Zinc finger nuclease-mediated transgene deletion

Petolino, J. F.; Worden, Andrew; Curlee, Krisi; Connell, Joseph H.; Moynahan, Tonya L. Strange; Larsen, C. A.; Russell, Sean

doi:10.1007/s11103-010-9641-4

Cited by 123 publications

(82 citation statements)

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“…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: 69%

“…Transgene deletion by ZFNs was recently reported by Petolino et al (2010), who flanked a transgene with the ZFN CCR5 (Perez et al, 2008). In their report, Petolino et al (2010) used a transgenic strategy in which transgenic target plants were crossed with CCR5 ZFNoverexpressing plants, giving rise to targeted offspring. Here, we expand the use of transgene delivery to the important model plant Arabidopsis with an efficiency of 28% (calculated for actual deletion events; Table I).…”

Section: Discussionmentioning

confidence: 99%

“…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). Since genomic DSBs can function as traps for the integration of A. tumefaciens transferred DNA (T-DNA) molecules via NHEJ (Salomon and Puchta, 1998;Chilton and Que, 2003;Tzfira et al, 2003), we decided to explore the possible use of the NHEJ DNA-repair pathway not only for site-specific mutagenesis and targeted gene insertion but also for gene replacement.…”

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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: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

2013

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“…The most obvious application of such an approach is the use of artificially constructed modular endonucleases, such as meganucleases (26), zinc finger nucleases (27), or transcription activator-like effector nucleases (28), that are designed to cut at a specific site in the gene of interest. Recent studies demonstrated that the aforementioned nucleases can be applied in the same way as I-SceI used in our study for different DSB-induced recombination reactions in plants (8,15,19,29). For commercial application, the donor vector might contain a GT cassette flanked by two recognition sites of a site-specific endonuclease that would also cut within the target locus (Fig.…”

Section: Discussionmentioning

confidence: 99%

In planta gene targeting

Fauser

Roth

Pacher

et al. 2012

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

197

176

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The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a sitespecific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.plant biotechnology | plant breeding | gene technology | double-strand-break repair S ince the first report on gene targeting (GT) in plants was published (1), various approaches were tested to improve the efficiency of the method (2-5), which has been summarized in recent reviews (6, 7). We were able to demonstrate that the integration of a transfer DNA (T-DNA) by homologous recombination (HR) into a specific locus could be enhanced by two orders of magnitude via double-strand-break (DSB) induction using a site-specific endonuclease (8). More recently, by the use of zinc finger nucleases (9), which, in principle, can be used to induce a DSB at any genomic site, endogenous loci have been targeted in Arabidopsis (10), tobacco (11), and maize (12) at high frequencies (13). Some time ago, a method for in vivo targeting was developed in Drosophila. A stably integrated donor precursor molecule is first circularized by the FLP recombinase and subsequently linearized via cutting a single I-SceI recognition site, generating the actual GT vector (14). However, such a technique has not been successfully transferred to plants. We have previously shown that DNA can be efficiently excised from the genome in planta by the use of a site-specific endonuclease (15). To test whether the combination of this approach with DSB-induced recombination might lead to an efficient GT system, that is independent of transformation, we performed a proof-of-concept (POC) experiment in Arabidopsis using I-SceI (16) as a sitespecific nuclease. ResultsGenerating Homozygous Single-Copy GT Lines. Our in planta GT system is based on three different constructs (two shown in Fig. 1A) that were transformed independently by floral dipping. The target locus contains a truncated β-glucuronidase (GUS) gene (uidA) that can be restored via GT. DSB induction at the two ISceI recognition sites flanking a kanamycin-resistance gene would result in excision of the kanamycin-resistance gene and in activation of the target locus for HR. Th...

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“…removal, replacement, and addition of expression cassettes by NHEJ) can potentially be achieved by transient ZFN expression in target cells, with or without the addition of a donor DNA molecule (Weinthal et al, 2010). Indeed, both I-SceI-and I-CeuI-mediated transgene addition (Salomon and Puchta, 1998;Chilton and Que, 2003;Tzfira et al, 2003) and ZFN-mediated transgene deletion (Petolino et al, 2010) have been reported in plant cells. In addition, we have recently demonstrated that ZFN-and NHEJ-mediated gene replacement is also feasible in plant species (D. Weinthal, T. Taylor, and T. Tzfira, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

Zeevi¹,

Liang²,

Arieli³

et al. 2011

Plant Physiol.

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Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.

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Zinc finger nuclease-mediated transgene deletion

Cited by 123 publications

References 39 publications

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

In planta gene targeting

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors

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