Targeted editing of the genomes of living organisms not only permits investigations into the understanding of the fundamental basis of biological systems but also allows to improve productively and quality of crops. This includes the creation of plants with valuable compositional properties and with traits that confer resistance to various biotic and abiotic stresses. Recently, several novel genome editing systems have been developed, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALNEs), and clustered regularly interspersed short palindromic repeats/Cas9 (CRISPER/Cas9). These exciting new methods have proved themselves as effective and reliable tools for the genetic improvement of plants. The genome editing systems can also be used to exploit the genetic diversity present in the semi-domesticated and wild relatives of the cultivated crops by targeting homologous domesticated genes through allele-mining. In this chapter various tools available for gene editing, their merits, and demerits have been discussed.
TopZinc-Finger Nucleases
Zinc finger nucleases (Z NFs) is considered to be the first generation genome editing tools that was developed through chemically engineered nucleases, having folded up to ββα configuration. The enzyme is derived from fusion of zinc-finger based DNA-recognition modules and the DNA-cleavage domain of the FokI restriction endonucleases (Figure 1a). The α-helix of the protein gets inserted to the DNA after binding of the protein into the major groove of the double helix of DNA. Each zinc finger recognizes and binds to a triplet nucleotide sequence and assembles into a group at the specific binding site(s). The monomer of ZFN has two different functional domains: an artificial ZF Cys2-His2 domain (N-terminal end) and a non-specific FokI DNA cleavage domain (C-terminal end). Dimerization of the FolI domain is critical for enzymatic activity of ZEN. The individual zinc finger domains are interchangeable and according to the order of the domain, they can bind to specific sites sequences in the genome. Several zinc finger domains capable of recognizing large numbers of triplet nucleotides has been generated with the objectives to target large sequences of interest.
ZFNs have so far been used to modify rice, maize, soybean, rapeseed, tobacco, apple, fig petunia, and Arabdiopsis. Compared to other tools, ZFN has been found to been efficient, high specificity, and minimal non-target effects. Although ZNFs have been used successfully for development of herbicide tolerance and stacking useful traits in maize and identification of safe regions for gene integration in rice, the technique has remained complicated and technically challenging for crop improvement. Therefore, the current focus is on to improve the design and delivery system of ZNF technology for wider acceptability for crop improvement.