1. Introduction

Sustainable agriculture is considered to be the key for improving plant crops through genetic engineering, since random mutagenesis processes are part of conventional biotechnology techniques used for most researchers in this field [1]. CRISPR/Cas9 systems (clustered regularly interspaced short palindromic repeats-CRISPR associated) are related to a well-known CRISPR array defined by series of 20–50 bp genomic locus (i.e., unique spacers separated by direct repeats).

On the other hand, these unique spacers usually have similar length with preceded AT-rich fragments [2]. CRISPR loci were identified for the first time about two decades ago when they found a series of short genomic sequences (i.e., spacers) in Escherichia coli originated by viral genomes and probably due to the presence of conjugative plasmids, and in this sense, the foreign genetic material allowed the bacteria to record a kind of memory (e.g., immune system) to counteract future infections. When foreign DNA sequences match these unique spacers, they are commonly known as "photospacers" [1, 3]. Thereby, the corresponding immunization is against a foreign phage (e.g.), and when a new infection of this nature takes place in the future, the array expansion of the CRISPR is unchained and in consequence, new spacers originate from the genetic material of the phage.

According to some authors [4–6], this interesting immune system (to call it that) may be divided into three metabolic stages: adaptation, crRNA (CRISPR-RNAs) biogenesis, and interference. When a foreign DNA introduction happens, there is a selective process through the machinery that selects protospacers, which will be inserted into the CRISPR locus (insertion takes place into the leader end of the system). In the first stage of crRNA biogenesis, a transcription of CRISPR locus is observed followed by a direct processing of sequence elements (pre-crRNAs–crRNAs), all of them with the corresponding single spacer. After this stage, Cas proteins interact the crRNAs by assembling an effector complex (Figure 1A) [4]. This is very important since these components are intermediary elements of the interference stage where recognition of foreign DNA happens upon future infections and consequently, its degradation. It is very important to mention that a spacer acquisition creates genetic records of previous infections; as mentioned above, CRISPR immunity happens when there is an imminent detection of strange nucleic acids and consequently, the integration of foreign genetic material into the host's cells (the DNA integration occurs in the chromosome).

sciences field have been mainly focused on plant domestication with economic and social interest. Thus, interspaced short palindromic repeats constantly open the doors to the generation and knowledge application in the area of functional genomics that jointly guide

Figure 1. Functional and organizational system of the CRISPR/Cas9. (A) The process of induced immunity is carried out in three stages consisting of an adaptation, crRNA biogenesis, and interference. In the first stage, the adaptive machinery performs a selection of photospacers, and they are leader-end inserted into CRISPR locus that is subsequently transcribed during crRNA biogenesis. In a complementary way, the pre-crRNA processing into crRNAs with simple spacers is developed. Finally, the effector complex is originated through the assembly of crRNA with Cas proteins, which interacts (interference stage) in a subsequent way to the infection, and consequent degradation of the foreign genetic material. (B) Structurally, the CRISPR system is divided into six representative types (operons). Dashed outlines represent genes in some subtypes. Pink sequences represent genes related to the interference process. Yellow sequences refer to crRNA biogenesis, and the green color refers to adaptation genes. Subtypes IV are characterized by the absence of CRISPR loci.

Understanding CRISPR/Cas9: A Magnificent Tool for Plant Genome Editing

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The action mode of CRISPR/Cas9 biological system is basically based on the participation of two distinct elements: the Cas9 protein (CRISPR-associated protein 9; RNA-guided DNA endonuclease enzyme associated with CRISPR) and sgRNA (single guide RNA) [8]. Nowadays, Cas9 proteins are found mainly in different bacteria species such as Brevibacillus laterosporus [9], Staphylococcus aureus [10], and other representative species within the genus Streptococcus [11]. Cas9 proteins have shown two representative domains: the first one is known as HNH (nuclease-associated proteins), which is responsible for cleaving and regrouping the complementary strand of crRNA. On the other hand, the second domain known as RuvC-like (nuclease domain that cleaves complementary DNA strands) has the purpose of carrying out the cleavage of the complementary strand of dsDNA (double stranded

researchers toward the implementation of theoretical and applied biostrategies [7].

2. CRISPR/Cas9 mechanism: brief overview of its nature

Adapted from Wright et al. [4].

CRISPR systems are highly complex and diverse, and nowadays, efforts have been made to classify them into six interesting types: Type I (eight different Cas representative operons); Type II (tracrRNA; trans-activating crRNA and four Cas); Type III (eight Cas and Csm/Cmr); Type IV (four DinG/Csf); Type V (four Cas/Cpf2); and Type VI (three Cas/C2c2). In this case, operon type IV shows an extensive presence in the lack of CRISPR loci (Figure 1B) [4].

Due to the endless background that precede the functionality and applications of CRISPR systems in the field of genetic engineering, it has been shown that this metabolic phenomenon is extremely attractive for the molecularly directed crop improvement as well as plant genomic research. In general, the efforts that outline research for CRISPR systems within the agronomic Understanding CRISPR/Cas9: A Magnificent Tool for Plant Genome Editing http://dx.doi.org/10.5772/intechopen.81080 43

1. Introduction

42 Transgenic Crops - Emerging Trends and Future Perspectives

direct repeats).

