Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template |
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Authors: | Michael J Metzger Audrey McConnell-Smith Barry L Stoddard A Dusty Miller |
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Institution: | 1.Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, 2.Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, WA 98195 and 3.Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA |
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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. |
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