**4. Applications**

CRISPR/Cas9-based genome editing has contributed a lot in improving various traits of crops for the past years. Still, new challenges are being faced by the scientific community for the enhancement of the quality of various edible plants. There are numerous applications of CRISPR/Cas-based gene editing, namely enhancement of yield, improvement of resistance to pathogens, diseases, resistance to herbicides, and improvement of stress tolerance. In this chapter, details of studies related to the applications mentioned above of CRISPR are given and summarized in **Table 1**. Most of the CRISPR-related work in vegetables is done in tomatoes. The earliest report based on CRISPR-based genome editing was done by editing the *ARGONAUTE7* (*SlAGO7*) gene which is involved in the development of leaves. Moreover, mutated *SlAGO7* caused a change in the morphology of leaves which turned them into wiry leaves [77]. Another interesting work is to edit *SELF PRUNING 5G (Sp5G)* and *SELF PRUNING (Sp)* genes that caused early flowering [78]. Similar editing in developmental genes was also carried out


#### **Table 1.**

*Applications of CRISPR/Cas9 genome editing in various crops for improved traits.*

*Brassica oleracea* [79] and *Lactuca sativa* [80]. Many genes involved in the biosynthesis of carotenoids such as *Anthocyanin 1* (*ANT1*) are involved in anthocyanin biosynthesis [81], *Phytoene desaturase* (*SlPDS*), *Phytochrome interacting factor* (*SlPIF4*) [82], and *Phytoene synthase* (*PSY1*) [83]. Mutant tomato plants with the knockout of the *Slagamous-like 6* (*SlAGL6*) gene produced parthenocarpic fruits under heat stress conditions that otherwise rigorously hinder fertilizationdependent fruit set [84]. Silencing the *eIF4E* gene in tomatoes and melons has attained resistance to RNA virus [85, 86]. Granule bound starch synthase (*GBSS*) gene using CRISPR/Cas9 that produced amylopectin and lacks amylose [87]. CRISPR/Cas9 mediated mutagenesis of genes involved in starch biosynthesis in sweet potato (*Ipomoea batatas*) was done for improving the quality of starch [88]. CRISPR/Cas9 induced mutation of CaERF28 conferred resistance to anthracnose in chili pepper (*Capsicum annuum* L.) [89].

Regulating homeostasis is a reliable way to enhance the yield of cereal. Genome editing of the C terminus of *LOGL5* in rice which codes for the cytokininactivation enzyme increases the yield in rice during all ecological conditions [90]. Production of a high-yielding wheat variety was done by editing the gene that encodes cytokinin oxidase/dehydrogenase (*CKX*) by a knock-out mechanism [91]. Similarly, knocking out another gene that codes for amino acid permease in rice which is actively involved in nutrient portioning, led to an increase in yield [60]. In addition, some genes in rice (*PIN5b, GS3, GW2*, *GW2*, and *GW5*) have been edited based on CRISPR/Cas9 technology, which has led to an increase in yield [61, 62]. To increase their yield, scientists have successfully edited genes in fruits such as *CLV* [63] and *ENO* [92]. The nutritional content of crops is an essential trait

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

for adding their commercial value to local and global markets. Genome editing of granule-bound starch synthase 1 *GBBS1* gene in maize produced low amylose content variety [93]. Gluten causes celiac disease in susceptible individuals and CRISPR/Cas9 editing techniques have successfully edited the conserved region in the loci of wheat, leading to 85% loss in immunoreaction [94]. Bacterial blight caused by *Xanthomonas oryzae* is a severe threat to rice production, and genome editing of *SWEET* genes using the CRISPR technique imparted high resistance to bacterial blight [64, 95]. In addition, editing of the *LOB1* gene in *Citrus sinensis* conferred resistance to *Xanthomonas citri* [65]. Powdery mildew caused by *Blumeri agraminis* fungi also leads to various growth defects in wheat plants. Targeting three wheat homologous genes (*EDR1*) by CRISPR/Cas9 editing improved resistance to these fungi [66]. Similarly, the tomato was also conferred resistance to *Oidium neolycopersici* that also causes powdery mildew, by mutating *Solanum lycopersicum MLO1* [67]*.*

