Keywords

16.1 Introduction

Food demand is the basic requirement of life and since the early ages of agriculture, farmers have attempted to enhance the quality and yield of plant species. However, agricultural products are threatened by biotic and abiotic stress factors. Global climate change and the emergence of new genotypes of plant pests and pathogens pose serious threats to the sustainable production of crops. Besides, the growing world population which is estimated to reach 9.7 billion by 2050 and the increase of 58–98% food demand during this time renders the conventional agricultural practices insufficient to secure the food supply (Valin et al. 2014). Therefore, the introduction of innovative technologies not only contributes to crop production by improving the tolerance of crop plants during stresses, but also improves the nutritional quality and yield of crops. The advent of recombinant DNA technology tackled many existing restrictions in plant breeding stages. Identification, characterization, and transformation of foreign genes into host plants, to confer desired features including resistance to biotic and abiotic stress factors, herbicide tolerance, improvement of nutritional qualities and yield, have been numerously reported (Pasquali et al. 2008; Tripathi 2012; Bakhsh et al. 2015, 2016; Khabbazi et al. 2016, 2018; Anayol et al. 2016). Modification of the plant genome using chemical and physical mutagenesis is another approach to achieve desired agricultural traits (Roychowdhury and Tah 2013). However, plant genetic modification through these mutagens has random effects on the host plant and consequently might lead to an unexpected outcome. To reduce the unwanted genome alterations, target-specific modifying tools are advantageous and could be exploited alternatively. Genome editing of plants has gained notable achievements since the advent of sequence-specific nucleases (SSN) (Shelake et al. 2019). These tools including zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, have encouraged the creation of plant cultivars with desired traits. ZNFs- and TALENs-based genome editing has low-efficiency, is technically complex and cumbersome, therefore despite the reported researches (Lor et al. 2014; Sawai et al. 2014; Clasen et al. 2016) and due to the existing limitations, these methods are not employed any further (Kumar et al. 2018). CRISPR/Cas9 technology on the other hand is more accurate, simple and simultaneously can modify several targets in the genome. Moreover, improvement of a trait in crops using genome editing-based breeding methods requires an average of 4–6 years, while crossbreeding, mutation breeding, and transgene breeding approaches require 8–12 years (Chen et al. 2019). The discovery of CRISPR/Cas9 and its application in the determination of gene function and plant genetic modification studies have opened a promising avenue for the increased food supply in current global conditions (Yan et al. 2016; Minkenberg et al. 2017). The importance of this system is highlighted especially when it induces heritable targeted mutagenesis and contributes to the development of transgene-free plants (Wang et al. 2014; Pan et al. 2016). The introduction of non-transgenic edited plants has an unprecedented advantage to develop plants with desired traits, however maintenance of these genotypes is crucial as well. Hence, in vitro multiplication and generation of synthetic seeds could ensure the conservation and availability of the valuable genotypes (Ergül et al. 2018; Khabbazi et al. 2017, 2019). This chapter reviews the major CRISPR/Cas9-based genetic modifications in different plant species with improved yield, nutritional quality, and response to biotic and abiotic stress factors.

