title: 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.
authors: Program in Molecular and Cellular Biology, University of Washington, Seattle, Washington, USA.
authors: Michael T
authors: MT
authors: Certo
authors: Byoung Y
authors: BY
authors: Ryu
authors: James E
authors: JE
authors: Annis
authors: Mikhail
authors: M
authors: Garibov
authors: Jordan
authors: J
authors: Jarjour
authors: David J
authors: DJ
authors: Rawlings
authors: Andrew M
authors: AM
authors: Scharenberg
mesh_terms: D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics*