Abstract
Gene targeting (GT) is more precise transformation technology compared to other methods of genetic modification. However, GT efficiency is extremely low in higher plants (0.01-0.1% of random integration events). Recently, synthetic site-specific endonucleases, named zinc finger nucleases (ZFNs), have been used for higher efficient GT for several plant species. Although ZFN-induced double-stranded DNA breaks (DSBs) enhanced GT in higher plants up to 1-3%, further improvement must be needed for more efficient GT to apply higher plant species showing lower transformation frequency.
Exonuclease-1 (EXO1) produces 3' cohesive single-strand DNA lesions, thereby facilitating later events of homologous recombination. To study the effect of EXO1 on DSB-induced GT, a GT assay was developed in rice. Using this system, we succeeded in high-frequency GT with EXO1 coupled with ZFNs as a DSB inducer up to 100-fold enhancement; our result therefore established a new strategy for plant genetic manipulation in basic science and agricultural application.