16.2 CRISPR/Cas System; Origin, Mechanism, and Its Use in Genome Editing

CRISPR/Cas is an adaptive immunity system naturally existing in bacteria and archaea and protects the host from invasive genetic elements like phages. Small nucleic acid fragments of invasive pathogens are incorporated into the host’s CRISPR loci (called spacer) and stored for future encounters (Amitai and Sorek 2016). Once the host cell is exposed to a new invasion, spacer sequences are transcribed, and as individual CRISPR RNAs (crRNAs) guide the Cas nuclease to the cognate nucleic acid sequences of the pathogen and cleave them (Barrangou et al. 2007). Based on the nature of interference and molecules involved, CRISPR/Cas systems have been divided into two classes and six types. CRISPR/Cas systems can target DNA (type I, II, V), RNA (type VI) or both DNA and RNA molecules (type III) (Koonin et al. 2017). Type II CRISPR/Cas9 isolated from Streptococcus pyogenes was the first demonstrated to show target-specific cleavage on the genome in eukaryotic cells and under in vitro conditions (Gasiunas et al. 2012; Jinek et al. 2012; Mali et al. 2013; Cong et al. 2013). This system is based on RNA-guided interference with DNA and has been employed in the development of plants with desired traits. CRISPR/Cas9 complex is comprised of Cas9 nuclease and a single guide RNA molecule (sgRNA). The sgRNA is an artificial fusion of a crRNA with a fixed transactivating crRNA (tracrRNA). A 20-nucleotide fragment at 5ʹ end of the sgRNA directs the complex to the target sites in genome adjoining to conserved protospacer-adjacent motifs (PAMs). The Cas9 enzyme induces double-strand breaks (DSBs) at specific genomic sites and exposes the broken DNA to either error-prone non-homologous end joining (NHEJ) repair or the precise homology-directed repair (HDR) mechanism. NHEJ may cause some unwanted indel mutations at the junction site of the repair whereas the HDR pathway stimulates accurate alteration of a gene sequence. HDR-mediated insertion or replacement of desired sequences in target sites of the genome provides an unprecedented tool for the genetic engineering of plants (Voytas and Gao 2014). Although, delivery of template DNA together with preparation of DSBs makes HDR mechanism more challenging than NHEJ, in vitro and in planta studies have reported the HDR-mediated genome editing in plants (Zhang et al. 2018a).

16.3 Effectiveness and Versatility of CRISPR/Cas9 System in Genome Editing of Plants

The use of CRISPR/Cas9 in genome editing of plants was first indicated in model plants tobacco (Nicotiana benthamiana) and Arabidopsis (Li et al. 2013; Nekrasov et al. 2013). The versatility of CRISPR/Cas9 based genome editing has been studied by knocking out the phytoene desaturase gene (PDS) in different crop species such as potato, grape, sweet orange, and watermelon. Disruption of the PDS gene causes albino phenotype in mutants and acts as a visual marker for CRISPR/Cas9-based mutagenesis (Jia et al. 2014; Pan et al. 2016; Tian et al. 2017; Nakajima et al. 2017). The inheritance pattern of the pds mutants was investigated by monitoring the albino phenotype, sequencing, and genotyping (Pan et al. 2016). The proportion of PDS mutated cells is correlated with the cell age so that lower leaves of the plant display higher rates of the PDS mutation (Nakajima et al. 2017). The higher rates of mutagenesis in older leaves could be due to inefficient repair mechanism of old cells or the repeated induction of double-strand breaks (DSBs). The induced PDS mutagenesis in watermelon plant caused the albino phenotype either as a clear or mosaic pattern with no off-target effects (Tian et al. 2017). Transient expression of CRISPR/Cas9-gRNA-PDS in sweet orange leaves also disrupted the PDS gene without detecting any off-target effects. Pretreatment of the sweet orange leaves with a culture of Xanthomonas citri ssp. citri facilitated the agro-infiltration and enhanced the protein expression level in leaves (Jia and Wang 2014). To increase the efficiency of mutagenesis, studies have been carried out to optimize the Cas9 gene expression. Comparing zCas9 (Xing et al. 2014), AteCas9 (Schiml et al. 2014; Fauser et al. 2014) and Cas9p (Ma et al. 2015), AteCas9 was identified as the most efficient codon-optimized Cas9 enzyme in knocking out the flavanone-3-hydroxylase gene in carrot cells (Klimek-Chodaka et al. 2018). Numerous reports have supported the use of CRISPR/Cas9 for accurate mutagenesis in a variety of crop species with different aims of nutritional quality improvements or biotic and abiotic factor tolerance (reviewed in Ricroch et al. 2017; Santosh Kumar et al. 2020).

