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Tracking genome engineering outcome at individual DNA breakpoints.

ABSTRACT: 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.

SUBMITTER: Certo MT 

PROVIDER: S-EPMC3415300 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Tracking genome engineering outcome at individual DNA breakpoints.

Certo Michael T MT   Ryu Byoung Y BY   Annis James E JE   Garibov Mikhail M   Jarjour Jordan J   Rawlings David J DJ   Scharenberg Andrew M AM  

Nature methods 20110710 8

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 nuclea  ...[more]