(tracrRNA) to activate and guide Cas9 nuclease (Barrangou et al. 2007). The Cas9 cleaves the homologs double‐stranded DNA sequence (protospacer) in the foreign DNA. The availability of PAM downstream of the target DNA is a key for the successful cleavage with frequent 5′‐NGG‐3′ and less frequent 5′‐NAG‐3′ (Hsu et al. 2013). Seed sequence approximately 12 bp upstream of the PAM are integral for pairing between RNA and target DNA (Bortesi and Fischer 2015). Multiplex editing by the introduction of double‐stranded breaks (DSBs) on various sites and ability to edit several genes at the same time is a unique feature of CRISPR/Cas system (Zhou et al. 2014). It only requires monomeric Cas9 protein with sequence‐specific gRNA meanwhile, ZFNs and TALENs requires dimeric proteins with reference to target site. In the CRISPR/Cas system, the recognition of target site is based on the Watson and Crick model enabling the off‐target detection through sequencing analysis along with off‐target mutation can be fixed by careful design of gRNA. On the contrary, ZFNs and TALENs target specificity depends on protein‐DNA interaction with unpredictable and function‐specific properties (Cho et al. 2014). Moreover, the scientific community equipped with CRISPR expertise had contributed a lot to disseminate the fresh information to newcomers which is opposite to ZFN proprietary nature. Several online platforms are now available and are assisting researchers for all concerns related to CRISPR (Table 3.1; Figure 3.1).
3.4 Application of CRISPR/Cas System for Crops Quality Improvement
The improvement in quality is as important as yield and plant breeding programs are focusing on the improvement of grain quality. The grain quality is multigenic in nature and controlled by a number of factors. The demand for better quality grain is increasing owing to improvement in grain‐processing techniques, environmental variations and consumers’ preferences. The breeding to develop semi‐dwarf cultivars and hybrids of various crops have markedly increased production with a compromise on quality. The advancement in genomics studies for grain quality has further improved the understanding of genes and biosynthesis pathways controlling essential features of grain quality. The complex network of genes controlling quality attributes can be resolved by understanding functional and comparative genomics of major genes and their faithful introgression across cultivars. The targeted mutagenesis through the CRISPR/Cas system has been successfully adopted in several crops (Fiaz et al. 2019) for traits including biotic and abiotic stresses, yield and quality improvement. The series of published articles on the following system is considered as proof of concept describing the application of CRISPR/Cas system for the knockout of genes controlling desirable phenotypes. A generalized protocol for the application of CRISPR/Cas system in several crop species has been described with the potential to be employed for improvement of quality of important crops (Figure 3.2).
Table 3.1 Online platform for tools of CRISPR/Cas system.
Source: Extracted from (Bortesi and Fischer 2015; Chira et al. 2017).
| sgRNA design platform | Webpage link | References |
|---|---|---|
| CRISPR Design | http://crispr.mit.edu | Hsu et al. (2013) |
| E‐CRISP | http://www.e‐crisp.org/E‐CRISP | Heigwer et al. (2014) |
| CRISPR Multi Targeter | http://www.multicrispr.net/basic_input.html | Prykhozhij et al. (2015) |
| sgRNA designer: CRISPRko | http://portals.broadinstitute.org/gpp/public/analysis‐tools/sgrna‐design | Doench et al. (2016) |
| Off‐Spoter | https://cm.jefferson.edu/Off‐Spotter | Pliatsika and Rigoutsos (2015) |
| CCTop | http://crispr.cos.uni‐heidelberg.de/index.html | Stemmer et al. (2015) |
| CHOPCHOP | http://chopchop.cbu.uib.no/index.php | Montague et al. (2014) |
| Plasmid free access | ||
| Add gene | https://www.addgene.org/search/advanced | Bortesi and Fischer (2015) |
| Expert discussion forum | ||
| OXfCRISPR | https://www.dpag.ox.ac.uk/research/liu‐group/liu‐group news/oxfcrispr/ | Bortesi and Fischer (2015) |
3.4.1 Rice
Rice (Oryzasativa L.) is a major cereal and a source of calories and protein to the vast population around the globe. It has a diploid genome (2n = 24); the small genome size makes it a model crop among monocots to investigate for research objectives (Fiaz et al. 2019). It has been reported that approximately, 499.37 million tons of rice was produced around the globe during year 2019 (FAO (Food and Agriculture Organization of The United Nations) Statistics 2017–18). The prime attributes influencing quality include cooking and eating, phytochemicals and micronutrients‐related characters (Lau et al. 2015). People from different ethnic groups and/or geographical regions have different grain‐quality preferences (Sabouri et al. 2012). Rice of premium quality like Indian basmati and Thai Jasmine are highly valued due to their typical aroma during cooking (Ferrero 2004). Rice grain quality parameters are very complex and easily affected by the environment. The key components which have impact on grain quality are (i) appearance quality (ii) milling quality (iii) eating‐cooking quality and (iv) nutritional quality which are further divided into many other categories (Lou et al. 2009). From the last few years, much research has demonstrated the successful application of CRISPR‐based targeted mutagenesis approach in rice to make efficient and reliable crop quality improvement.
