Use of Zinc-Finger Nucleases for Crop Improvement

Over the past two decades, new technologies enabling targeted modification of plant genomes have been developed. Among these are zinc-finger nucleases (ZFNs) which are composed of engineered zinc-finger DNA-binding domains fused with a nuclease, generally the FokI nuclease. The zinc-finger domains are composed of a series of four to six 30 amino acid domains that can bind to trinucleotide sequences giving the entire DNA-binding domain specificity to 12-18 nucleotides. Since the FokI nuclease functions as a dimer, pairs of zinc-finger domains are designed to bind upstream and downstream of the cut site which increases the specificity of the complete ZFN to 24-36 nucleotides. The ability of these engineered nucleases to create targeted double-stranded breaks at designated locations throughout the genome has enabled precise deletion, addition, and editing of genes. These techniques are being used to create new genetic variation by deleting or editing endogenous gene sequences and enhancing the efficiency of transgenic product development through targeted insertion of transgenes to specific genomic locations and to sequentially add and/or delete transgenes from existing transgenic events.