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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...

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Detalles Bibliográficos
Autores principales: Certo, Michael T., Ryu, Byoung Y., Annis, James E., Garibov, Mikhail, Jarjour, Jordan V., Rawlings, David J., Scharenberg, Andrew M.
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
Descripción
Sumario: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.