*3.3. Genome Editing Mechanism of CRISPR*/*Cas9 System*

The mode of GE is established by the healing process of the genome. After the identification of the target site, Cas9 allows sgRNA to pair with the target DNA sequence. The Cas9 endonuclease is composed of the HNH and RucV-like domain, which cuts the target DNA strands three to four bases upstream of the PAM site. The HNH domain cuts the complementary DNA strands while the RuvC domain cleaves the non-complementary to gRNA. The blunt-ended DSBs can be repaired by the HDR and NHEJ repair pathways (Figure 5D). The NHEJ is error prone and causes DNA insertion or deletion at the target sequence [23]. The expression of sgRNA as pair, NHEJ mechanism came up with large deletions. The large deletions in chromosomes were attained by the NHEJ mechanism utilizing co-expressed sgRNAs. The HDR repair mechanism is only operational when a specific homologous target site is available with respect to the DSB site. In plants, through GE, many outstanding repairs were achieved via HDR, such as gene replacement, DNA correction, and targeted knock-in [93,94].

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 13 of 49

**Figure 5.** Illustration of CRISPR/Cas9-mediated GE. The CRISPR/Cas9 system is composed of sgRNA and Cas9. (**A**) sgRNA with a guide sequence (colored pink) is developed by the combination of protospacer with crRNA and tracrRNA. (**B**) Cas9 machinery combines with sgRNA to form a complex to trigger CRISPR/Cas9 editing. The Cas9 nuclease consists of two parts, depending on its function and structure. The recognition site identifies the target DNA and interacts with sgRNA. The nuclease site contains two domains RuvC-like and HNH which cleave the target DNA site non-complementary by the RuvC domain and complementary by the HNH domain to the gRNA. (**C**) The Cas9 nuclease detects the genomic target site (indicated with blue color) having a 20 bp target sequence that is homologous to seed or guide sequence (indicated with pink color), which is crucial for Cas9 activity and specificity. The specific PAM sequence (indicated with red color) is detected by Cas9: sgRNA complex and DSBs created by the Cas9 endonuclease three base pairs upstream of the PAM sequence. (**D**) Targeted mutagenesis of a desired gene is achieved by filling the DSB (indicated with black color) by means of the HDR or NHEJ mechanism. The NHEJ repair mechanism generally produces insertion (indicated with yellow color), deletion (indicated with brown color) or indels (indicated with black line) at the break point, generating targeted mutants. The HDR repair mechanism uses a template DNA sequence for homologous recombination to produce gene replacement or gene insertion (indicated with green color). **Figure 5.** Illustration of CRISPR/Cas9-mediated GE. The CRISPR/Cas9 system is composed of sgRNA and Cas9. (**A**) sgRNA with a guide sequence (colored pink) is developed by the combination of protospacer with crRNA and tracrRNA. (**B**) Cas9 machinery combines with sgRNA to form a complex to trigger CRISPR/Cas9 editing. The Cas9 nuclease consists of two parts, depending on its function and structure. The recognition site identifies the target DNA and interacts with sgRNA. The nuclease site contains two domains RuvC-like and HNH which cleave the target DNA site non-complementary by the RuvC domain and complementary by the HNH domain to the gRNA. (**C**) The Cas9 nuclease detects the genomic target site (indicated with blue color) having a 20 bp target sequence that is homologous to seed or guide sequence (indicated with pink color), which is crucial for Cas9 activity and specificity. The specific PAM sequence (indicated with red color) is detected by Cas9: sgRNA complex and DSBs created by the Cas9 endonuclease three base pairs upstream of the PAM sequence. (**D**) Targeted mutagenesis of a desired gene is achieved by filling the DSB (indicated with black color) by means of the HDR or NHEJ mechanism. The NHEJ repair mechanism generally produces insertion (indicated with yellow color), deletion (indicated with brown color) or indels (indicated with black line) at the break point, generating targeted mutants. The HDR repair mechanism uses a template DNA sequence for homologous recombination to produce gene replacement or gene insertion (indicated with green color).

*3.3. Genome Editing Mechanism of CRISPR/Cas9 System*  Biolistic and *Agrobacterium*-mediated transformation can be applied to transfer the sgRNA and Cas9 protein into desired cells [95]. GE by CRISPR/Cas9 is heavily dependent on the choice of sgRNA promoters and ubiquitous expression of the Cas9 enzyme. Universal *CaMV35S* RNA polymerase II promoters have been extensively used for Cas9 expression in plants. Similarly, for sgRNA expression, U3 or U6 RNA pol III promoters are applied [96]. The expression level of sgRNAs is significantly greater in endogenous promoters as compared to exogenous promoters [97]. Moreover, sgRNA expression is guided by U6 promoters which were derived from monocotyledonous or dicotyledonous varieties and can only be used in monocot or dicot plants [98]. For successful integration of CRISPR/Cas9 machinery in plant nuclei, Cas9 proteins must join with nuclear localization signals [96].

To bind the target DNA by synthetically developed short gRNA sequences of approximately 20 nucleotides, the mechanism of CRISPR/Cas editing demands the PAM 50 NGG motif for the Cas9 enzyme to cleave 3–4 bases in the target DNA sequence after the generation of the protospacer [9]. There are two domains of Cas nucleases which have the ability to cut one strand of DNA like the HNH domain and RuvC-like domain. Simple steps involving the execution of the CRISPR mechanism are recognition of the PAM sequence; sgRNA development; cloning of sgRNA; transformation into the host cell; selection of transformed individual organisms; and edited lines confirmation, as described in Figure 6. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 19 of 49

**Figure 6.** Basic steps in the workflow of CRISPR/Cas9-based genome editing. **Figure 6.** Basic steps in the workflow of CRISPR/Cas9-based genome editing.

To overcome the limitations associated with mutant screening, different techniques have been developed, including annealing at critical temperature polymerase chain reaction (ACT-PCR) [136],

genotyping differences. A mutant can be detected rapidly when it has a clear evident phenotype. For example, a visible albino phenotype was observed when a gene phytoen desaturase mutated via the CRISPR/Cas9 system. It was applied as a phenotypic marker to detect rice- and tobacco-edited plants [141,142]. Additionally, transgenic plants can also be screened using some herbicide/antibiotic selectable markers [141,143]. But making a connection among visual phenotypes and targeted genes is the only challenge associated with phenotyping-mediated screening [135]. In other approaches,
