**2.2 Zinc finger nuclease-based engineering**

Artificial sequential nucleases known as zinc finger nucleases (ZFNs) have transformed the area of programmable nucleases. ZFNs were created by attaching numerous zinc finger DNA-binding domains to the restriction endonuclease FokI's nonspecific cutting pattern [21]. The protein molecules can enlighten the difference between two DNA sequences separated by only a few nucleotides. This allows the two endonucleases to form a dimer, breaking double-stranded DNA [22]. Furthermore, because each motif in the zinc finger array reads a distinct

#### *Enhancement of Agricultural Crops: A CRISPR/Cas9-Based Approach DOI: http://dx.doi.org/10.5772/intechopen.100641*

3-nucleotide complementary strand, the domain composition variable sequence can always be chosen to fit the particular destination. ZFNs were originally used for sequence-specific mutagenesis in tobacco in the early 2000s, which was most likely the first time a designed endonuclease identified and fractured chromosomal DNA [23, 24]. With these remarkable achievements, ZFN usage in agriculture has indeed been limited due to factors such as the technical complexity of manufacturing and the scarcity of aiming places in genomes in comparison to more recently established functional genomics approaches.
