| Site-specific genome engineering technologies are an increasingly important tool in the post-genomic era, as many biotechnological objectives require the generation of precisely genetically modified organisms. Rare cutting endonucleases, through their capacity to create a targeted DNA strand break, can engage and exploit endogenous repair mechanisms to alter a genomic sequence at a defined locus. However, different DNA repair pathways can yield distinct engineering outcomes. For example, the non-homologous end joining pathway can result in gene disruption ("knock-out"), while engaging the homology-directed repair pathway with an exogenous donor results in gene targeting ("knock-in"). Therefore, it is crucial to understand how experimental variables influence DNA repair pathway choice, and to develop methods that enable a genome engineer to bias break resolution towards a desired outcome. Towards this goal, we constructed a reporter system, Traffic Light, that provides a flow cytometric readout of endonuclease-induced genome engineering outcome at individual DNA breakpoints, and allows quantitative tracking of nuclease and donor template levels. Using the Traffic Light system, we have identified key experimental variables influencing DNA repair pathway choice, and developed several molecular strategies to bias the engineering outcome; including the induction of single vs. double strand breaks to minimize mutagenic outcomes, application of siRNA's identified in high-throughput screens to increase gene targeting rates, and coupling endonucleases with exonucleases to enhance gene disruption rates. These strategies will help facilitate efficient and precise site-specific genomic engineering in a variety of organisms. |