16.4 Genome Editing Technologies Contribute to Pathogen Resistance in Crops

Pathogens along with insect pests threaten sustainable crop production worldwide. Bacteria, viruses, and fungi are the most devastating pathogens causing serious economic losses (FAO 2017). Approximately 20–40% of global agricultural losses are caused by these pathogens (Savary et al. 2012). In certain crops such as rice, diseases caused by pathogens are the main reason for the yield losses (Heinrichs and Muniappan 2017). To meet the food demands of the growing global population, chemicals have traditionally been used to combat pathogenic diseases. Different aspects of chemical control from consumer health to imposed costs have brought the need for alternative approaches. Using classical plant breeding methods, resistant cultivars have been developed, however, it is a time-consuming and tough process. In the last two decades, the transformation of resistance genes has successfully enhanced the resistance of crops to pathogens. The gradual acquisition of pathogen resistance to chemicals and resistant varieties can render these approaches ineffective. Genome editing-based technologies have opened new avenues to control agricultural product losses and superseded the limitations of conventional breeding methods. Employment of engineered SSNs such as ZFNs, TALENs, and CRISPR/Cas9 has facilitated the genome modifications toward biotic stresses. Unlike ZFNs and TALENs which are low efficient and technically complex, CRISPR/Cas9 is highly effective and target-specific; however, the efficiency of the entire process remains species- and genotype-dependent. Moreover, off-target mutations, as well as unexpected damages, could be limiting in plant genetic manipulations. PEG-, gene gun- and Agrobacterium-mediated transformation methods and lately a nanodot based transformation method have been used to develop different strategies for stable and transient expression of Cas9/sgRNA constructs in crops (reviewed in Borrelli et al. 2018; Doyle et al. 2019). Employment of gene bombardment method ensures the availability of a sufficient amount of template DNA in host cells, however, the existence of excessive foreign genes and vector sequences might be contaminant for the recipient cell. A. tumefaciens-mediated transformations of plants results in the stable transformation of gene constructs and screening of the mutant plants containing foreign gene sequences in subsequent stages (Baltes et al. 2017). In the last few years, CRISPR/Cas9 has been widely used to improve the resistance of crops to different biotic stresses. These studies have mainly addressed viral, fungal, and bacterial diseases in plants (Table 16.1).

Table 16.1 CRISPR/Cas-based editing of plant genome and development of plants with resistance/tolerance to biotic stress factors
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16.4.1 CRISPR/Cas-Based Engineering of Crops for Virus Resistance

Most of the biotic resistance studies in CRISPR-based edited plants are related to viruses (Borrelli et al. 2018). Based on their genome nature, plant viruses are classified into five groups: single-stranded DNA, single-stranded RNA, double-stranded RNA, positive-sense single-stranded RNA, negative-sense single-stranded RNA, and reverse-transcribing viruses. Virus-resistant plant species could be created through either targeting the viral genetic material or editing plant genome. Resistance obtained by disruption of viral genes requires the integration and permanent expression of Cas9/sgRNA constructs, therefore, such modifications subject the plants generated to biosafety regulations of genetically modified organisms (GMOs). In contrast, modifying plant susceptibility genes such as eukaryotic translation initiation factors necessary for the RNA virus life cycle, release non-transgenic virus-resistant plants (Sanfcon 2015).

CRISPR/Cas edited virus-resistant plants have been mostly developed for resistance to geminiviruses (Ji et al. 2015; Baltes et al. 2015; Ali et al. 2015, 2016). Geminiviruses cause substantial destructions in important families of plants including Fabaceae, Cucurbitaceae, Solanaceae, etc. (Zaidi et al. 2016). Most of these studies have been performed on model plants Arabidopsis thaliana and Nicotiana benthamiana (Table 16.1). Expression of Cas9/sgRNA constructs in host plants targeting the coding and non-coding regions of the virus reduced the virus accumulation. Targeting the virus coat protein (CP), replication protein (Rep) and intergenic regions (IR) effectively attenuates viral symptoms; however, mutations disrupting the virus in the non-coding IRs such as the stem-loop sequence within the origin of replication resulted in more reduction of virus replication and accumulation (Ali et al. 2015). Furthermore, targeting the virus in the IR inhibits the creation of new variants of the mutated virus which can potentially replicate and escape the CRISPR/Cas9 machinery (Ali et al. 2016). Targeting plant susceptibility genes such as eIF4E, eIF(iso)4E, and eIF4G has resulted in the development of non-transgenic plants resistant to RNA viruses of Potyviridae (Chandrasekaran et al. 2016; Pyott et al. 2016; Macovei et al. 2018).

