EXZACT TM Precision Technology: Scientific and Regulatory Advancements in Plant-Genome Editing with ZFNs

Rudgers and Sastry-Dent Approaches to improving agricultural crops have relied on either mutational or traditional breeding and selection methods or through the random introduction of novel genes into crops by transformation (e.g. using Agrobacterium). These approaches are inherently time-consuming, inefficient and expensive, especially for developing complex, multi-trait products that are now standard for the industry. To better serve the market, new technologies have been developed that focus on targeted genome editing. One such technology is Dow AgroSciences’ EXZACTTM Precision Technology that facilitates precise changes in plant genomes including DNA deletions, edits (point mutations) and targeted gene additions (trans-, cis-, intra-). Using EXZACTTM Precision Technology, developers and breeders can now introduce genetic traits more efficiently and precisely than ever before. EXZACTTM Precision Technology is based on zinc-finger nucleases (ZFNs) that are composed of a DNA-binding domain (zinc-finger proteins) and a nuclease domain derived from the restriction endonuclease Fok1. DNA binding is mediated by ZF modules, where each ZF recognizes a specific three-base-pair DNA sequence. Typically four to six individual ZFs are linked together for specific recognition of a unique DNA sequence (12–18 nucleotides) (Figure 1). The Fok1 nuclease that generates the specific doublestranded DNA cleavage at the targeted DNA sequence requires dimerization of the two Fok1 domains for cleavage activity. Therefore, functional ZFNs are dimeric proteins consisting of two unique zinc-finger proteins (Figure 1) that bind to two unique DNA EXZACT TM Precision Technology: Scientific and Regulatory Advancements in Plant-Genome Editing with ZFNs