*4.3. Bioinformatics Tools for Designing the CRISPR*/*Cas9 Construct*

One of the most crucial steps for highly precise GE is to design an sgRNA construct for CRISPR/Cas9. To date, numerous bioinformatics tools have been developed and are available online for sgRNA designing. There are many accessible online tools with plant databases and which permit the design of sgRNA for identification of new target sites [100], as shown in Table 2. For example CRISPR Design (http: //www.genome-engineering.org) was developed by Zhang and colleagues for designing sgRNA and it also assists in assessing off-target mutation [101]. In 2014, Xie and coworkers successfully developed a web tool named CRISPR-PLANT (http://www.genome.arizona.edu/CRISPR) in order to design efficient sgRNA constructs for CRISPR/Cas9-based GE [102]. For example, a novel web tool was developed by Michano and colleagues for rapid detection of target loci in soybean for CRISPR/Cas9-mediated GE [103]. Similarly, CRISPR-P (http://cbi.hzau.edu.cn/crispr/) eases the designing of sgRNA for every plant having an available sequenced genome and also helps to evaluate off-targets [104].


**Table 2.** List of various single guide RNA (sgRNA) designing bioinformatics tools for the CRISPR/Cas9 system.

## *4.4. Construction of the sgRNA Expression Cassette*

Construction of a unique sgRNA expression cassette is the most important step in CRISPR/Cas9-mediated gene editing, and it works as a guide system for the Cas9/sgRNA complex consisting of 98 nucleotides with a 20 nucleotide target sequence [92]. In plants, RNA polymerase III is used to transcribe sgRNA and its expression is mostly governed by U3 or U6 promoters [93]. As the expression cassettes of sgRNA:U3/U6 promoters are very small in length, approximately ~300–600 bp, overlapping PCR or adaptor ligation can be applied to construct these expression cassettes [123]. In 2015, Ma and colleagues developed a robust cloning-free approach for sgRNA expression cassette development based on the PCR technique. Gibson assembly or the Golden Gate cloning strategy was used for direct cloning of sgRNA expression cassette into binary vectors for the CRISPR/Cas9 system [24]. In another approach, Gao and Zhao utilized a ribozyme mechanism to generate sgRNA by transcription of pre-RNA through RNA polymerase II, whereby inducible or constitutive promoters can be ligated to obtain the desired function of sgRNA [124].

#### *4.5. Construction of Cas9 Expression Cassettes*

Cas9 is composed of 4107 bp of coding sequence. For Cas9 nuclear localization in eukaryotes, the Cas9 coding sequence must be fused with the nuclear localization signal. Plant usage–bias codons have been used to design highly efficient and optimized Cas9 expression cassettes for improved GE in plants [125]. For example, utilization of codon-optimized Cas9p in rice and Gramineae family has been improved by enhancing GC contents [24], which imitate the genes from the Gramineae family [126]. Commonly, constitutive promoters like 35S *Cauliflower mosaic virus* (CaMV) and ubiquitin from *A. thaliana*, rice, and maize can govern the expression of Cas9 in dicots and monocots for highly targeted gene editing using callus-based transformation approaches.

## *4.6. Transformation Approaches for CRISPR*/*Cas9-Based Vector Delivery into Plants*

For CRISPR/Cas9-mediated GE, cargo–vector harboring the expression cassettes of both sgRNA and the *Cas9* gene must be carried to targeted sites in plant cells. For cargo–vector transformation, floral dip and biolistic approaches are generally executed. Nowadays, advanced strategies like ribonucleo-protein complex, plasmid delivery, and virus-mediated delivery systems are applied for plant transformation. There are certain limitations in using the virus-mediated delivery system, but several studies have been carried out in plants using the virus delivery system [127,128]. A transient expression system is commonly used by researchers to transfer the vector into protoplast for analyzing the efficiency and feasibility of the CRISPR/Cas9 toolkit [129]. Biolistic and PEG-mediated transformation techniques can be used for direct delivery of *Cas9* gene expression cassettes [130]. However, it is difficult to regenerate plants from protoplast due to the heritable targeted mutations, which poses a major drawback associated with this approach in many plant species. *Agrobacterium*-mediated transformation is a highly efficient approach for stable transformation of the CRISPR/Cas9 system in dicot and monocots [131,132].

#### *4.7. Strategies for Mutant Screening*

The CRISPR/Cas9 system is a groundbreaking innovation in GE technology for developing desired mutants and numerous mutant libraries have been created by the CRISPR/Cas9 system so far, such as the genomic-scale mutant library for tomato [133] and rice [134,135]. As the applications for GE approaches are increasing day by day, scientists are required to screen huge numbers of mutants, using a strategy that includes the detection of off-target and on-target edits and which later removes the transgenes in edited plant off-springs.

To overcome the limitations associated with mutant screening, different techniques have been developed, including annealing at critical temperature polymerase chain reaction (ACT-PCR) [136], high-resolution melting analysis (HRMA) [137], polyacrylamide gel electrophoresis (PAGE)-mediated genotyping [138], T7 endonuclease I (T7EI) approach [139], and restriction enzyme site loss technique [140]. There are certain pros and cons for each technique and they are centered on 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, high-throughput sequencing is highly efficient and precise strategy to screen all the mutants generated by the CRISPR/Cas9 system [144]. For the detection of DNA-free plants edited by CRISPR/Cas9, whole genome sequencing is quite beneficial and helpful [145].
