**1.1. Site-specific nucleases (SSNs)**

SSNs have two major components, which are the engineered nonspecific endonucleases and the sequence-specific DNA-binding domains. The nonspecific nucleases have the ability to produce DSBs in DNA but they are very random as they lack specificity. On the other hand, DNA-binding domains are proteins that can specifically bind to DNA sequences that are complementary to them [8]. Hence, when DNA-binding domains synergize with the endonucleases, the paired components are able to introduce breaks at any specific target site [9, 10].

As shown in **Figure 1**, after the DNA break is induced, it will trigger the native DNA repair mechanisms of the cell to fix the break either by the error-prone nonhomologous end joining (NHEJ) mechanism or by the homology-directed repair (HDR) mechanism [11]. The NHEJ mechanism can occur during any phases of the cell cycle and due to its high erroneous repair rate, it occasionally forms frameshift mutation. Hence, this phenomenon can be exploited to form what is known as "knocking out" of a specific gene where the functionality of the protein encoded by that gene is lost due to random insertion, deletion or inversion. However, if provided an engineered DNA repair template that is homologous to the upstream and downstream of the target sequence, the HDR pathway can then be activated instead. The HDR mechanism functions to repair the break differently from which it will insert a new strand of DNA as a form of a desired mutation that resembles the DNA repair template [7]. This is also known as gene "knocking in" where the genome now has a new gain-of-function to encode a specific protein.

**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].

## *1.1.1. Zinc finger nuclease (ZFN)*

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 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

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 agricul-

SSNs have two major components, which are the engineered nonspecific endonucleases and the sequence-specific DNA-binding domains. The nonspecific nucleases have the ability to produce DSBs in DNA but they are very random as they lack specificity. On the other hand, DNA-binding domains are proteins that can specifically bind to DNA sequences that are complementary to them [8]. Hence, when DNA-binding domains synergize with the endonucleases, the paired components are able to introduce breaks at any specific target site [9, 10].

As shown in **Figure 1**, after the DNA break is induced, it will trigger the native DNA repair mechanisms of the cell to fix the break either by the error-prone nonhomologous end joining (NHEJ) mechanism or by the homology-directed repair (HDR) mechanism [11]. The NHEJ mechanism can occur during any phases of the cell cycle and due to its high erroneous repair rate, it occasionally forms frameshift mutation. Hence, this phenomenon can be exploited to form what is known as "knocking out" of a specific gene where the functionality of the protein encoded by that gene is lost due to random insertion, deletion or inversion. However, if provided an engineered DNA repair template that is homologous to the upstream and downstream of the target sequence, the HDR pathway can then be activated instead. The HDR mechanism functions to repair the break differently from which it will insert a new strand of DNA as a form of a desired mutation that resembles the DNA repair template [7]. This is also known as gene "knocking in" where the genome now has a new gain-of-function to encode

(SSNs) [1].

130 Next Generation Plant Breeding

ture industry.

a specific protein.

**1.1. Site-specific nucleases (SSNs)**

can be generated through this methodology [4–6].

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 sequences [10].

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

**Figure 2.** ZFN dimer binding to DNA at each side of the sequence. Different shaded boxes represent the different zinc fingers and each of them binds to a specific codon. Short dotted lines indicate 3-bp long codons. Shaded ellipses indicate the Fok1 endonuclease domain with the ability to induce DNA double-stranded breaks and they are coupled by the zinc fingers to guide them to their cleavage site. The cleavage site will be between the dimer and it is about 4-bp long. Adapted from Carroll [9].

known that the Fok1 endonuclease domain must dimerize to produce the breaks on DNA [13, 16]. However, the interaction between the dimer ZFN can be weak if it is not optimally designed. Thus, the optimal design for ZFN to achieve DNA cleavage is to direct two sets of fingers to neighboring sequences and join each to a Fok1 DNA-cleaving domain. Once the dimers bind to their respective sequences, adjacent cleavage factors will facilitate the dimerization and cleavage of the DNA strand [9]. Nevertheless, the construction of ZFN has proven to be difficult as there are many complicated interference in the interaction between the ZFPs despite many attempts made in the past to simplify them [17–23].

TALE proteins have a different method to detect DNA sequences. They recognize a single nucleotide instead of DNA triplets. In fact, TALE proteins can also be assembled in a way to recognize virtually any DNA sequence that is desired by their users [26]. This attribute enables TALENs to have a higher range of target sites to suit user specifications, making them significantly more flexible and generally more straightforward. Like ZFN, the DNA-cleaving domain for TALENs is the nonspecific Fok1 endonuclease domain, which is highly dependent on the DNA-binding domains to achieve higher specificity of DSBs [1]. In a nut shell, both ZFNs and TALENs function as genome editing tools but they are considerably complex, a challenge to be constructed and less efficient compared to newly developed genome editing tool like CRISP/Cas9 system.

**Figure 3.** Transcription activator-like effector nuclease (TALEN) dimer bound to DNA. Like the ZFN, it has a Fok1 endonuclease domain that can cleave DNA. Indicated by small dotted lines, each TALE protein can specifically detect a single nucleotide base pair and it can be linked with more TALE proteins to detect longer sequences. By coupling the Fok1 endonuclease domain with the TALE proteins, it is now known as TALEN and it can specifically detect and cleave

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

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The most recently developed SSN in genome editing technology is known as the CRISPR/ Cas9 system. The CRISPR/Cas9 system was developed based on the bacterial type-II CRISPR/ Cas adaptive immune system that is deployed by the hosts to recognize and eliminate any invading phage or plasmid DNA [27–29]. The three major components of the immune system are the protospacer-containing CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) and

During the invasion of foreign DNA, the bacterial type-II CRISPR/Cas system will integrate a short fragment of the foreign DNA, called "spacers," into the CRISPR genomic loci [31]. These spacers act as a form of an acquired immunity memory for the host. Then, the spacers will be transcribed and processed in the form of crRNA. Thus, the CRISPR genome loci act as the library where it can store information to enable the bacteria CRISPR/Cas9 system to retarget any known foreign DNA. In case of another attack by the invader, the crRNAs will then bind to the tracrRNAs that have the ability to trigger the direction of the Cas9 endonuclease

*1.1.3. CRISPR/Cas9*

DNA. Adapted from Gupta [73].

the Cas9 endonuclease [27, 30].
