxyribonucleases