Beit Jann, Israel
Beit Jann, Israel

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Tzfira T.,Ben - Gurion University of the Negev | Weinthal D.,Ben - Gurion University of the Negev | Marton I.,Danziger Innovations Ltd. | Marton I.,Hebrew University of Jerusalem | And 3 more authors.
Plant Biotechnology Journal | Year: 2012

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.


Vainstein A.,Hebrew University of Jerusalem | Marton I.,Hebrew University of Jerusalem | Marton I.,Danziger Innovations Ltd. | Zuker A.,Danziger Innovations Ltd. | And 2 more authors.
Trends in Biotechnology | Year: 2011

Endonuclease-mediated induction of genomic double-strand breaks has enabled genome editing in living cells. However, deploying this technology for the induction of gene disruption in plant cells often relies on direct gene transfer of endonuclease (i.e. zinc finger nuclease or homing endonuclease) expression constructs into the targeted cell, followed by regeneration of a mutated plant. Such mutants, even when they have no detectable traces of foreign DNA, might still be classified as transgenic because of the transgenic nature of the endonuclease delivery method. Indirect delivery of endonucleases into target cells by viral vectors provides a unique non-transgenic approach to the production of mutated plants. Furthermore, viral vectors can spread into the growing and developing tissues of infected plants, which could provide a unique opportunity to bypass the regeneration step that is often required in direct gene-transfer methods. © 2011 Elsevier Ltd.


Vainstein A.,Hebrew University of Jerusalem | Marton I.,Danziger Innovations Ltd. | Zipin Rotman A.,Danziger Innovations Ltd. | De Costa N.,Danziger Innovations Ltd. | And 4 more authors.
Acta Horticulturae | Year: 2012

The ability to modify genome sequences in plant cells is fundamental to modern agriculture. Naturally occurring and artificial rare-cutting endonucleases (i.e., zinc finger nuclease, homing endonuclease or TAL effector nuclease) have been used for targeted mutagenesis in model and crop species. However, stable transformation is the preferred method for gene expression in plant species, and nuclease-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. We developed an alternative, non-transgenic approach for nuclease delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of nucleases into a variety of tissues and cells of intact plants. Since viral vectors can spread into the growing and developing tissues of infected plants, the novel approach of genomeediting provides a unique opportunity to bypass the regeneration step that is often required in direct gene-transfer methods.


Marton I.,Danziger Innovations Ltd | Marton I.,Hebrew University of Jerusalem | Zuker A.,Danziger Innovations Ltd | Shklarman E.,Danziger Innovations Ltd | And 6 more authors.
Plant Physiology | Year: 2010

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNAvirus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods. © 2010 American Society of Plant Biologists.


Marton I.,Danziger Innovations Ltd | Honig A.,Danziger Innovations Ltd | Omid A.,Danziger Innovations Ltd | De Costa N.,Danziger Innovations Ltd | And 4 more authors.
International Journal of Developmental Biology | Year: 2013

Researchers and biotechnologists require methods to accurately modify the genome of higher eukaryotic cells. Such modifications include, but are not limited to, site-specific mutagenesis, site-specific insertion of foreign DNA, and replacement and deletion of native sequences. Accurate genome modifications in plant species have been rather limited, with only a handful of plant species and genes being modified through the use of early genome-editing techniques. The development of rare-cutting restriction enzymes as a tool for the induction of site-specific genomic double-strand breaks and their introduction as a reliable tool for genome modification in animals, animal cells and human cell lines have paved the way for the adaptation of rare-cutting restriction enzymes to genome editing in plant cells. Indeed, the number of plant species and genes which have been successfully edited using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered homing endonucleases is on the rise. In our review, we discuss the basics of rare-cutting restriction enzyme-mediated genome-editing technology with an emphasis on its application in plant species. © 2013 UBC Press.


Patent
Danziger Innovations Ltd. and Hebrew University of Jerusalem | Date: 2014-07-28

A method of generating genotypic variation in a genome of a plant is disclosed. The method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant.


Patent
Hebrew University of Jerusalem and Danziger Innovations Ltd. | Date: 2010-10-21

A method of generating genotypic variation in a genome of a plant is disclosed. The method comprising introducing into a gamete or a gamete producing tissue of the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, wherein the introducing is performed such that the gamete or gamete producing tissue expresses the chimeric nuclease but not all plant tissues express the chimeric nuclease, thereby generating genotypic variation in the genome of the plant.


Patent
Danziger Innovations Ltd. and Hebrew University of Jerusalem | Date: 2015-10-28

A pTRV based expression vector comprising a nucleic acid sequence encoding a heterologous polypeptide, said nucleic acid sequence being devoid of the 2b sequence as set forth in SEQ ID NO: 43, and a method of generating a transgenic plant, the method comprising introducing into one or more cells of the plant at least one of said pTRV based expression vector.


Avin-Wittenberg T.,Weizmann Institute of Science | Michaeli S.,Weizmann Institute of Science | Honig A.,Weizmann Institute of Science | Honig A.,Danziger Innovations Ltd | Galili G.,Weizmann Institute of Science
Plant Signaling and Behavior | Year: 2012

Autophagy is a mechanism used for the transport of macromolecules to the vacuole for degradation. It can be either non-selective or selective, resulting from the specific binding of target proteins to Atg8, an essential autophagyrelated protein. Nine Atg8 homologs exist in the model plant Arabidopsis thaliana, suggesting possible different roles for different homologs. In a previous report published in the Plant Cell, our group identified two plant-specific proteins, termed ATI1 and ATI2, which bind Atg8f, as a representative of the nine Atg8 homologs. The proteins were shown to associate with novel starvationinduced bodies that move on the ER network and reach the lytic vacuole. Altered expression level of the proteins was also shown to affect the ability of seeds to germinate in the presence of the germination inhibiting hormone ABA. In the present addendum article, we demonstrate that, in addition to Atg8f, ATI1 binds Atg8h, an Atg8 homolog from a different sub-family, indicating that ATI1 is not a specific target of Atg8f. © 2012 Landes Bioscience.


PubMed | Danziger Innovations Ltd.
Type: Journal Article | Journal: Plant physiology | Year: 2010

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.

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