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Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases

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 d...

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Detalles Bibliográficos
Autores principales: Gupta, Ankit, Meng, Xiangdong, Zhu, Lihua J., Lawson, Nathan D., Wolfe, Scot A.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017618/
https://www.ncbi.nlm.nih.gov/pubmed/20843781
http://dx.doi.org/10.1093/nar/gkq787
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author Gupta, Ankit
Meng, Xiangdong
Zhu, Lihua J.
Lawson, Nathan D.
Wolfe, Scot A.
author_facet Gupta, Ankit
Meng, Xiangdong
Zhu, Lihua J.
Lawson, Nathan D.
Wolfe, Scot A.
author_sort Gupta, Ankit
collection PubMed
description 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.
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spelling pubmed-30176182011-01-10 Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases Gupta, Ankit Meng, Xiangdong Zhu, Lihua J. Lawson, Nathan D. Wolfe, Scot A. Nucleic Acids Res Synthetic Biology and Chemistry 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. Oxford University Press 2011-01 2010-09-14 /pmc/articles/PMC3017618/ /pubmed/20843781 http://dx.doi.org/10.1093/nar/gkq787 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Synthetic Biology and Chemistry
Gupta, Ankit
Meng, Xiangdong
Zhu, Lihua J.
Lawson, Nathan D.
Wolfe, Scot A.
Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases
title Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases
title_full Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases
title_fullStr Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases
title_full_unstemmed Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases
title_short Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases
title_sort zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases
topic Synthetic Biology and Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017618/
https://www.ncbi.nlm.nih.gov/pubmed/20843781
http://dx.doi.org/10.1093/nar/gkq787
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