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Zinc‐finger nucleases are chimeric fusion proteins consisting of a DNA‐binding domain and a DNA‐cleavage domain. The DNA‐binding domain is composed of a set of Cys2His2 zinc fingers (usually three to six). Each zinc finger primarily contacts 3 bp of DNA and a set of three to six fingers recognize 9–18 bp, respectively. The DNA‐cleavage domain is derived from the cleavage domain of the FokI restriction enzyme. FokI activity requires dimerization; therefore, to site‐specifically cleave DNA, two zinc‐finger nucleases are designed in a tail‐to‐tail orientation (Kim et al. 1996).

Zinc‐finger nucleases can be remodified to recognize different DNA sequences. However, one limitation with redirecting targeting is that it depends on the context of the host. For example, a zinc finger that recognizes GGG may not recognize this sequence when fused to other zinc fingers. As a result, the modular assembly of zinc fingers has had limited success (Ramirez et al. 2008). One of the more successful methods for redirecting targeting involves generating a library of three zinc‐finger variants from a pre‐selected pool of zinc‐finger monomers (Maeder et al. 2008). The resulting library of zinc‐finger arrays can then be interrogated using a bacterial two‐ hybrid screen, where binding of the zinc‐finger array to a pre‐determined sequence results in the expression of a selectable marker gene. This method has generated highly‐active zinc‐finger nuclease (ZFN) pairs for sites within animal and plant genomes. Since the development of ZFN technology, several studies have been done to engineer specific zinc‐finger modules for each of the 64 codon triplets (Bae et al. 2003; Dreier et al. 2001; Pabo et al. 2001). Until now, several ZFNs have been designed and used in numerous species. The developments for more specific and efficient technologies also gave rise to fewer off‐target effects. There are three most commonly available tools for engineering the ZF domains: context‐dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly (MA). Several softwares are available for designing engineered ZFs (ZiFiT), containing the database of ZFs (ZiFDB) and identification of potential targets for ZFNs in several model organisms (ZFNGenome) (Kim et al. 2009; Mandell and Barbas 2006; Sander et al. 2007).

Figure 1.1 (A) Diagrammatic representation of (a) Zinc‐finger nucleases (ZFNs), (b) Transcription activator‐like effector nucleases (TALENs) and (c) Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediates DSBs formation. (B) dCas9‐based targeted genome regulation by (a) activation of gene expression, (b) repression of gene expression and (c) DNA methylation.

Source: Adapted from Mahfouz et al. (2014) © 2014. Reproduced with the permission of John Wiley & Sons.

Zinc‐finger nucleases have been widely used for plant genome engineering. Plant species that have been modified using zinc‐finger nucleases include, Arabidopsis, maize, soybean, tobacco, etc. (Ainley et al. 2013; Cai et al. 2009; Curtin et al. 2011; Lloyd et al. 2005; Marton et al. 2010; Osakabe et al. 2010; Shukla et al. 2009; Townsend et al. 2009; Wright et al. 2005; Zhang et al. 2010). With their relatively small size (~300 amino acids per zinc‐finger nuclease monomer), and the further advancements in methods for redirecting targeting (Sander et al. 2011a), zinc‐finger nucleases should continue to be an effective technology for editing plant.