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: 580; 570; 0303 health sciences; Deoxyribonucleases; Base Sequence; DNA Repair; DNA, Plant; Arabidopsis Proteins; Molecular Sequence Data; Arabidopsis; DNA Helicases; Genes, Plant; Plants, Genetically Modified; Protein Engineering; Recombinant Proteins; 03 medical and health sciences; Mutation; Mutagenesis, Site-Directed; DNA Breaks, Double-Stranded; Amino Acid Sequence; Promoter Regions, Genetic; Transcription Factors

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