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, 107 (26), 12034-9

Site-directed Mutagenesis in Arabidopsis Using Custom-Designed Zinc Finger Nucleases

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Site-directed Mutagenesis in Arabidopsis Using Custom-Designed Zinc Finger Nucleases

Keishi Osakabe et al. Proc Natl Acad Sci U S A.

Erratum in

  • Proc Natl Acad Sci U S A. 2011 Jan 4;108(1):433

Abstract

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Consensus ZFN target sites in the Arabidopsis ABA-INSENSITIVE4 (ABI4) gene. The schematic representation of the ABI4 gene is shown at the top. Asterisk indicates the position of the mutation in the abi4 mutant. AP, AP2 domain; S/T, serine- and threonine-rich domain; Q, glutamine-rich domain; Acid, activation domain. Target sites of ZFN monomers are highlighted with gray bars. The putative cleavage sites are shown by arrows. FokI-DD, mutated Fok I nuclease domain (R487D); FokI-RR, mutated Fok I nuclease domain (D483R).
Fig. 2.
Fig. 2.
Alteration of amino acid sequences after ZFN digestion in the ABI4 gene. (A) Structure of T-DNA for the ZFN expression binary vector. LB, left border sequence of T-DNA; RB, right border sequence of T-DNA; gfbsd2, GFP + blasticidin S-resistance gene fusion expression cassette; Phsp18.2, Arabidopsis HSP18.2 gene promoter; AAA, ZFN_AAA; 2A, self-cleaving 2A peptide derived from Thosea asigna (27); TCC, ZFN_TCC; Tp5, Arabidopsis polyA-binding protein PAB5 gene terminator. The gfbsd2 expression cassette is driven by 2× cauliflower mosaic virus 35S gene promoter and the nopaline synthase gene terminator. (B) Examples of repaired DNA sequences at ZFN target sites (bold) of the ABI4 genes in transgenic lines zfn_abi4-1 and zfn_abi4-9. The putative cleavage region is shown in lowercase letters. Mutations are indicated in magenta-colored characters and deletions are shown as hyphens. (C) Alteration of amino acid sequences after ZFNs digestion of the ABI4 gene. Two lines (zfn_abi4-1–1 and zfn_abi4-9-1) were recovered from initially detected mutated T1 plants for the ABI4 gene. After establishing the T3 generation, mutation sites were reconfirmed. Target sites of ZFN monomers are underlined. The putative site of the double-strand break is double-underlined. Predicted amino acid sequences of this region of the ABI4 gene in the two mutant lines are shown at bottom.
Fig. 3.
Fig. 3.
The ZFN targeted mutation in ABI4 in Arabidopsis confers ABA-insensitive and stress tolerance phenotypes. (A and B) abi4, zfn-abi4, and WT plants were grown for 10 d on medium containing ABA (A) and glucose (B). (C) Seed germination rates of abi4, zfn_abi4, and WT plants at various concentrations of ABA. Germination was assessed at d 2 after sowing. SD values were calculated from three individual experiments. n = 30 seeds per experiment. WT (■), abi4 (□), zfn_abi4-1–1 (○), zfn_abi4-9–1 (▲).
Fig. 4.
Fig. 4.
Efficiency and fidelity of NHEJ repaired DNA after ZFN cleavage in the ABI4 gene in atku80 cells. (A) The frequency of end-joining after induction of DSBs by ZFNs. The relative error-prone end-joining frequency was derived from the number of DNA clones positive for the Surveyor nuclease assay per number of DNA clones tested (n = 480 for each experiment). Experiments were performed with three independent lines, and data are presented as mean ± SD. (B) Distribution of length of deletions at individual junctions. Deletions are defined as the sum of base pairs lost at both sides of the DSB. (C) Examples of repaired DNA sequences obtained from genomic DNA of WT and atku80 plants after ZFN cleavage. ZFN recognition sites are depicted in bold. Putative cleavage regions are shown in lowercase characters. Mutations are shown in magenta-colored characters and deletion are shown as hyphens.

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