Compared to conventional methods, EXZACT™ Precision Technology offers a robust molecular toolkit for enhancing crops through targeted, precise genome modification. Conventional agricultural biotechnology is based on methods that engineer changes at random sequences, or locations, in the plant genome. Researchers must then screen and select for plants that are modified at the gene- or sequence-of-interest, determine whether any unintended genome modification has occurred (which might lead to undesirable outcomes) and assess whether the modification resulted in the improved trait properties.
EXZACT™ Mechanics: Zinc-Finger Nucleases
EXZACT™ is based on the use of proprietary, designed enzymes known as zinc-finger nucleases (ZFNs). ZFNs are engineered fusions of a DNA recognition domain and a nuclease domain. The DNA recognition domain can be designed to bind novel DNA sequences with high sequence-specificity; therefore, ZFNs that bind to any gene can be created. The nuclease domain is used to induce a double-stranded break in the DNA of living plant cells, which invokes the cell's naturally occurring DNA repair mechanisms. These repair mechanisms include non-homologous end-joining and homology-directed repair, both of which are highly conserved across eukaryotic systems including plant species. Non-homologous end-joining repair of double-stranded breaks leads to small insertions or deletions of DNA sequence at the cleavage site. Because the break is induced at a precise, pre-specified sequence, this "imperfect" repair is a method for targeted disruption of native genes. Homology-directed repair uses a donor DNA as the template for repairing a break. By providing the cell with a donor DNA molecule that contains segments of sequence homology to the cleavage site, it is possible to drive highly efficient and precise insertion of that donor into the break. Donor molecules may contain novel transgenes, edits of the native gene, or a combination of sequences that will insert into the pre-specified genome target, leading to targeted modifications.
Innovation in Plant Genome Modification Realized (publications)
EXZACT™ Precision Technology's targeted approach represents a new way of thinking about agricultural biotechnology. One practical example of how EXZACT™ has been used is illustrated in a scientific paper published in the May 21, 2009, issue of the journal Nature. In this study, scientists from Dow AgroSciences used the EXZACT™ platform to insert an herbicide tolerance gene into a native site in the corn genome. ZmlPK1 encodes an enzyme that is involved in biosynthesis of phytate, a component of feed corn that is indigestible by certain animals and causes excess phosphate in the environment. The herbicide tolerance gene was precisely inserted into the ZmlPK1 gene in corn, thereby disrupting that gene. Results from this research confirmed that the addition of the herbicide tolerance gene occurred only at the desired target location and without insertion of other DNA. This insertion was precise and reproducible. The scientific team showed that the herbicide tolerance gene was faithfully transmitted to the next generation of corn plants, which carried both herbicide tolerance and reduced phytate levels (a stacked trait).
To learn more about the research published in Nature, view a press release or a slide presentation summarizing EXZACT’s features and benefits. You can view the full article if you have access privileges to Nature online or request an electronic copy of the article.
In a recent report in the journal Plant Molecular Biology (2010), scientists at Dow AgroSciences have demonstrated transgene removal using EXZACT™ technology. Based on the ability to design zinc-fingers to virtually any DNA sequence, this study explores the strategy for removing undesirable or unwanted genes and transgenes from the genome of plants. You can view the full article on-line if you have access privileges to PMB online.
Plant Molecular Biology (2009) also features a research report describing the use of EXZACT™ in tobacco, an important model that has been used to study plant transformation for many years. The results of this study are further proof of the flexibility and versatility that EXZACT™ offers to basic researchers and crop developers alike.
You can view the full article if you have access privileges to PMB online.
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