Sustainable agriculture is considered to be the key for improving plant crops through genetic engineering, since random mutagenesis processes are part of conventional biotechnology techniques used for most researchers in this field [1]. CRISPR/Cas9 systems (clustered regularly interspaced short palindromic repeats-CRISPR associated) are related to a well-known CRISPR array defined by series of 20–50 bp genomic locus (i.e., unique spacers separated by

On the other hand, these unique spacers usually have similar length with preceded AT-rich fragments [2]. CRISPR loci were identified for the first time about two decades ago when they found a series of short genomic sequences (i.e., spacers) in Escherichia coli originated by viral genomes and probably due to the presence of conjugative plasmids, and in this sense, the foreign genetic material allowed the bacteria to record a kind of memory (e.g., immune system) to counteract future infections. When foreign DNA sequences match these unique spacers, they are commonly known as "photospacers" [1, 3]. Thereby, the corresponding immunization is against a foreign phage (e.g.), and when a new infection of this nature takes place in the future, the array expansion of the CRISPR is unchained and in consequence, new

According to some authors [4–6], this interesting immune system (to call it that) may be divided into three metabolic stages: adaptation, crRNA (CRISPR-RNAs) biogenesis, and interference. When a foreign DNA introduction happens, there is a selective process through the machinery that selects protospacers, which will be inserted into the CRISPR locus (insertion takes place into the leader end of the system). In the first stage of crRNA biogenesis, a transcription of CRISPR locus is observed followed by a direct processing of sequence elements (pre-crRNAs–crRNAs), all of them with the corresponding single spacer. After this stage, Cas proteins interact the crRNAs by assembling an effector complex (Figure 1A) [4]. This is very important since these components are intermediary elements of the interference stage where recognition of foreign DNA happens upon future infections and consequently, its degradation. It is very important to mention that a spacer acquisition creates genetic records of previous infections; as mentioned above, CRISPR immunity happens when there is an imminent detection of strange nucleic acids and consequently, the integration of foreign genetic

material into the host's cells (the DNA integration occurs in the chromosome).

CRISPR systems are highly complex and diverse, and nowadays, efforts have been made to classify them into six interesting types: Type I (eight different Cas representative operons); Type II (tracrRNA; trans-activating crRNA and four Cas); Type III (eight Cas and Csm/Cmr); Type IV (four DinG/Csf); Type V (four Cas/Cpf2); and Type VI (three Cas/C2c2). In this case,

Due to the endless background that precede the functionality and applications of CRISPR systems in the field of genetic engineering, it has been shown that this metabolic phenomenon is extremely attractive for the molecularly directed crop improvement as well as plant genomic research. In general, the efforts that outline research for CRISPR systems within the agronomic

operon type IV shows an extensive presence in the lack of CRISPR loci (Figure 1B) [4].

spacers originate from the genetic material of the phage.

Figure 1. Functional and organizational system of the CRISPR/Cas9. (A) The process of induced immunity is carried out in three stages consisting of an adaptation, crRNA biogenesis, and interference. In the first stage, the adaptive machinery performs a selection of photospacers, and they are leader-end inserted into CRISPR locus that is subsequently transcribed during crRNA biogenesis. In a complementary way, the pre-crRNA processing into crRNAs with simple spacers is developed. Finally, the effector complex is originated through the assembly of crRNA with Cas proteins, which interacts (interference stage) in a subsequent way to the infection, and consequent degradation of the foreign genetic material. (B) Structurally, the CRISPR system is divided into six representative types (operons). Dashed outlines represent genes in some subtypes. Pink sequences represent genes related to the interference process. Yellow sequences refer to crRNA biogenesis, and the green color refers to adaptation genes. Subtypes IV are characterized by the absence of CRISPR loci. Adapted from Wright et al. [4].

sciences field have been mainly focused on plant domestication with economic and social interest. Thus, interspaced short palindromic repeats constantly open the doors to the generation and knowledge application in the area of functional genomics that jointly guide researchers toward the implementation of theoretical and applied biostrategies [7].

## 2. CRISPR/Cas9 mechanism: brief overview of its nature

The action mode of CRISPR/Cas9 biological system is basically based on the participation of two distinct elements: the Cas9 protein (CRISPR-associated protein 9; RNA-guided DNA endonuclease enzyme associated with CRISPR) and sgRNA (single guide RNA) [8]. Nowadays, Cas9 proteins are found mainly in different bacteria species such as Brevibacillus laterosporus [9], Staphylococcus aureus [10], and other representative species within the genus Streptococcus [11]. Cas9 proteins have shown two representative domains: the first one is known as HNH (nuclease-associated proteins), which is responsible for cleaving and regrouping the complementary strand of crRNA. On the other hand, the second domain known as RuvC-like (nuclease domain that cleaves complementary DNA strands) has the purpose of carrying out the cleavage of the complementary strand of dsDNA (double stranded DNA). The nature of the sgRNA is extremely curious, since this is a kind of synthetic RNA with a length not greater than 100 bp and whose structure owns a 20 bp sequence coupled to the 5<sup>0</sup> -end that works as a guide allowing the identification of target sequences through specific adjacent motifs (i.e., PAM sequences; protospacer adjacent motifs) [8].

