**2.4 Clustered regularly interspaced short palindromic repeat/CRISPR/Cas9 nuclease system**

The CRISPR-Cas system, like TALENS, draws its inspiration from biology. The CRISPR-Cas nucleases had first been found in the adaptive immune system function of archaea and bacteria. CRISPR codes for "spacer" RNA molecules that create associations between CRISPR-associated (Cas) nucleases and instruct them to break down external nucleic acids. The spacer segments within those biological systems were obtained from bacteriophage components caused by a bacterium the prokaryote progenitor. The system's aiming specialization is based upon basic genetic platform principles. A lateral or segmental short sequencespecific element is necessary for a target sequence site to be accepted and bisected; the above offers assurance that the prokaryote utilizing the CRISPR/ Cas system will not focus its genome; these representations are just not available among exogenously linked patterns. The *Streptococcus pyogenes* CRISPR-Cas9 system (CRISPR-*Sp*Cas9) was the first to be discovered for genetic manipulation, and also the term "CRISPR-Cas9" is generally used to describe this technology [32–34]. To avoid misunderstanding, CRISPR-Cas9 can only be used to relate to properties that CRISPR-SpCas9 and its orthologs share. The designed CRISPR-Cas9 system is made up of two parts—(1) the Cas9 nuclease and (2) a single guide RNA (sgRNA), which is made up of two RNA molecules—the spacer-containing CRISPR RNA (crRNA), and they serve to enhance crRNA (tracrRNA), which itself is needed again for retired moiety's growth and development. The sgRNA

#### **Figure 1.**

*Home page of CHOPCHOP bioinformatics tool for designing of sgRNA.*


#### **Figure 2.**

*Preliminary stage for the identification of off target regions of our desired genomic sequence.*

#### **Figure 3.**

*Intermediate stage for identification of off target regions of our desired genomic sequence. In this view, we can identify the %GC, levels of off targets and primer sequences.*

leads the nuclease combination to a specific DNA location, causing the identical nucleotide sequence to be cleaved [20, 21]. Cas9 has a composite morphology, having dual nuclease domains, RuvC and HNH [14, 20, 35]. The CRISPRCas9 system has gone through several modifications in the area of plant genome engineering methods [36], including simplicity of design, cloning, and transport into *Enhancement of Agricultural Crops: A CRISPR/Cas9-Based Approach DOI: http://dx.doi.org/10.5772/intechopen.100641*


**Figure 4.**

*Final stage of off target analysis or our desired genomic sequence.*

plant cells, which also has resulted in a significant success rate in genome editing technology. The design of guide RNAs using the CHOPCHOP software tool is summarized in **Figures 1–4**.
