Inactivating a herbicide-resistance transgene in Nicotiana tabacum plants using CRISPR/Cas9

Herbicide and antibiotic tolerance genes serve as useful selectable markers for the development of transgenic plants expressing other transgenes. It may be desirable for regulatory or safety reasons to silence the herbicide tolerance trait after transformants have been selected. However, because the genes of interest and the marker gene are usually tightly linked, traditional segregation-based strategies for elimination of undesirable transgenes are usually unsuccessful. Here, we created Nicotiana tabacum plants that carry a single copy of a Cas9 gene, a nuclease in the clustered regularly interspaced short palindromic repeats (CRISPR) system, physically linked to the selectable marker gene bar for tolerance to the herbicide glufosinate (Basta, Liberty). Here, bar was targeted within the genome by introducing bar-specific single guide RNAs (sgRNAs) to the N. tabacum line in vitro, resulting in abolishment of the glufosinate-tolerance trait in mature plants. Sequence analysis of the bar gene revealed a frame-shift mutation at a sgRNA target site, confirming efficacy of the strategy.