Cargando…
Tracking genome engineering outcome at individual DNA breakpoints
Site-specific genome engineering technologies are increasingly important tools in the post-genomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2011
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3415300/ https://www.ncbi.nlm.nih.gov/pubmed/21743461 http://dx.doi.org/10.1038/nmeth.1648 |
_version_ | 1782240345967296512 |
---|---|
author | Certo, Michael T. Ryu, Byoung Y. Annis, James E. Garibov, Mikhail Jarjour, Jordan V. Rawlings, David J. Scharenberg, Andrew M. |
author_facet | Certo, Michael T. Ryu, Byoung Y. Annis, James E. Garibov, Mikhail Jarjour, Jordan V. Rawlings, David J. Scharenberg, Andrew M. |
author_sort | Certo, Michael T. |
collection | PubMed |
description | Site-specific genome engineering technologies are increasingly important tools in the post-genomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated Traffic Light, that supports rapid flow cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high throughput screens for factors that bias the engineering outcome. We applied the Traffic Light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred. |
format | Online Article Text |
id | pubmed-3415300 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
record_format | MEDLINE/PubMed |
spelling | pubmed-34153002012-08-09 Tracking genome engineering outcome at individual DNA breakpoints Certo, Michael T. Ryu, Byoung Y. Annis, James E. Garibov, Mikhail Jarjour, Jordan V. Rawlings, David J. Scharenberg, Andrew M. Nat Methods Article Site-specific genome engineering technologies are increasingly important tools in the post-genomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated Traffic Light, that supports rapid flow cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high throughput screens for factors that bias the engineering outcome. We applied the Traffic Light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred. 2011-07-10 /pmc/articles/PMC3415300/ /pubmed/21743461 http://dx.doi.org/10.1038/nmeth.1648 Text en Users may view, print, copy, download and 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 Certo, Michael T. Ryu, Byoung Y. Annis, James E. Garibov, Mikhail Jarjour, Jordan V. Rawlings, David J. Scharenberg, Andrew M. Tracking genome engineering outcome at individual DNA breakpoints |
title | Tracking genome engineering outcome at individual DNA breakpoints |
title_full | Tracking genome engineering outcome at individual DNA breakpoints |
title_fullStr | Tracking genome engineering outcome at individual DNA breakpoints |
title_full_unstemmed | Tracking genome engineering outcome at individual DNA breakpoints |
title_short | Tracking genome engineering outcome at individual DNA breakpoints |
title_sort | tracking genome engineering outcome at individual dna breakpoints |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3415300/ https://www.ncbi.nlm.nih.gov/pubmed/21743461 http://dx.doi.org/10.1038/nmeth.1648 |
work_keys_str_mv | AT certomichaelt trackinggenomeengineeringoutcomeatindividualdnabreakpoints AT ryubyoungy trackinggenomeengineeringoutcomeatindividualdnabreakpoints AT annisjamese trackinggenomeengineeringoutcomeatindividualdnabreakpoints AT garibovmikhail trackinggenomeengineeringoutcomeatindividualdnabreakpoints AT jarjourjordanv trackinggenomeengineeringoutcomeatindividualdnabreakpoints AT rawlingsdavidj trackinggenomeengineeringoutcomeatindividualdnabreakpoints AT scharenbergandrewm trackinggenomeengineeringoutcomeatindividualdnabreakpoints |