**3. CRISPR/Cas9 exotic variants and challenges**

CRISPR/Cas9 genome editing utilizing the SpCas9 enzyme from *Streptococcus pyogenes* continuously transforming the area of genome editing by offering very accurate, simple, and highly efficient gene alterations by creating nicks on the double-stranded genome of the targeted organism. Since 2013 scientists have extensively used and still exploring its vast possibilities in genome editing. Even though the efficiency of Cas9 is still high, there are some setbacks regarding their use in gene editing. One of the limitations of using them in plant biotechnology is the indistinct regulations using CRISPR/Cas9 edited plants. Another drawback is the unavailability of a standardized transformation protocol to deliver the CRISPR/Cas9 construct to some plants. These problems are now solved by the availability of novel exotic variants of genome editing enzymes that have been tested as equally efficient or perhaps more efficient than *Sp*Cas9 [37]. Scientists have identified and characterized many other kinds of microbial communities; CRISPR-RNA-guided adaptive immune systems are found. Two primary classes are distinguished, with five types and 16 subclasses [38]. These enzymes require multisubunit proteins to bind to crRNA and cleave the target genome, which is all found in class 1. "Class 2" consists mostly of two types of effectors—Type II and Type VI. Each of these types of effectors binds and cleaves crRNA and target nucleic acids. Cpf1 [38, 39], C2c1 or C2c3 [40], and C2c2 with two HEPN RNase (higher eukaryotes and prokaryotes nucleotide-binding) domains are used in Class 2 Type V and Class 2 Type VI, respectively. In contrast, Class 2 Type II is characterized by Cas9, RuvC, and HNH nuclease domains, while Class 2 Type V uses a single Ruv [41]. Some of these effectors have experimented with some plant species.

The discovery of RNA-dependent RNase enzyme systems from Class 2 Type II (FnCas9) and Class 2 Type VI (C2c2) cleared the path for novel approaches to genome editing. The bacterium Leptotrichiashahii Class 2 Type II C2c2 is directed by a single crRNA and may be trained to cleave any ssRNA with corresponding protospacers. These effectors, which are composed of two HEPN domains containing catalytic residues, preferentially cleave ssRNAs at varying distances from the crRNA binding site rather than adenine targets. C2c2 binding is controlled by a crRNA secondary structure with at least one 24-nt stem-loop structure and a 22–28-nt complementary sequence to these RNA protospacers. The latter must be flanked at the 3'end by a mononucleotide protospacer-flanking site (PFS) comprised of adenine, uracil, or cysteine [41, 42]. Another RNasebased system was identified in 2013 [43] from microbe *Francisellanovicida* (*Fn*Cas9), which could target bacterial mRNA and lead to altered gene expression and is PAM independent. This enzyme successfully inhibited the hepatitis C virus (HCV) in Huh-7.5 cells by RNA inhibition method. This enzyme targets

both positive and negative strands of the virus, thus paralyzing RNA translation and replication. It was shown that mismatches up to three to six base pairs at 3'or 5′ end were tolerated by *Fn*Cas9 whereas more than six mismatches led to complete loss of activity. This enzyme is also capable of targeting DNA [44]. The above studies suggest the feasibility of developing viral infections resistant crops. The regulatory policies related to the usage of transgenic plants are still going very strong in many countries. To overcome this problem, smaller versions of genome editing enzymes are developed that can be used along with viral vectors to transform plants with desired traits. Virus vectors allow high and transient expression of heterologous genes for editing. This is proved in the case of targeted mutagenesis of *Nicotiana benthamiana* and *Petunia hybrida* using tobacco rattle virus (TRV) [45].

