title: Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template.
abstract: Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI 'cleavase', but no evidence of toxicity with the I-AniI 'nickase.' Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs.
authors.affiliation: Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Metzger
authors.forename: Audrey
authors.initials: A
authors.lastname: McConnell-Smith
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
authors.forename: A Dusty
authors.initials: AD
authors.lastname: Miller
mesh_terms: D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D000229:Dependovirus / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D011995:Recombination, Genetic; D013698:Templates, Genetic
chemical_list: D004706:Endodeoxyribonucleases