In most of the studies, CRISPR-based edited plants have been developed using the SpCas9 enzyme isolated from Streptococcus pyogenes (reviewed in Ricroch et al. 2017). However, this enzyme only cuts double-stranded DNA molecules, therefore, remains inefficient for RNA viruses. Later studies isolated other CRISPR associated enzymes from Francisella novicida (FnCas9) and Leptotrichia wadei (LwaCas13a) which could target RNA molecules. FnCas9 and LwaCas13a have been used to develop plants resistant to cucumber mosaic virus, tobacco mosaic virus and turnip mosaic virus (Zhang et al. 2018b; Aman et al. 2018). Endonuclease activity of FnCas9 is not essential for enzymatic interference; therefore, FnCas9 could be utilized as a CRISPR interference tool (CRISPRi) (Zhang et al. 2018b).

16.4.2 CRISPR/Cas-Based Genetic Modification of Plants Against Fungal Disease

Studies over the plant-pathogen interactions have elucidated the molecular mechanisms underlying pathogen infection and plant immune system. The presence of plant genes serving pathogens can lead to the emergence of diseases. Targeting the susceptibility genes corresponding powdery mildew (MLO-A1, MLO-1, and MLO-7), transcription factors involved in stress responses (WRKY52 and ERF922), regulators associated with host immune system (NPR3) and subunits of the exocyst protein complex (SEC3A), has conferred resistance to fungal diseases in a variety of annual and perennial plants (reviewed in Borrelli et al. 2018).

Till date, resistance against powdery mildew (Wang et al. 2014; Malnoy et al. 2016; Nekrasov et al. 2017), gray mold (Wang et al. 2018), black pod disease (Fister et al. 2018), and blast disease of rice (Wang et al. 2016; Ma et al. 2018) has been improved in several crops such as wheat, rice, tomato, grapevine, and cacao tree. Self-pollination and subsequent selection of individuals with on-target mutations but lacking Cas9/sgRNA constructs introduce edited non-transgenic plants (Nekrasov et al. 2017). Generally, CRISPR/Cas constructs are transferred to host plants through vector plasmids however this method is intercepted by the biosafety regulations of transgenic crops. Implementing genome editing of plants without using DNA plasmid attempts to reduce the social distrust related to the transformations of foreign genes. Transient expression of plasmid-free genome editing constructs is critical particularly in perennial fruit crops due to the longer time required for segregation and backcrossing. In this regard, Malnoy et al (2016) delivered the purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of apple and grape cultivars. This method could increase the public acceptance of the genetically modified (GM) crops and thereby could be exempted from the existing GMO regulations (Waltz 2012; Jones 2015).

16.4.3 Bacterial Resistance Achieved Through CRISPR/Cas9

Unlike other pests and pathogens, bacterial diseases of plants cannot be controlled by chemicals, and the only way to cope is to prevent the disease by using different approaches such as efficient agricultural practices, cultivating healthy plants, and developing resistant varieties (Janse 2001). CRISPR/Cas9-based targeted mutagenesis on plant susceptibility genes has conferred resistance to bacterial infection in host plants. Canker is one of the serious diseases of citrus cultivars around the world. The disease is caused by Xanthomonas citri subsp. citri (Xcc) and CsLOB1 is the susceptibility gene in the host induced by the pathogenicity factor PthA4 (Hu et al. 2014). Modifying the PthA4 effector binding elements (EBEs) in the promoter region of CsLOB1 gene in Duncan grapefruit variety interfered with the binding of PthA4, therefore, decreased the typical symptoms of bacterial canker (Jia et al. 2016). Later CRISPR/Cas9-mediated disruption of EBEPthA4 confirmed the presence of the link between CsLOB1 promoter activity and the susceptibility against Xcc in Wanjincheng orange (Citrus sinensis Osbeck) (Peng et al. 2017).

