Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases

• DOI: 10.1073/pnas.1000234107
• Publication Date: 2010-05-28T02:22:57Z
• Authors: Keishi Osakabe; Yuriko Osakabe; Seiichi Toki
• Keywords: Zinc finger nuclease; Gene targeting

Site-directed mutagenesis in higher plants remains a significant technical challenge for basic research and molecular breeding. Here, we demonstrate targeted-gene inactivation an endogenous gene Arabidopsis using zinc finger nucleases (ZFNs). Engineered ZFNs stress-response regulator, the ABA-INSENSITIVE4 ( ABI4 ) gene, cleaved their recognition sequences specifically vitro, ZFN genes driven by heat-shock promoter were introduced into genome. After induction, mutations with deletion substitution generated via ZFN-mediated cleavage observed somatic cells at frequencies as high 3%. The homozygote mutant line zfn_abi4-1–1 exhibited expected phenotypes, i.e., ABA glucose insensitivity. In addition, was applied to DNA repair-deficient plant, atku80 . We found that lack of AtKu80, which plays role end-protection dsDNA breaks, increased error-prone rejoining frequency 2.6-fold, end-degradation. These data approach can be used efficient production precision reverse genetics.