Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut.

Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.

Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes.

Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective low-cost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops.