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

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

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 ZFNs and TALENs, the CRISPR/Cas9 system is potentially more efficient and

**1.** *Target design simplicity*: the DNA sequence targeting system of the CRISPR/Cas9 system is based on a formation of RNAs rather than protein or DNA recognition. RNA is much more accessible as it is simple and readily produced to be used to target any sequence in

**2.** *Efficiency*: the CRISPR/Cas9 system is a straightforward tool for genome editing. Modification of the target genome can be carried out by directly introducing RNAs that encode the Cas9 protein and gRNA to the host. This method allows researchers to skip all the long and

**3.** Multiplexed mutation: several targeted mutations can be carried out in multiple genes at different locations at once by introducing multiple gRNAs at once. For example, Dr. Yang H. and his team have successfully simultaneously introduced mutation in five different

The CRISPR/Cas9 system is a type II adaptive immune system in bacteria and archaea, protecting them against invading nucleic acid such as virus by cleaving the foreign DNA through specific sequence recognition [29]. The immunity is acquired through the integration of short fragment of the foreign DNA as spacer between two adjacent repeats at the proximal end of a CRISPR locus [29]. The bacterial CRISPR/Cas9 system involves three stepwise processes,

The defense mechanism of CRISPR/Cas9 system can be divided into three stages, which are

**2.1. Bacterial adaptive immunity: acquisition, biogenesis and interference**

spacer acquisition or adaptation, crRNA biogenesis and interference (**Figure 4**).

laborious processes of the classical homologous recombination techniques.

of the crRNA-tracrRNA-Cas9 complex [32–34].

compared to the combination of crRNA and tracrRNA [38, 39].

effective with three main advantages [40], which are:

genes in mouse embryonic stem cells [41–43].

**2. Mechanism of CRISPR/Cas9 system**

namely acquisition, biogenesis and interference.

a genome.

134 Next Generation Plant Breeding

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 into a vector.

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

in the activation of the Cas9 protein from a non–DNA-binding conformation into an active DNA-binding conformation. Once the Cas9 protein is activated, it recognizes DNA target by binding with DNA sequence that matches its PAM. Once the Cas9 protein finds a potential target sequence with complementary PAM, it melts the bases immediately upstream of the PAM and pairs them with the complementary sequence on the gRNA. RuvC and HNH nuclease domains will then cleave the DNA target after the third nucleotide base upstream of the

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

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To date, the most common Cas9 protein used in plants is from the bacteria *Streptococcus pyrogens* (SpCAs9), which recognizes the NGG-type PAM [46]. Even though this PAM sequence is widely distributed across plant genomes, it does not cover the entire genome of the plant [46]. Many naturally occurring Cas9 orthologues impose distinct crRNA-tracrRNA duplex and PAM requirements [30]. Therefore, exploring orthologous Cas9 proteins with cognate gRNA and PAM sequences would greatly expand possible target sequences in a given genome and add new Cas9 orthologues with unique properties into the CRISPR/Cas arsenal [30]. As the alternative orthologous Cas9 requires different PAM sequences, the total number of possible target sites within a plant genome can be increased. Hence, the CRISPR/ Cas9 system is modified by cointroducing multiple Cas9 orthologue-based platforms with different effectors such as nuclease, transcription activator or repressor into the same cell, where they are guided by a specific group of gRNAs to carry out multiplex and complex

Today, the application of the CRISPR/Cas9 system-based genome editing for crop improvement has already begun its movement despite still being in its infancy. As a matter of fact, there are already numerous publications that reported that this technology has been success-

The applications of the CRISPR/Cas9 system in plants can be classified into three types, which

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

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

small deletion or insertion via NHEJ repair mechanism in CRISPR/Cas9 system [5, 7].

fully implemented in a broad range of plant species (as shown in **Table 1**).

are the gene disruption, gene insertion and gene regulation.

PAM to generate blunt-ended DSBs.

**2.4. Orthologues of CRISPR/Cas9**

manipulation of gene activities [30]

**3.1. Gene disruption**

**3. Application and recent advances**

**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 target, resulting in the formation of DSBs.

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].

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

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 in the activation of the Cas9 protein from a non–DNA-binding conformation into an active DNA-binding conformation. Once the Cas9 protein is activated, it recognizes DNA target by binding with DNA sequence that matches its PAM. Once the Cas9 protein finds a potential target sequence with complementary PAM, it melts the bases immediately upstream of the PAM and pairs them with the complementary sequence on the gRNA. RuvC and HNH nuclease domains will then cleave the DNA target after the third nucleotide base upstream of the PAM to generate blunt-ended DSBs.
