Site-directed mutagenesis in
            <i>Arabidopsis</i>
            using custom-designed zinc finger nucleases

Osakabe, Keishi; Osakabe, Yuriko; Toki, Seiichi

doi:10.1073/pnas.1000234107

Cited by 289 publications

(165 citation statements)

References 46 publications

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“…However, Osakabe et al (2010), showed that the lack of KU80, which functions in protecting exposed DNA ends in DSBs, leads to larger deletions at the ZFN-induced break site as compared with shorter mutations in the wild-type plants. It was thus suggested that an alternative NHEJ pathway might exist in Arabidopsis (Osakabe et al, 2010). Our site-specific mutagenesis rate (19% and 26% for Arabidopsis and tobacco, respectively, when calculated for two individual mutations per event; Table I) was similar to those reported by others (Lloyd et al, 2005;de Pater et al, 2009;Tovkach et al, 2009).…”

Section: Discussionsupporting

confidence: 73%

“…In previous studies, HR-mediated gene-replacement strategies have been the preferred mode of gene replacement in plants (Wright et al, 2005;Shukla et al, 2009;Townsend et al, 2009;de Pater et al, 2013), while NHEJ-mediated repair of ZFN-induced DSBs has been limited to targeted mutagenesis and transgene removal (Lloyd et al, 2005;Morton et al, 2006;Maeder et al, 2008;de Pater et al, 2009;Tovkach et al, 2009;Osakabe et al, 2010;Petolino et al, 2010;Zhang et al, 2010). Our findings show that the NHEJ DNA-repair pathway can be harnessed for gene replacement in plant species.…”

Section: Discussionmentioning

confidence: 94%

“…de Pater et al (2009) reported that the rate of sitespecific mutagenesis in ZFN-overexpressing transgenic plants is much lower than expected, and Osakabe et al (2010) discovered that the ZFN-mediated site-specific mutagenesis rate is similar in wild-type and KU80-deficient plants. However, Osakabe et al (2010), showed that the lack of KU80, which functions in protecting exposed DNA ends in DSBs, leads to larger deletions at the ZFN-induced break site as compared with shorter mutations in the wild-type plants.…”

Section: Discussionmentioning

confidence: 99%

“…However, it is important to note that while ZFNinduced DSBs can indeed induce the HR repair machinery, most of these breaks are repaired by the cell's NHEJ DNA-repair machinery in plant and other species. Thus, ZFNs have also been used for the induction of site-specific mutagenesis in many eukaryotic cells, including plant cells (Lloyd et al, 2005;Tovkach et al, 2009;Marton et al, 2010;Osakabe et al, 2010;Zhang et al, 2010;Curtin et al, 2011;Even-Faitelson et al, 2011). More recently, ZFNs have been used for NHEJmediated targeted chromosomal deletions (Lee et al, 2010;Söllü et al, 2010), transgene removal (Petolino et al, 2010), and even NHEJ-mediated targeted DNA integration in mammalian genomes (Orlando et al, 2010).…”

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confidence: 99%

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Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

2013

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Stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) by zinc finger nucleases (ZFNs) has been deployed for gene replacement in plant cells. Nonhomologous end joining (NHEJ)-mediated repair of DSBs, on the other hand, has been utilized for the induction of site-specific mutagenesis in plants. Since NHEJ is the dominant DSB repair pathway and can also lead to the capture of foreign DNA molecules, we suggest that it can also be deployed for gene replacement. An acceptor DNA molecule in which a green fluorescent protein (GFP) coding sequence (gfp) was flanked by ZFN recognition sequences was used to produce transgenic target plants. A donor DNA molecule in which a promoterless hygromycin B phosphotransferase-encoding gene (hpt) was flanked by ZFN recognition sequences was constructed. The donor DNA molecule and ZFN expression cassette were delivered into target plants. ZFN-mediated sitespecific mutagenesis and complete removal of the GFP coding sequence resulted in the recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was unlinked to the acceptor DNA. More importantly, ZFNmediated digestion of both donor and acceptor DNA molecules resulted in NHEJ-mediated replacement of the gfp with hpt and recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was physically linked to the acceptor DNA. Sequence and phenotypical analyses, and transmission of the replacement events to the next generation, confirmed the stability of the NHEJ-induced gene exchange, suggesting its use as a novel method for transgene replacement and gene stacking in plants.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

2013

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“…Zinc finger nuclease (ZFN) and, more recently, transcription activator-like effector nuclease (TALEN) have been tested as tools for targeted plant gene editing. Several publications have reported successful modifications in native plant genes, but both methods are tedious, expensive, and with only a small number of genes modified so far (18)(19)(20)(21)(22)(23)(24)(25). In sharp contrast, we report specific modifications of seven genes in this study in the span of just a few months, demonstrating the extremely easy-to-use feature of the CRISPR/Cas system.…”

Section: Discussionmentioning

confidence: 69%

Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

Feng

Mao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

617

453

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The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/ Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.

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Switchgrass Genetics and Breeding Challenges

Bartley

Saathoff

et al. 2013

Bioenergy Feedstocks

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

Cited by 289 publications

References 46 publications

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

Switchgrass Genetics and Breeding Challenges

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