Expression of OsSWEET13, a member of sucrose transporters family proteins, is required for bacteria-host interactions. OsSWEET13 activity is induced by Xanthomonas oryzae pv. Oryzae PthXo2 effector protein and CRISPR/Cas9-mediated knockout of OsSWEET13 susceptibility gene better explored the role of PthXo2 in the emergence of the bacterial blight of rice (Zhou et al. 2015). Recently, CRISPR-based genome-edited SWEET gene promoters (SWEET11/SWEET13/SWEET14) introduced rice lines with broad-spectrum resistance to bacterial blight disease (Oliva et al. 2019).

16.5 Tolerance to Herbicides and Abiotic Stress Factors via CRISPR/Cas9

Weeds are the biotic restraint that causes significant losses in agricultural production and if not controlled timely and effectively reduces the crop yield by up to 50% (Oerke 2006). Transgenic soybean, canola, cotton, and corn were the first glyphosate and glufosinate tolerant crops released to the market in 1996–1997. According to a report, more than 100 million hectares worldwide are cultivated with genetically modified (GM) crops with at least one herbicide tolerance gene (ISAAA 2012). Adoption of herbicide-tolerant (HT) transgenic crops reduces the use of chemicals and greenhouse gas emissions resulting from agricultural practices; moreover, it increases the product yield, farmer income (Cerdeira and Duke 2006; Martino-Catt and Sachs 2008). However, the expression pattern of the transgene is influenced by genetic elements such as the gene promoter and intron fragments (reviewed by Huang et al. 2015). The repeated cultivation of a single herbicide-tolerant crop causes the evolution of weeds resistant to the frequently used chemicals (James 2014). To address this issue, gene stacking has contributed successfully.

Herbicides bind to vital proteins in the host and affect the normal functioning of the cell, inhibit plant growth and consequently make weeds unable to survive (Schonbrunn et al. 2001). Modifying the structure of the target proteins such that their functions are not affected but at the same time it disables the binding of herbicides, is an efficient method to confer tolerance to herbicides. Precise mutagenesis in phosphoenol pyruvate binding site within 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is an example in this regard which inhibits the binding of glyphosate to phosphoenol pyruvate. Employment of CRISPR/Cas9 tool enables the accurate modification of target sites in the plant genome (Table 16.2). An example in this regard is the substitutions in T178I and P182A within phosphoenol pyruvate binding (PEP) binding site in Linum usitatissimum (Sauer et al. 2016). Comparing the edited plants with wild type flax indicated at the higher levels of tolerance to glyphosate. Likewise, induction of such mutations in EPSPS gene of rice plant confirmed the efficiency of the CRISPR/Cas9, however, based on results, only those individuals containing heterozygous gene were viable and showed tolerance to glyphosate (Li et al. 2016a). In addition to EPSPS, acetolactate synthase (ALS) gene has been targeted by CRISPR/Cas system to apply mutagenesis with the aim of increasing tolerance to herbicides. Acetolactate synthase is a conserved protein required for biosynthesis of branched-chain amino acids such as valine, leucine, and isoleucine. This protein is a primary target for some classes of herbicides including pyrimidinyl thiobenzoates, imidazolinones, triazolopyrimidines, sulfonylureas, and sulfonyl-aminocarbonyl triazolinones (Baltes et al. 2017). CRISPR-based ALS mutations conferred tolerance to such herbicides in Solanum tuberosum, Oryza sativa, Zea mays, and Glycine max species (Svitashev et al. 2015; Li et al. 2015; Butler et al. 2016; Sun et al. 2016; Endo et al. 2016; Butt et al. 2017) (Table 16.2).

