Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases.
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.
Osakabe
Keishi
K
Plant Genetic Engineering Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.
Osakabe
Yuriko
Y
Toki
Seiichi
S
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D017343:Genes, Plant; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
C470421:ABI4 protein, Arabidopsis; D029681:Arabidopsis Proteins; D018744:DNA, Plant; D011994:Recombinant Proteins; D014157:Transcription Factors; D003851:Deoxyribonucleases; D004265:DNA Helicases; C469228:Ku80 protein, Arabidopsis