As *Sp*Cas9 is having a larger size (4.2 kb), the tobacco rattle virus cannot be used to express *Sp*Cas9 in plants. To resolve this problem, small genome editing enzymes were identified from different microbes such as *Staphylococcus aureus* (*Sa*Cas9, 3.2 kb), *Streptococcus thermophilus* (*St*1Cas9, 3.4 kb), and *Neisseria meningitidis* (*Nm*Cas9, 3.2 kb). These enzymes belong to the Class 2 Type II immune system and cleave double-stranded DNA using RuvC and HNH domains. Moreover, this group of enzymes cuts DNA at a specific target region, usually 21- to 24-nt long near 5′-NNGRRT-3′ or 5′-NNNRRT-3′ 5′-NNAGAAW-3′and 5′-NNNNGMTT-3′ PAM motifs, respectively. Here, in the PAM sequence, N signifies any nucleotide, R signifies A or G, M signifies A or C, and W signifies A or T [46–49]. In addition, research suggest that while using *Sa*Cas9, a greater rate of mutation (80%) was obtained by targeting the 5′-NNNGGT-3′ PAM sequence and induced homologous recombination in the selected lines. The above enzymes target a much longer PAM sequence for genome editing purposes. As an alternative, a new set of single crRNA-guided DNase enzymes with shorter PAM motifs have been recently identified again from the microbial community. This also belongs to Class 2 Type V CRISPR effectors Cpf1 from *Francisellanovicida* U112 (*Fn*Cpf1), *Acidaminococcus* sp. (*As*Cpf1), and *Lachnospiraceae* bacterium (*Lb*Cpf1) and have been successfully tried in rice and tobacco. *Fn*Cpf1 uses a single short RNA guide molecule, 42- to 44-ntcrRNA, which begins with 19 nt of the direct repeat followed by 23–25 nt of the spacer sequence. *Fn*Cpf1 identifies5′- TTN-3′region, a short T- rich PAM upstream of the 5′end. Further, it cuts the double-stranded DNA in a staggered way after the 18th base on the nontargeted (+) strand and after the 23rd base on the targeted (˗) strand [50]. Targeted mutations were observed in *NtPDS* and *NtSTF1* of *N. benthamiana* and *Os*DL, *Os*ALS, *Os*NCED1–3, and *Os*AO1–5 loci of *Oryzasativa* when codon-optimized *Fn*Cpf1 and crRNA were expressed in rice and tobacco. Interestingly, deletions were observed in both the transgenic plants as well as in transformed progenies, and mutation efficiency in rice and tobacco was around 47.2 and 28.2%, respectively [50].

Many new versions of *Sp*Cas9 have been developed with the core objective to enhance their specificity. One of the limitations that have come across was offtargeting, which will cause undesired mutations in the target. The next drawback is that some plant species have larger genomes with many duplicate genes, making genome editing technology less precise. The first *Sp*Cas9 variant that was obtained by mutating one of its domains (HNH or RuvC) was single-stranded DNA cleavage *Sp*Cas9-nickases [35, 51]. SpCas9-nickases are employed in pairs to carry out nonhomologous repair of double-stranded breaks (DSBs) using properly offset (>100 bp long) guide RNAs [47]. This strategy decreases off-target mutagenesis by extending the recognized DNA target area from 23 to 2 9 23 bp while

#### *Enhancement of Agricultural Crops: A CRISPR/Cas9-Based Approach DOI: http://dx.doi.org/10.5772/intechopen.100641*

maintaining an on-target cleavage rate comparable to that of wild-type SpCas9 [47]. In *Arabidopsis*, a single SpCas9 D10A nickase was equally efficient at initiating homologous recombination as a nuclease or homing endonuclease I, SceI, [52]. On the other hand, coupled SpCas9 nickases generated alterations comparable to those induced by SpCas9 nuclease. Furthermore, deletions were detected, not insertions, which occurred at a lower frequency [53]. Slaymaker et al. enhanced the specificity of SpCas9 by decreasing its helicase activity and created an improved form of SpCas9 (eSpCas9) [54]. Wright et al. created a split-SpCas9 system, a binary SpCas9 system, to enhance SpCas9 specificity. This was accomplished by overexpressing the nuclease and a-helical lobes in *Escherichia coli* as distinct polypeptides [55]. Komor et al. used a different approach to enhance the specificity of SpCas9, combining SpCas9-nickase with cytidine deaminase to create SpCas9-CD. While all other SpCas9 variations cause deletions or insertions in the DNA sequence, this variant enables the direct conversion of cytidine to uridine, which has the same base-pairing properties as thymine [56].

Another variant of *Sp*Cas9 is termed dead Cas9 (d*Sp*Cas9), which is developed by mutating both cleavage domains of *Sp*Cas9, and this enzyme is an RNA-guided DNA binding protein without cleavage activity [14]. In addition, it is fused with fluorescent or other types of markers and can be used in several biotechnological applications. This kind of fusion creates catalytically inactive and dead *Sp*Cas9 having the FokI nuclease domain at the N-terminus [57]. Compared to monomeric *Sp*Cas9, homodimer FokI enzymes are more precise in cleaving the target genome and can induce lesser off-targets. Piatek et al. demonstrated the fusion of synthetic transcriptional activators with the C terminus of d*Sp*Cas9 to the EDLL domain or the TAL activation domain. They developed d*Sp*Cas9—EDLL and d*Sp*Cas9—TAD synthetic transcriptional activators. This effector, guide RNA, and target molecules were transformed to *tobacco* through the agroinfiltration method. Though there were no stably transformed lines, the strong transcriptional activity of EDLL and TAD was proved in transgenic plants [58]. Fusion of d*Sp*Cas9 with methylated or demethylated promoters can lead to activation or inactivation of a gene. Some of the examples of this type of fused protein are d*Sp*Cas9-Tet1 and d*Sp*Cas9-Dnmt3a [59]. The deletion and insertion of methylases using CRISPR/Cas9 technology will allow modifications at the genetic level in living organisms [1].
