title: Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells.
abstract: CRISPR/Cas9-derived RNA-guided nucleases (RGNs) are DNA targeting systems, which are rapidly being harnessed for gene regulation and gene editing purposes in model organisms and cell lines. As bona fide gene delivery vehicles, viral vectors may be particularly fit to broaden the applicability of RGNs to other cell types including dividing and quiescent primary cells. Here, the suitability of adenoviral vectors (AdVs) for delivering RGN components into various cell types is investigated. We demonstrate that AdVs, namely second-generation fiber-modified AdVs encoding Cas9 or single guide RNA (gRNA) molecules addressing the Cas9 nuclease to the AAVS1 "safe harbor" locus or to a recombinant model allele can be produced to high-titers (up to 20 × 10(10) transducing units/ml). Importantly, AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells yields rates of targeted mutagenesis similar to or approaching those achieved by isogenic AdVs encoding TALENs targeting the same AAVS1 chromosomal region. RGN-induced gene disruption frequencies in the various cell types ranged from 18% to 65%. We conclude that AdVs constitute a valuable platform for introducing RGNs into human somatic cells regardless of their transformation status. This approach should aid investigating the potential and limitations of RGNs in numerous experimental settings.
authors.affiliation: 1] Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC 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, Einthovenweg 20, 2333 ZC Leiden, The Netherlands [2].
authors.forename: Maarten
authors.initials: M
authors.lastname: Holkers
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Josephine M
authors.initials: JM
authors.lastname: Janssen
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Manuel A F V
authors.initials: MA
authors.lastname: Gonçalves
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016296:Mutagenesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems