Metzger, M. J. and McConnell-Smith, A. and Stoddard, B. L. and Miller, A. D. (2011) Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template. Nucleic Acids Research, 39 (3). pp. 926-935. ISSN 0305-1048
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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 DSBinduced 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.
| Item Type: | Article or Abstract |
|---|---|
| DOI: | doi:10.1093/nar/gkq826 |
| PubMed ID: | 20876694 |
| PMCID: | PMC3035452 |
| Depositing User: | Library Staff |
| Date Deposited: | 16 Mar 2012 18:34 |
| Last Modified: | 16 Mar 2012 18:34 |
| URI: | http://authors.fhcrc.org/id/eprint/552 |
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