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• W2102693281 abstract "Zinc finger nucleases (ZFNs) facilitate tailor-made genomic modifications in vivo through the creation of targeted double-stranded breaks. They have been employed to modify the genomes of plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double-stranded breaks at off-target sites. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites using Illumina sequencing. Only a minority of these off-target sites accumulated lesions, where the thermodynamics of zinc finger–DNA recognition appear to be a defining feature of active sites. Surprisingly, we observed that both the specificity of the incorporated zinc fingers and the choice of the engineered nuclease domain could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both zinc finger-dependent and -independent characteristics that can be tailored to create ZFNs with greater precision." @default.
• W2102693281 created "2016-06-24" @default.
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• W2102693281 date "2010-09-14" @default.
• W2102693281 modified "2023-10-03" @default.
• W2102693281 title "Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases" @default.
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• W2102693281 doi "https://doi.org/10.1093/nar/gkq787" @default.
• W2102693281 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3017618" @default.
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