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Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing
Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identific...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814844/ https://www.ncbi.nlm.nih.gov/pubmed/27030102 http://dx.doi.org/10.1038/srep23549 |
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author | Miyaoka, Yuichiro Berman, Jennifer R. Cooper, Samantha B. Mayerl, Steven J. Chan, Amanda H. Zhang, Bin Karlin-Neumann, George A. Conklin, Bruce R. |
author_facet | Miyaoka, Yuichiro Berman, Jennifer R. Cooper, Samantha B. Mayerl, Steven J. Chan, Amanda H. Zhang, Bin Karlin-Neumann, George A. Conklin, Bruce R. |
author_sort | Miyaoka, Yuichiro |
collection | PubMed |
description | Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator–like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology. |
format | Online Article Text |
id | pubmed-4814844 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-48148442016-04-04 Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing Miyaoka, Yuichiro Berman, Jennifer R. Cooper, Samantha B. Mayerl, Steven J. Chan, Amanda H. Zhang, Bin Karlin-Neumann, George A. Conklin, Bruce R. Sci Rep Article Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator–like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology. Nature Publishing Group 2016-03-31 /pmc/articles/PMC4814844/ /pubmed/27030102 http://dx.doi.org/10.1038/srep23549 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Miyaoka, Yuichiro Berman, Jennifer R. Cooper, Samantha B. Mayerl, Steven J. Chan, Amanda H. Zhang, Bin Karlin-Neumann, George A. Conklin, Bruce R. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing |
title | Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing |
title_full | Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing |
title_fullStr | Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing |
title_full_unstemmed | Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing |
title_short | Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing |
title_sort | systematic quantification of hdr and nhej reveals effects of locus, nuclease, and cell type on genome-editing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814844/ https://www.ncbi.nlm.nih.gov/pubmed/27030102 http://dx.doi.org/10.1038/srep23549 |
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