**3.3. Gene regulation**

involved in the kernel maintenance [48]. Today, there is a known mutant maize that has a deletion in the coding sequence of the WX1 allele [49, 50] that causes it to have an altered grain starch composition [51]. Waxy corns are highly sought after in the commercial market because it provides a variety of benefit such as improved uniformity, stability and texture despite its lower yield compared to elite corns [52]. Up until recently, there have been attempts to introduce the mutant WX1 allele by crossbreeding a nonelite Waxy corn with an elite plant with excellent agronomic qualities. However, this method was unsuccessful as some of the nonelite alleles near the mutant *WX1* gene may be carried along during the introgression process in

Recently, an agricultural company known as DuPoint took this matter with an alternative solution through gene disruption by using the CRISPR/Cas9 system [53]. The gene disruption via CRISPR/Cas9 system is cheap, fast and, most importantly, precise as *WX1* deletions can now be generated directly in the genome of the elite plant to overcome the imperfections that are associated with trait introgression. The gene disruption via CRISPR/Cas9 system works by deleting the entire *WX1* gene with the usage of two Cas9-gRNAs. Each of the Cas9-gRNAs will target two sites, which are the upstream of the transcriptional start site and the downstream of the stop codon. Then, the region is excised and the remaining DNA damage is repaired through the NHEJ, which will bring about the *WX1* null allele with the Waxy phenotype [52]. Another study that utilized the ability of gene disruption of CRISPR/Cas9 system was carried out in wheat, an important staple food in many parts of the world [54]. The team reported that the *inositol oxygenase* (*INOX*) and *phytoene desaturase* (*PDS*) gene of the wheat plant was successfully deactivated at the same time, making it a multiplex mutagenesis. The application of the CRISPR/Cas9 system to the gene causes the gene to have random insertion into its sequence, resulting in gene disruption. Consequently, the phenotype of the wheat changes to

Gene insertion or addition is another famous technique where more than one nucleotide base pairs are added into a DNA sequence. The newly inserted sequence can be designed in such a way where it can specifically encode proteins that bring crucial benefits. However, designing the inserted-to-be DNA sequence is not an easy task as imperfection could pose a risk to the health of the host cell or organism, or it can be simply nulled as it fails to function as predicted [55]. In the terms of gene addition for crop improvement, the desired goal is usually the addi-

In fact, adding multiple genes that confer different trait improvement in a single plant is a common practice to produce elite cultivar. For instance, the TC1507 maize that contains both the *Bacillus thuringiensis* (*Bt*) gene that confers insect resistance and *acetyltransferase* gene that is herbicide tolerance [56]. To date, the most common and efficient method used is to collocate all the desired genes into a single molecular stack, whereby all of the genes will now behave as a single locus or better known as gene stacking [52]. The colocation is no easy task as it has two major limitations. First, each of the genes may potentially affect each other if they are placed adjacently too close [57]. Second, once the transgenes are collocated, they can no longer be moved as they are placed too close to each other to the point where it acts as a single locus.

tion of beneficial traits such as pest resistance, high yield or quality.

addition to increased time requirements [52].

express albinism or etiolated leaves.

**3.2. Gene insertion**

140 Next Generation Plant Breeding

Gene regulation is a technique whereby the gene encoding for its transcription factors is altered to induce changes in its gene expression level [62]. Consequently, plant traits such as the fruit color, size and shape can be controlled and adjusted according to the consumer demands.

The CRISPR/Cas9 system can also be used to regulate the expression of genes for plants [63]. It was carried out by the usage of a catalytically inactive Cas9 known as dead Cas9 (dCas9). The deactivation occurs when rare bacteriophages with anti-CRISPR protein AcrIIA4 binds to the Cas9 of a gRNA that causes its cleaving activity to be disabled [64]. Consequently, the dCas9 is unable to cleave DNAs but it can still bind to specific DNA sequences with gRNA. To be used in gene regulation, the dCas9 must be fused with either a transcriptional activator or a repressor.

For transcriptional activation, dCas9 will be fused with a transcription activator domain such as VP64. For example, there is a study that reported that the paired dCas9-VP64 couple successfully activates the *anthocyanin pigment 1* (*AtPAP1*) gene from *Arabidopsis thaliana,* which encodes the protein involved in the production of anthocyanin pigment 1 [65]. Meanwhile, for transcriptional repression, dCas9 will be fused with a transcription repressor domain such as SRDX instead. Consistently, a study had reported the usage of dCas9-SRDX pair to successfully repress the *A. thaliana cleavage stimulating factor 64* (*AtCSTF64*) gene of a plant of the same species. This technique is still new compared to the previously mentioned gene disruption and gene addition techniques.
