BASICS OF ZINC FINGER TECHNOLOGY

What are Zinc Finger Proteins? Zinc finger proteins (ZFPs) are one of the largest classes of naturally-occurring DNA binding proteins. Found in most eukaryotic organisms , including plants, the name “zinc finger protein” was coined to describe the zinc finger protein structure which resembles a finger-like shape and includes a zinc ion. EXZACT technology uses ZFPs that are specifically designed to bind to unique three base pair DNA sequences (triplets). The EXZACT™ ZFP library consists of 2000+ characterized ZFP modules with multiple ZF binding domains for any given DNA triplet. Read more on What Is EXZACT? 

What is a Zinc Finger Nuclease? A Zinc Finger Nuclease (ZFN) is a fusion between a ZFP DNA binding domain and an endonuclease (FokI for example). ZFP DNA binding domains can be designed to bind any plant DNA sequence and the endonuclease domain is used to induce a double-stranded break in the DNA. The FokI cleavage domain must dimerize (combine with a similar molecule) in order to cleave DNA. Thus, a pair of ZFPs must bind their corresponding DNA sequence and be present at the same time in order for the FokI endonuclease to dimerize and cleave the DNA sequence. See “EXZACT™ Mechanics: Zinc-Finger Nucleases” on The Science of EXZACT for more details.

QUESTIONS SPECIFIC TO EXZACT™ PRECISION TECHNOLOGY

What makes “EXZACT™ ZFNs” superior to other plant ZFNs used in scientific research? Several lines of evidence show that EXZACT™ ZFN designs are highly specific and can discriminate between intended- and off-target sequences by as little as a single nucleotide of DNA sequence. For example, it has been shown in maize (Shukla et. al .) that EXZACT™ ZFNs targeting the maize IPK1 gene can differentiate between IPK1 and a highly similar (paralogous) gene (IPK2) which differs by only a single nucleotide in the ZFP binding domains. See EXZACT™ ZFNs publications for more information.

Can all DNA sequences be targeted using EXZACT™ ZFNs? Due to the complex structure and chemistry of DNA, certain DNA sequences may be more difficult to target than others (e.g. highly repetitive DNA sequences, protein bound DNA sequences). However, the EXZACT™ zinc-finger library consist of 2000+ characterized zinc-finger modules with multiple ZFPs for any given DNA triplet. In most cases, by linking four to six zinc-finger modules together, EXZACT™ ZFPs can be designed to most plant DNA sequences.

What genomic modifications can EXZACT™ ZFNs create? EXZACT™ ZFNs allow scientists to Add, Delete and Edit plant genomic DNA. Using EXZACT™ Add, a single gene or a stack of genes can be added to a predetermined genetic location within the plant genome. EXZACT™ Edit allows scientists to rewrite gene sequences to produce novel crops and products while EXZACT™ Delete is used to remove undesirable DNA sequences and traits from the plant genome. (Read more about how EXZACT Precision Technology improves on traditional plant breeding techniques.)

In the case of EXZACT™ Add and Edit a repair template is required. This repair template consists of the desired genomic modification (nucleotide change, gene, or stack of genes) to be inserted, in addition to homologous genomic sequence matching the sequencing surrounding the intended ZFN cut site. Once the ZFN cleaves the targeted DNA sequence, a natural plant DNA repair mechanism (homology directed repair, HDR) repairs the DNA break using the repair template and, in the process, encodes the modifications present on the template into the repair site.

Using EXZACT™ Delete , no repair template is needed. Once the targeted DNA sequence has been cleaved by EXZACT™ ZFNs, the cell will use another DNA-repair process known as Non-Homologous End-Joining (NHEJ) to resolve the double stranded break. This repair process sometimes results in short nucleotide deletions or insertions at the break site allowing the inactivation of genes and removal of undesirable plant traits.

Additional details about DNA repair mechanisms can be found on the Science of EXZACT page.

What is the difference between EXZACT Edit and Add? Although EXZACT™ Edit and Add rely on the same general principles for repairing DNA breaks (HDR), the end products of these applications are very different. In the case of EXZACT™ Add, a new DNA sequence (e.g. gene, promoter, terminator…) or multiple DNA sequences (e.g. gene stacks) are added to the plant genome; this may lead to the expression of new genes and traits, such as insect or herbicide tolerance. EXZACT™: Accelerating Agriculture Biotechnology provides additional detail. Or view our publications describing targeted transgene insertion in plants using designed zinc-finger nucleases.

In the case of EXZACT™ Edit, small changes (single base or small deletions / additions) are made to the native plant DNA, resulting in altered gene sequences. These types of changes are similar to those that occur through traditional or mutation breeding, which are long-standing practices in conventional agriculture. The end products are plants and crops with improved characteristics for both growers and consumers. For more details on EXZACT™ Edit see EXZACT™ Mechanics: Zinc-Finger Nucleases.

What crops and model plant species have EXZACT™ ZFNs been validated in?  ZFNs have been designed and tested against a wide range of genomic target sequences in a diverse variety of crop and plant species including maize, canola, wheat, soybean, tomato, tobacco and Arabidopsis. The number of crops and plant species continues to grow and it is anticipated that ZFNs will function in virtually any plant species.  See EXZACT ZFNs publications for more information. 

Are ZFNs present or absent in the end product?  Depending on the goal, ZFNs can either be absent or present in the end product.  In cases where ZFNs are not required or desired in the end product, ZFN DNA is transiently introduced into plant cells, allowing just enough time for the ZFNs to be expressed, bind and cleave the target DNA sequence.  Depending on the method of ZFN DNA introduction, the ZFN DNA can be either naturally degraded by the plant cell or segregated away. In the end, the final product is absent of ZFN DNA and contains only the desired change in the plant genome.  See EXZACT™ ZFNs publications  for more information.

EXZACT AND REGULATORY

Are products developed using EXZACT™ ZFNs considered GMO? EXZACT™ Delete and Edit result in mutational products.  There is debate on whether mutagenesis is considered GMO or non-GMO, however, traditionally mutational agricultural products have a long history of safe use and have been regulated as non-GM products or exempt from regulation.  In the case of EXZACT™ Add, in which new genes or traits are introduced into the plant, it is anticipated that these products would be regulated like traditional transgenic crops.

What are the regulatory agencies’ opinions about the regulation status of EXZACT™ ZFN products?  The USDA has determined that plants containing targeted deletions (EXZACT Delete ), caused by naturally-occurring DNA repair after the targeted break is made by EXZACT zinc-finger nucleases, and in which no genetic material is inserted into the plant genome, are not regulated articles under USDA regulatory guidelines (CFR part 340) .  In addition, the USDA believes that changes to the plant genome generated by the deletion process would not result in a product requiring regulatory oversight as long as no DNA was inserted into the plant genome during the process.

The Australian Government, Office of the Gene Technology Regulator (OGTR) has responded to Dow AgroSciences stating that crops developed using EXZACT Delete technology would not contain introduced foreign nucleic acid, once the ZFN genes are no longer present, and would not be considered GMO and not subject to regulation under the Gene Technology Act 2000.

In the case of EXZACT Edits, the USDA and OGTR will consider case-by-case inquires regarding the regulatory status of plants produced using this method.  It is anticipated that as long as the Edit poses no regulatory risks as outlined in CFR part 340, EXZACT Edits will not require USDA oversight.

How do EXZACT™ Edit or Delete mutations differ from those that occur through breeding or classical mutagenesis? EXZACT™ mutations cannot be distinguished, even at the molecular level, from mutations derived using classical mutagenesis or traditional breeding methods.  However, unlike classical mutagenesis methods which result in the introduction of hundreds to thousands of random mutations in the plant genome, EXZACT™ mutations are predetermined and targeted to a specific location, allowing one to significantly reduce the probability of unintentional and undesirable alterations at other genetic locations.