3. Cas9 enzyme: multifunctional DNA endonuclease

The Cas9 enzyme has a very peculiar structure since it consists of two distinct nodes complemented each other: an alpha-helical recognition site (REC) and an endonuclease structure (NUC) containing HNH proteins. On the other hand, Cas9 structure has RuvC-like and Cterminal domains with variable structures [18]. Both nodes are linked through anchoring-like structures mostly conformed by arginine-rich bridges and other less complex structures generally observed between residues 712–720. In this sense, there are three very important domains with alpha-helical structure that integrate the REC node (Hel-I, II, and III) and as an interesting fact; Hel domains show no structural similarity with some other known proteins to date [19].

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

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

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

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

The Cas9 enzyme has a very peculiar structure since it consists of two distinct nodes complemented each other: an alpha-helical recognition site (REC) and an endonuclease structure (NUC) containing HNH proteins. On the other hand, Cas9 structure has RuvC-like and Cterminal domains with variable structures [18]. Both nodes are linked through anchoring-like structures mostly conformed by arginine-rich bridges and other less complex structures generally observed between residues 712–720. In this sense, there are three very important domains with alpha-helical structure that integrate the REC node (Hel-I, II, and III) and as an interesting fact; Hel domains show no structural similarity with some other known proteins to date [19].

specific adjacent motifs (i.e., PAM sequences; protospacer adjacent motifs) [8].



the 5<sup>0</sup>

It is important to mention that 3<sup>0</sup>

44 Transgenic Crops - Emerging Trends and Future Perspectives

this process [13, 14].

within the living cells [16, 17].

3. Cas9 enzyme: multifunctional DNA endonuclease

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, respectively [21, 22].

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 peptides [24].

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 tolerated at the 5<sup>0</sup> -ends or at a greater distance from the corresponding MAMP sequences. It 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 metabolic pathway.