CRISPR/Cas-based genome editing technology has to produce doublestranded breaks efficiently in the genome. Hence, scientific communities have also very well utilized their ability to edit plant viruses that attack plants and cause various diseases by employing various RNA-targeting versions of Cas enzymes such as Cas13a, Cas13b, Cas13d, and *Fn*Cas9 [68, 69]. Another problem that CRISPR has solved is to produce herbicide-resistant crops for controlling weeds that adversely affect crops' growth and decrease soil fertility and directly affect the yield from various crops. Acetolactate synthase (*ALS*) is a critical enzyme in producing branched-chain amino acids that are specifically targeted by herbicides such as sulfonylurea and imidazolinone. Using cytosine base editing, we impart herbicide resistance to *Oryza sativa ALS* by creating base transitions [70, 71]. Additionally, acetyl coenzyme A carboxylase (*ACCase*) is a critical enzyme in the production of lipids and a valuable target for genome editing with herbicides. Adenine base editing of the rice ACCase gene resulted in establishing a C2186R substitution, resulting in a mutant rice strain that is tolerant to haloxyfop-R-methyl [72]. Similarly, quizalofop-resistant wheat has been developed by adding an A1992V mutation into wheat ACCase [73]. Additionally, it has been demonstrated that altering *EPS* [96], *PPO* [97], *TubA2* [98], and *SF3B1* [99] mediates resistance to glyphosate, butafenacil, trifluralin, and herboxidiene (GEX1A). In addition to their numerous agricultural applications, these herbicide-resistant alleles can be used as selective markers to enhance gene editing processes [70]. CRISPR-based technology has been very effective in tackling stress conditions in plants and many literature surveys define the higher rate of success based on CRISPR genome editing. Xie and Yang [74] demonstrated targeted mutagenesis of the *Oryza sativa* gene (*OsMPK5*), which negatively regulates both biotic and abiotic stresses in rice.

Moreover, minimum low off-target efficiency was proved using specific guide RNAs and two appropriate vectors pRGE3 and pRGE6 [74]. Mitogen-activated protein kinases (MAPKs) are important signaling molecules that respond to drought stress in tomatoes by defending the cell membrane against oxidative damage and regulating the transcription of drought-stress-related genes. Slmapk3 mutants produced by CRISPR editing are fewer droughts tolerant and display increased wilting symptoms [75]. CRISPR/Cas9 genome editing has been used to manipulate the synthesis of secondary metabolites in plants. Zhang et al. recently showed multiplex CRISPR genome editing to enhance the isoflavone content of soybean and strengthen its resistance to soybean mosaic virus by altering the *GmF3H1*, *GmF3H2*, and *GmFNSII-1* genes [76].

**Plants with desired traits**

**Flowchart showing the production of plants with the desired trait.**

## **5. Safety regulations in genome editing**

Plant genome editing methods are critical for developing crops that can withstand biotic, abiotic, and extreme climatic changes and resolving global policy and governance issues. Apart from remarks on principle-based biotechnology adoption and the ethical, social, and biological issues raised by the CRISPR/Cas system, the current state of agriculture is examined. As a result of the technology's limitations, moral concerns about CRISPR have developed, necessitating intra and international attention to discover solutions that benefit the broader public interest. However, there is a public debate regarding the direction and control of new methods' utility inside the industrialized world [100]. Governments in developing

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

nations expect to give an alternative that promotes the alleviation of famine and poverty [101]. However, the uncertainty of agricultural biotechnology safety leads to suspicion of the regulatory framework and implies that the biological safety regulations [102] to preserve biological variety, including environmental protection and health safety, must be rejected. The unpredictability of agricultural biotechnology safety results in mistrust of the regulatory framework and implies that biosafety regulation should be rejected. Global hunger and poverty have been significantly reduced because of the green revolution. Malnutrition and the occurrence of certain chronic illnesses among otherwise healthy people have been exacerbated by inadequate amounts of specific amino acids, minerals, vitamins, and lipid acids in staple crops, as well as animal diets derived from them (the so-called diseases of civilization). The green revolution has made major contributions to the decrease of world hunger and poverty. Certain amino acids, minerals, vitamins, and fatty acids are deficient in staple crops and animal diets generated from them, contributing to malnutrition and the growing incidence of certain chronic illnesses in otherwise well-nourished people (the so-called diseases of civilization). Nevertheless, a recent trend demonstrates according to research conducted by the United Nations Food and Agricultural Organization (FAO), worldwide hunger and malnutrition have increased since 2014, with an estimated 821 million people lacking access to sufficient food in 2017 [103]. The advent of transgenic plants with insect-resistant, herbicide-tolerant, and nutritional characteristics has led to an explosion in the number of genetically modified crops grown on a wide scale. According to the latest report, 18 million farmers planted GMO crops on 181.5 million hectares in 28 countries in 2014, representing a 3–4% increase over the 2013 figures [104]. Recent marketed genetically modified crops include tomato, corn, soybean, cotton, canola, rice, potato, squash, melon, and papaya, with soybean, corn, and cotton being the most important because of their widespread cultivation critical role in the agricultural economy in many nations. America, Argentina, and Canada lead the world in producing and exporting genetically modified goods [105]. A good portion of the countries battling against GMOs in Africa are the same ones that struggled with them in Europe. Only local civil society organizations and a few NGOs have stood in the way of Uganda's effort to combat banana leaf wilt with its genetically modified bananas. All GMO-related activities in India must be regulated by the Environmental Protection Act (EPA) [106], which was passed in 1986. The Ministry of Environment, Forests and Climate Change (MoEF&CC) is responsible for this regulation [107]. Indian genetically modified crops are subject to a multitiered regulatory system controlled by the Ministry for Environment and Forest and Department of Biotechnology, which is part of the Ministry of Science and Technology. Six competent authorities comprise this system—the Recombinant DNA Advisory Committee (RDAC), the Review Committee on Genetic Manipulation (RCGM), the Genetic Engineering Appraisal Committee (GEAC), the Institutional Biosafety Committees (IBSC), the State Biotechnology Coordination Committees (SBCC), and District Level Committees (DLC). All the various committees' tasks and responsibilities are defined in the Rules 1989 [108]. Genetic Engineering Appraisal Committee (GEAC) is a board within the Ministry of Environment, Forests and Climate Change that regulates the manufacture, import, export, and storage of hazardous microorganisms and genetically engineered organisms or cells under the Environment Protection Act 1986. Indian lawmakers have charged the Government Environmental Assessment Council (GEAC) with performing environmental evaluations of operations using GMOs and their products in research, industrial manufacturing, field application, and environmental discharge. For the production, environmental release, and