Several studies have shown that Cas9/RuvC domains have very similar structures to retroviral integrases (virally encoded; specialized recombinases capable of catalyzing the recombination of viral DNA particles into the genome's host cell). In contrast to the above, the HNH nuclease domains show a fold-type ββα structure linked to a metallic cofactor that in the same way, it is linked to another HNH domain from a different endonuclease that allows the recognition through metallic ions in order to locate cleavage sites on the target DNA sequence [18]. Metallic ion-dependent restriction enzymes show highly conserved structures formed by

Understanding CRISPR/Cas9: A Magnificent Tool for Plant Genome Editing

http://dx.doi.org/10.5772/intechopen.81080

45

In basal conditions, the nature of Cas9 enables the enzyme to be inactive and when a recombination between sgRNA and the corresponding REC lobe is observed, it is precisely that this natural state changes. Thus, the previous complex performs a specific search for PAMs (trinucleotide NGG) in order to identify target sequences into the double DNA strand. When a linkage has happened within the respective PAMPS, a cleavage of the hybrid DNA-RNA complex takes place thanks to HNH domain and, jointly, RuvC assists the structuring of dsSDBs (double-stranded SDBs) by cleaving the corresponding complementary sequence [21]. It is important to mention that both eukaryotic and prokaryotic cells show NHEJ and HDR mechanisms capable of repairing DSBs through the intervention of DNA ligase IV, whose nature helps regroup damaged nucleotide ends (Indels; introduction or deletion of mutations) as well as the use of complementary homologous DNA templates,

Within the group of Cas9 proteins, there are repression effectors that are fused with a transcription activation system called dCas9 (CRISPR tool based on a modified version of the Cas9 protein). The dCas9 systems are usually combined with effector protein domains that regroup functional peptides that will target specific regions of genome loci. Thus, the resulting complex performs activation and shutdown mechanisms of gene repression, thereby; it is considered an efficient regulator of genetic information flow. This is why CRISPR/dCas9 system may be a modular platform in several cellular processes to control transcription [21, 23]. Another important fact about the dCas9 complexes is that they are able to combine with different epigenetic nature molecules, such as methylation and histone

On the other hand, specificity is a considerable element in genome editing tools. In the case of Cas9/gRNA (guide RNA) complexes, they have an extremely precise capacity to develop cleavages in DNA sequences, even when small mismatches may be observed into the guide template [25]. When this type of phenomenon happens, nonspecific cleavages are mostly

has been noticed that short gRNAs (a.c. 20 bp) confer better specificity to Cas9 proteins at the target cleavage sites within the genetic editing processes [26]. Likewise, when the inactivation of Cas9 conserved domains is observed, a specific break occurs in one of the DNA strands (nickase), which leads to a splitting and loss of its double-stranded native structure [27]. In general, this type of nicks usually causes no mutations since they can be repaired in a very simple way by eliminating damaged bases through a specific repair


aspartate residues and in less quantity by histidine [20].

respectively [21, 22].

peptides [24].

tolerated at the 5<sup>0</sup>

metabolic pathway.

It is important to mention that 3<sup>0</sup> -end of the sgRNA resembles a loop structure that allows it to develop a very precise linkage with the target sequence. This is the way to structure a new complex that will be associated with Cas9 and in this sense, to perform the dsDNA cleavage that will cause double-stranded breaks (DSBs) [12]. Generally, a DSB is the result of the continuous DNA damage at chromosome level, although this is considered a completely normal phenomenon within the cell. However, the resulting by-products generated by the cellular metabolism itself such as reactive oxygen species (ROS) may interfere in the replication process due to the damage caused in the DNA. Also, environmental selective pressures as different chemical agents or UV light itself are considered other important factors involved in this process [13, 14].

When the above phenomenon has been carried out within the cell, the presence of DSBs activates the repair mechanism of damaged DNA through nonhomologous end joining (NHEJ) or homology-directed repair (HDR). In most cases, the repair of DBSs is carried out by NHEJ although the main reason why this happens is because it is the best way to make genetic insertions or deletions and consequently to give rise to a gene knockout (gene knockout is a genetic phenomenon through which an organism's gene becomes inoperative). In general, HDR is originated by the presence of an oligo template, and it activates the elimination of specific genes as well as foreign DNA (the mechanism involves the substitution of DNA sequences in a specific locus, or well, fragment sequences not found within this locus) [8, 12–15]. In addition to CRISPR, there are other methods currently used for genome editing which include the participation of peculiar endonucleases such as transcription activator-link effectors (TALEN) and zinc fingers. Through these mechanisms, a fusion between DNA-binding domains of transcription factors and the nuclease domain FokI (restriction enzyme) takes place. Beyond the application of CRISPR systems in genome editing and their regulation processes, these types of endonucleases may be used to be fused with fluorescent proteins in order to allow more specific loci location within the living cells [16, 17].
