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Extended-TAQing system for large-scale plant genome reorganization.

We previously developed a large-scale genome restructuring technology called the TAQing system. It can induce genome rearrangements by introducing transient and conditional formation of DNA double strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5'-TCGA-3' sequences in the genome at higher temperature (37-42°C). Such heat treatment sometimes confers lethal damages in certain plant species and TaqI cannot induce rearrangements in AT-rich regions. To overcome such weakness, we developed an Extended-TAQing (Ex-TAQing) system, which enables the utilization of a wider range of restriction enzymes active at standard plant-growing temperatures. We established the Ex-TAQing system using MseI that can efficiently cleave DNA at room temperature and the 5'-TTAA-3' sequence which is highly abundant in Arabidopsis genome. A synthetic intron-spanning MseI gene which was placed downstream of a heat shock-inducible promoter was conditionally expressed upon a milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genome rearrangements, including copy number variations, translocation, and direct end-joining at MseI cleavage sites. Ex-TAQing system could induce large-scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosome rearrangements associated with beneficial phenotypes, such as high-salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex-TAQing system, allowing more diverse patterns of genome rearrangements, by employing various types of endonucleases, and have opened a way to expand the capability for artificial genome reorganization.