marketing of GM crops, the Indian Parliament passed three essential laws. Included in these laws were the Environment Protection Act of 1986, which the Ministry of Environment manages, Forests and Climate Change (MoEF&CC), the Seeds Act of 1966, and the Seeds (Control) Order, which is administered by the Ministry of Agriculture [109]. It is generally recognized that transgenic crops offer significant advantages to society in solving concerns of food and nutrition security. Adding to the benefits of improved nutrition, herbicide tolerance, viral resistance, and tolerance to different environmental stressors, many fruits will last longer on the shelf, helping farmers in the marketplace. To accomplish its goal of food and nutrition security, India must continue its research into genetically modified crops. Despite the lack of concrete proof against the safety of genetically modified foods, the argument over whether or not they are safe will continue. Even while few public sector intuitions share worries about genetically modified foods, it is surprising that the same is true in regards to genetically modified animals. Government of India–funded intuitions should follow the same general principles as the Indian government, demonstrating their value by cooperating with the government to battle poverty and hunger. The members of the Technical Expert Committee constituted by the Supreme Court of India for Safety and Guidelines for genetically modified agricultural research debate the argument presented here, with some believing that it is significant and others seeing it as unimportant. However, while India has the inadequate infrastructure and lacks standards for genetically modified agricultural research and risk assessment, the initiative is of utmost importance given India's dire need. To prepare for future deregulation, India must continue researching genetically modified crops and construct basic infrastructural facilities while developing strict marketing and biosafety rules. Although portals such as the GEAC, the IGMORIS, and Biosafety Clearing-House play a role in biosafety assessment and regulations on GM plants, there is an urgent need to develop a single-window system and an online portal for the assessment, control, regulation, and approval of GM plants. The government should require every firm and public institution to register with this platform before starting any transgenic event or field testing, regardless of whether or not they are seeking clearance. To commercialize transgenic products, each new genetically modified organism (GMO) being developed must have a registration number, and registration date displayed online on a site or portal particularly intended for approval in any nation. This portal should include a publishing list that documents any genetically modified crop development activity so that any person interested in any genetically modified crop development event may access the entire information in one location. A gateway of this sort will be invaluable and broadly accessible for the public good by promoting the beneficial benefits of genetically modified food research, food safety, and the current state of genetically modified foods.

#### **6. Demonstration for CRISPR-CHOPCHOP for sgRNA designing**

In just 3 years, CRISPR genome editing dramatically changed biology, but also its acceptance and utility continue to expand. New CRISPR mechanisms and criteria for choosing ideal targets are being published all the time, highlighting the need for computerized CRISPR targeting tools to combine these guidelines to make target appropriate selection quicker. Among the most popular website software for genome editing with CRISPR and TALEN is CHOPCHOP (https://chopchop. cbu.uib.no/). It provides a user-friendly online environment for target selection, primer development, and restriction site identification, all based on the most

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

recent large-scale investigations. In both protein-coding and noncoding genes, CHOPCHOP allows accurate localization of subsections, including coding regions, UTRs, splice sites, and particular exons. For all sgRNAs, the program detects probable off-target sites, generates primers for target sites automatically, and shows all elements in a variable graphical interface that incorporates restriction relevant details for subsequent confirmation [109].

