**3.1. Gene disruption**

plant cell. The CRISPR/Cas9 system can be delivered via *Agrobacterium*-mediated transformation or particle bombardment [46]. Up to this stage, the expression cassettes are stably integrated into plant genome. Finally, the transformed plants with targeted mutations are screened by polymerase chain reaction (PCR) genotyping and confirmed by sequencing [46].

**Figure 4.** Type II adaptive immunity system by CRISPR/Cas9 in bacteria. The type II adaptive immunity system by CRISPR/Cas9 involves three stages, which are acquisition, crRNA biogenesis and interference to cleavage the DNA

The Cas9 protein consists of six domains, which are REC I, REC II, Bridge Helix, PAM interacting, HNH and RuvC [47]. It remains inactive in the absence of gRNA. The gRNA binds to Cas9 protein and induces a conformational change to form a riboprotein complex. This results

**2.3. RNA-guided DNA cleavage by Cas9**

target, resulting in the formation of DSBs.

136 Next Generation Plant Breeding

Gene disruption or simply known as gene knockout is a genetic technique that turns one of the genes in an organism to become inoperative. This technique is very powerful as it can inactivate any potential harmful or nonbeneficial gene that downgrades the quality of a plant. Gene disruption is the most applied technique as it can knockout genes by simply introducing small deletion or insertion via NHEJ repair mechanism in CRISPR/Cas9 system [5, 7].

A good example that employed full use of the gene knockout mechanism is the *Waxy* (*WX1*) gene of a maize plant. The maize *WX1* gene encodes a starch-synthesizing protein that is


**Species name Target gene(s) Gene function Description Mode of** 

Carotenoid biosynthesis, growth

biosynthesis

biosynthesis

biosynthesis

and carotenoid biosynthesis

biosynthesis

biosynthetic pathway catalyst

biogenesis regulation

*SlAGO7* Involved in RNA

*Sorghum bicolor DsRED2* Fluorescence Transgenic plants

regulator

*Oryza sativa OsPDS*, *OsMPK2*,

*Solanum lycopersicum* *OsBADH2*, *etc.*

*Petunia hybrid PDS* Carotenoid

*Populus tomentosa PtoPDS* Carotenoid

*Solanum tuberosum StALS1* Acetolactate

*Triticum aestivum TaINOX*, *TaPDS* Inositol metabolism

*Vitis vinifera IdnDH* Tartaric acid

*Zea mays ZmIPK* Phytic acid

**Table 1.** List of CRISPR/Cas9 system-based genome-edited plants.

**action**

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

Gene disruption

Gene disruption

Gene disruption

Gene disruption

Gene disruption

Gene disruption

Gene disruption

Gene insertion [64]

Gene insertion [65]

Transgenic plants displayed albinism and dwarfism after being subjected to targeted mutagenesis

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

Transgenic plants displayed albinism expression after being subjected to targeted mutagenesis

Transgenic plants displayed albinism expression after being subjected to targeted mutagenesis

Transgenic plants displayed needle-like or lacking lamina leaves after being subjected to targeted mutagenesis

Transgenic plants showed increased resistance on herbicides after being subjected to targeted mutagenesis

showed signs of red fluorescence after being subjected to targeted mutagenesis

Transgenic plants displayed albinism expression after being subjected to targeted mutagenesis

Transgenic plants showed no signs of tartaric acid in their fruits after being subjected to targeted mutagenesis

Transgenic plants showed reduction of phytic acid level after being subjected to targeted mutagenesis

**Ref.**

139

[60]

[61]

[62]

[63]

[66]

[67]

[68]


**Table 1.** List of CRISPR/Cas9 system-based genome-edited plants.

**Species name Target gene(s) Gene function Description Mode of** 

biosynthesis

biosynthesis

factor

factors

regulator

*ARF1* Auxin response factor

*Medicago truncatula GUS* Fluorescence Transgenic plants

biosynthesis

biosynthesis

*NbPDS* Carotenoid

*Nicotiana tabacum NtPDS* Carotenoid

Root hair growth

*Arabidopsis thaliana BR11*, *JAZ1*, *GAI* Growth regulators Transgenic plants

*Brassica oleracea BolC.GA4.a* Gibberellin

138 Next Generation Plant Breeding

*Citrus sinensis CsPDS* Carotenoid

*Glycine max Bar*, *GmFE11*,

*Marchantia polymorpha*

*Nicotiana benthamiana*

*Cucumis sativus eIF4E* Translation initiation

*GmFE12*, etc.

*Hordeum vulgare HvPM19* Grain dormancy

**action**

Gene disruption

Gene disruption

Gene regulation

Gene disruption

Gene regulation

Gene disruption

Gene disruption

Gene disruption

Gene disruption

Gene insertion [58]

displayed retarded growth after being subjected to targeted mutagenesis

Transgenic plants displayed dwarf phenotype after being subjected to targeted mutagenesis

Transgenic plants displayed albinism expression after being subjected to targeted mutagenesis

Transgenic plants developed resistance toward a broad range

Transgenic plants displayed higher root hair growth induction after being subjected to targeted mutagenesis

Transgenic plants displayed signs of dormancy after being subjected to targeted mutagenesis

Transgenic plants showed no response toward auxins after being subjected to targeted mutagenesis

displayed no signs of staining after being subjected to targeted mutagenesis

Transgenic plants displayed albinism expression after being subjected to targeted mutagenesis

Transgenic plants displayed albinism expression after being subjected to targeted mutagenesis

of virus

**Ref.**

[51]

[52]

[53]

[54]

[55]

[52]

[56]

[57]

[59]

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 addition to increased time requirements [52].

There is another unconventional substitute to molecular stacks where it can only be generated through the CRISPR/Cas9 system. That substitute is known as complex trait loci (CTL) or quantitative trait loci (QTL) and where transgenes can also be genetically collocated [58, 59]. An example of CTLs is constructed through the CRISPR/Cas9 system by specifically inserting the transgenes into the desired region in the genome through HDR. To start, the transgenes in the CTL can be separated by a larger distance (50 kb to more than 1 Mb) compared to the molecular stacks (few hundred or thousand bp) while retaining their genetic linkage [60]. The changes of distance solves both the limitations of the molecular stacks as adjacent transgene will no longer

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141

affect each of their function and they can now be individually moved and swapped.

immature sorghum embryo [61]. As a result, the plant now displays red fluorescence.

**3.3. Gene regulation**

and gene addition techniques.

the agriculture industry.

**3.4. CRISPR/Cas9 system-based genome-edited plants**

Similarly, with the help of the CRISPR/Cas9 system, the DsRED2 gene, which encodes a protein that expresses red fluorescence, was also successfully inserted into the genome of an

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

As the aforementioned plants are successfully genetically modified in the lab, there are actually some of them that are almost readily available in the commercial market. These plants may be new to the market but it is undeniable that they will eventually be able to monopolize the market as they have much more improved traits compared to their relative wild-type plants. As shown in **Table 2**, most of the plants such as the wheat and Ranger Russet potato are important food staples in many parts of the world and this proves that the CRISPR/Cas9 system-based genome editing for crop improvement is definitely on its way to revolutionize

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 express albinism or etiolated leaves.
