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Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases

Although great progress has been made in the characterization of off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated...

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Autores principales: Frock, Richard L., Hu, Jiazhi, Meyers, Robin M., Ho, Yu-Jui, Kii, Erina, Alt, Frederick W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320661/
https://www.ncbi.nlm.nih.gov/pubmed/25503383
http://dx.doi.org/10.1038/nbt.3101
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author Frock, Richard L.
Hu, Jiazhi
Meyers, Robin M.
Ho, Yu-Jui
Kii, Erina
Alt, Frederick W.
author_facet Frock, Richard L.
Hu, Jiazhi
Meyers, Robin M.
Ho, Yu-Jui
Kii, Erina
Alt, Frederick W.
author_sort Frock, Richard L.
collection PubMed
description Although great progress has been made in the characterization of off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN-nucleases revealed off-target hotspots for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases by more than 10-fold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.
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spelling pubmed-43206612015-08-01 Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases Frock, Richard L. Hu, Jiazhi Meyers, Robin M. Ho, Yu-Jui Kii, Erina Alt, Frederick W. Nat Biotechnol Article Although great progress has been made in the characterization of off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN-nucleases revealed off-target hotspots for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases by more than 10-fold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide. 2014-12-15 2015-02 /pmc/articles/PMC4320661/ /pubmed/25503383 http://dx.doi.org/10.1038/nbt.3101 Text en http://www.nature.com/authors/editorial_policies/license.html#terms Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
spellingShingle Article
Frock, Richard L.
Hu, Jiazhi
Meyers, Robin M.
Ho, Yu-Jui
Kii, Erina
Alt, Frederick W.
Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
title Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
title_full Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
title_fullStr Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
title_full_unstemmed Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
title_short Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
title_sort genome-wide detection of dna double-stranded breaks induced by engineered nucleases
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320661/
https://www.ncbi.nlm.nih.gov/pubmed/25503383
http://dx.doi.org/10.1038/nbt.3101
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