Journal
NATURE METHODS
Volume 8, Issue 8, Pages 671-U102Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.1648
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Funding
- Public Health Service
- National Research Service from the US National Institute of General Medical Sciences [T32 GM07270]
- US National Institutes of Health [RL1CA133832, UL1DE019582, R01-HL075453, PL1-HL092557, RL1-HL092553]
- Seattle Children's Center for Immunity and Immunotherapies
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Site-specific genome engineering technologies are increasingly important tools in the postgenomic 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.
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