*1.1.2. Transcription activator-like effector nucleases (TALENs)*

It may seem that ZFN is the most practical method; nevertheless, the challenge lies in the construction as mentioned in addition to less popularity as the success rate for the DNA repair pathway via HDR is still considerably low [7]. Therefore, a more recent genome editing tool known as transcription activator-like effector nucleases (TALENs) had been developed with better modularity [24, 25]. TALENs are quite similar to ZFN in terms of the idea of directing the same nonspecific endonuclease to a specific site with the help of DNA-binding motifs (**Figure 3**).

Unlike ZFN, the DNA-binding domains for TALENs are known as the transcription activatorlike effector (TALE) proteins rather than ZFPs and they are found in pathogenic plant bacteria (specifically of genus *Xanthomonas*) instead of in a eukaryotic cell. TALE protein is comprised of a repetitive sequence of a series of 34 amino acid residues, where each TALE protein has the ability to selectively bind to one nucleotide in the DNA target site [1]. Specifically, the pair residue at the 12th and 13th position is the one that determines the nucleotide specificity of the TALE proteins and they are known as the repeat variable domain (RVD).

**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 DNA. Adapted from Gupta [73].

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.

## *1.1.3. CRISPR/Cas9*

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

**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.

It may seem that ZFN is the most practical method; nevertheless, the challenge lies in the construction as mentioned in addition to less popularity as the success rate for the DNA repair pathway via HDR is still considerably low [7]. Therefore, a more recent genome editing tool known as transcription activator-like effector nucleases (TALENs) had been developed with better modularity [24, 25]. TALENs are quite similar to ZFN in terms of the idea of directing the same nonspecific endonuclease to a specific site with the help of DNA-binding motifs

Unlike ZFN, the DNA-binding domains for TALENs are known as the transcription activatorlike effector (TALE) proteins rather than ZFPs and they are found in pathogenic plant bacteria (specifically of genus *Xanthomonas*) instead of in a eukaryotic cell. TALE protein is comprised of a repetitive sequence of a series of 34 amino acid residues, where each TALE protein has the ability to selectively bind to one nucleotide in the DNA target site [1]. Specifically, the pair residue at the 12th and 13th position is the one that determines the nucleotide specificity of

the TALE proteins and they are known as the repeat variable domain (RVD).

despite many attempts made in the past to simplify them [17–23].

*1.1.2. Transcription activator-like effector nucleases (TALENs)*

(**Figure 3**).

Adapted from Carroll [9].

132 Next Generation Plant Breeding

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 the Cas9 endonuclease [27, 30].

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 to the target site of the foreign DNA. After that, it will induce DSBs on the foreign DNA that carries the same protospacer sequence, which is accompanied by the protospacer adjacent motif (PAM) to disable the virus [7, 30]. As a matter of fact, recent studies have shown that the specificity of Cas9 protein is highly dependent to the PAM sequence as it licenses the landing of the crRNA-tracrRNA-Cas9 complex [32–34].

Conceptually, the spacer acquisition process can be further divided into two steps, which are protospacer selection and integration of the spacer into the CRISPR array to synthesize new repeat [44]. The protospacer selection step of the spacer acquisition stage is guided by a conserved DNA sequence element, namely protospacer adjacent motif (PAM), that is located downstream of the DNA target, which has the sequence of 5'-NGG-3 [44]. Next, the selected protospacer will be integrated into the leader-repeat boundary of CRISPR array as new spacer and is duplicated in order to synthesize new repeats. Each repeat is a 29 nucleotide sequence, and repeats are interspaced by five intervening 32-nucleotide nonrepetitive sequences [30].

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

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Then, the CRISPR will be transcribed into a long precursor crRNA (pre-crRNA) with the help of Cas proteins (Cas1, Cas2, Cas9 and Cas4/casn2) or simply known as biogenesis. During this process, the tracrRNA will be transcribed from the bacterial genome as it is needed for processing of the pre-crRNA into a mature guide crRNA. Next, the antirepeat sequence of tracrRNA will enable the complementary base pairing with each pre-crRNA repeats, result-

During the target interference stage, the crRNA-tracrRNA duplex recognizes the PAM sequence located downstream of the protospacer in the foreign DNA and triggers the 'non– self-activation,' which prevents self-targeting of CRISPR array. After that, the duplex guides the Cas9 endonuclease to bind and cleave the DNA target, resulting in the formation of DSBs. Additionally, the DNA target specificity duplex is provided by what is called the 'seed sequence,' which is located at approximately 12 bases upstream of the PAM sequence that

The CRISPR/Cas9 system for plant genome editing comprises four steps. First, a gene-specific gRNA is designed and constructed by fusing crRNA and tracrRNA. Many online tools have been developed for computer-based design of gRNA [46]. However, the in silico design of gRNA has not been fully adapted for plants, and large-scale data collection and systematic study of gRNA efficiencies in plant cells are needed to increase the accuracy of computational gRNA selection [46]. Both gRNA and Cas9 expression cassettes are constructed separately. The expression of gRNA is driven by U3 or U6 small nuclear RNA gene promoters with defined initiation and termination site, facilitating the transcription of gRNA by RNA polymerase III. The first nucleotide in the guide sequence is a 'G' if U6 promoter is used or an 'A' if U3 promoter is used [47]. Guide sequence should match the target, except for the first nucleotide (5′ G or A) that does not have to match [47]. In Cas9 expression, nuclear localization of Cas9 requires fusion of a single or dual nuclear localization signal (NLS) to the Cas9 coding sequence, which is 4107-bp in length. Both expression cassettes are then assembled

The next step involves the transformation of protoplasts with CRISPR in which the activity of gRNA is best validated before being used in genome editing [46]. This step is followed by the selection of active CRISPR using polymerase chain reaction (PCR) or restriction enzyme digestion. Then, the CRISPR/Cas9 system in the form of vector is delivered into the target

ing in the formation of a crRNA-tracrRNA duplex [45].

matches the RNA with the DNA target [29] (**Figure 4**).

**2.2. Engineered CRISPR/Cas9 in plant genome editing**

into a vector.

The CRISPR/Cas9 system has been redesigned to work more efficiently by fusing the Cas9 endonuclease with an artificial crRNA-tracrRNA chimera known as guide RNA (gRNA) [35–37]. The gRNA maintains the function of both crRNA and tracrRNA where just by itself is able to recognize the PAM-containing target sequence to direct the Cas9 protein for DNA cleaving activity. In fact, studies have shown that the gRNA works more efficiently compared to the combination of crRNA and tracrRNA [38, 39].

Compared to ZFNs and TALENs, the CRISPR/Cas9 system is potentially more efficient and effective with three main advantages [40], which are:

