**1. Introduction**

As the agricultural commercial market continues to grow, development of new techniques for crop improvement is always in high demand; conversely, traditional breeding practices for crop improvement are phasing out as some techniques are far too time-consuming and

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

laborious and usually result in little yield. Examples of such traditional breeding are the selective crossbreeding between plants with the desired trait or classical breeding with induced mutation via radiation or chemicals. Currently, a new methodology that involves genetic engineering was developed and had paved the way to improve the quality of plants with high specificity for the attribute of interest; the application of site-specific nucleases (SSNs) [1].

SSNs are programmable nucleases that have the ability to produce DNA single-stranded or double-stranded breaks (DSBs) that activate the endogenous DNA repair pathways of the cells to repair the DNA damage and this usually leads to targeted mutagenesis [1, 2]. This technology empowers plant scientists to precisely regulate any genes in any plant while directly evaluating the function of that specific gene in the plant [3]. As a result, plants that express characteristics such as higher yield, disease resistance or shorter maturation periods can be generated through this methodology [4–6].

SSN-based genome editing system can be classified into three categories that are the zinc finger nuclease (ZFN), transcription-like effector nucleases (TALENs) and, the recently developed, clustered regularly interspaced short palindromic repeats that are associated with the RNA-guided Cas9 double-stranded DNA-binding protein (CRISPR/Cas9) [1]. The main differences between the categories lie in their mechanism of the double-stranded break induction and their efficiency in targeting their desired sequences [7]. These SSN-based genome editing systems are very powerful and they have undoubtedly revolutionized the agriculture industry.

*1.1.1. Zinc finger nuclease (ZFN)*

sequences [10].

One of the earliest SSNs developed for genome editing is known as the zinc finger nuclease (ZFN). ZFN is constructed through the coupling of two major domains, which are the eukaryotic-based DNA-binding domain known as the zinc finger protein (ZFP) and the endonuclease domain of the Fok1 restriction enzyme (**Figure 2**). Consequently, this coupling combines the quality of the DNA-binding specificity of the ZFP and cleaving activity of the Fok1 endonuclease into a single system, thus making ZFN a useful tool for genome editing [10]. Zinc finger proteins (ZFPs) have the ability to specifically bind to a discrete 3-base pairs (bp) sequence of DNA known as codons [12]. In fact, recent studies have shown that each ZFNs can use 3 zinc fingers to bind a 9-bp target sequence, and when made into a ZFN dimer, it can recognize up to 18-bp of a DNA [9, 13]. Furthermore, there are up to 64 possible types of ZFPs that can be produced as there are 64 codon combinations known today [10]. As a result, by determining which type of ZFPs are to be linked into the ZFN, researchers are able to design ZFNs in a way that they would only target desirable sequences. Additionally, studies have also shown that more fingers (up to six per ZFN) can be added to recognize longer and rarer target

**Figure 1.** Different DNA repair mechanisms for DSBs. Left: if a DNA repair template is provided, the repair will proceed via the homology-directed repair mechanism where a desired mutation that is based on the template is introduced to the DNA. Right: the double-stranded break can be repaired via error-prone nonhomologous end joining mechanism where random mutation will be introduced to the DNA in the form of deletion or insertion. Adapted from Ott de Bruin et al. [12].

The CRISPR/Cas9 System for Crop Improvement: Progress and Prospects

http://dx.doi.org/10.5772/intechopen.75024

131

Paired with the zinc fingers is the nonspecific Fok1 endonuclease domain, which is an enzyme that can be found naturally in *Flavobacterium okeanokoites* and it has the ability to induce DNA double-stranded breaks [14, 15]. By linking both domains together, they will now be known as the zinc finger nuclease, and the ZFP domain will lead the Fok1 endonuclease domain to the desired DNA sequence to cleave the target site, which is adjacent to it; this equips ZFN with the ability to precisely cut any targeted DNA sequences. In early studies, it has been