Table 16.2 CRISPR/Cas-based editing of plant genome and development of plants with resistance/tolerance to herbicides and abiotic stress factors
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In addition to the crop losses imposed by weeds, factors such as unfavorable drought, salinity, and temperature induce metabolic stresses that affect the growth of plants. CRISPR/Cas-mediated precise disruption of target sites in the genome has been employed to confer tolerance to stress factors (Table 16.2). Ethylene plays an important role in regulating plant response to water deficits and high temperatures (Hays et al. 2007; Kawakami et al. 2010, 2013). Reducing plants’ ethylene biosynthesis or decreasing the sensitivity of plants to ethylene can improve grain yield under drought conditions (Habben et al. 2014; Shi et al. 2015). ARGOS8 gene is a negative regulator of ethylene responses. In maize, the expression of ARGOS8 gene is relatively low and therefore a constitutive overexpression enhanced the expression level of ARGOS8 and consequently led to the increase in grain yield under drought stress (Shi et al. 2015). Utilization of CRISPR/Cas system via the HDR pathway contributed to precise allelic variation in ARGOS8 thereby enhancing the grain yield under drought conditions (Shi et al. 2017). HDR-dependent mutation of mir169a improved the drought tolerance in model plant A. thaliana such that more than 50% of mutant plants exhibited tolerance to drought stress whereas no wild individual survived (Zhao et al. 2016).

CRISPR/Cas9 tool has also been used to characterize the transcriptional response of rice RAV genes (OsRAVs) to salt stress. In this study, the expression patterns of the five members of OsRAVs were examined under salt stress, and it was observed that only OsRAV2 was stably induced by salt treatment. Further analysis on the OsRAV2 promoter region elucidated that pOsRAV2 is induced by salt. The GT-1 element located at pOsRAV2 is necessary for salt induction of the promoter (Duan et al. 2016).

16.6 Improvement of Crop Yield, Nutritional Quality and Storage Using CRISPR/Cas9

The effectiveness of the CRISPR/Cas9 system in the disruption of negative regulators of undesirable traits in plants has led to the improvement of yield, nutritional value, and shelf-life of crops, such that these researches share the highest proportion among the CRISPR-based studies (Table 16.3 and Fig. 16.1). Improving the yield is a crucial step to ensure food supply for the growing population. Crop yield is assessed considering various factors such as grain number, size, and weight as well as tiller number and panicle size. As a quantitative trait, yield depends upon many regulating factors. Disruption of genes responsible for the negative regulation of the aforementioned yield factors has contributed to the development of crops with improved yield quality (reviewed by Chen et al. 2019). Knocking out the OsGn1a, OsGS3, TaGW2, OsGW5, OsGLW2, TaGASR7, OsDEP1, TaDEP1, and OsAAP3 genes has indicated the effectiveness of CRISPR/Cas system in improving the crop yield by the creation of targeted loss-of-function mutations in plants (Li et al. 2016d; Liu et al. 2017; Lu et al. 2018; Zhang et al. 2016, 2018c). Simultaneous knock out of three genes including GW2, GW5, and TGW6 increased the grain weight in rice which demonstrated the effectiveness of CRISPR/Cas use in trait pyramiding in plants (Xu et al. 2016).

Table 16.3 CRISPR/Cas-based editing of plant genome and development of plants with improved nutritional value, yield, and other improvements
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Fig. 16.1
figure 1

The relative proportion of CRISPR/Cas-based studies in plants

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Crop quality traits such as starch, oil, and secondary metabolite content have also been improved by the CRISPR/Cas-based gene editing. Starch produced from crops like potato has many applications in the food and industrial sectors. Starch has two components including amylose and amylopectin and modification of amylose or amylopectin alters the properties of starch (Zeeman et al. 2010). Granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in many plant species. In potato plant (Solanum tuberosum), the GBSS enzyme is encoded by a single locus (GBSS1) with four alleles. High amylopectin potato genotypes (Waxy potato) have been developed by gene silencing technologies and traditional mutational breeding methods (Andersson et al. 2003; Muth et al. 2008). Recently, CRISPR/Cas-based multiallelic indel mutations of potato GBSS gene reduced the amylose, and increased the amylopectin content of the starch (Andersson et al. 2017, 2018). In Zea mays, knock out of the endogenous waxy gene led to the production of grains with high amylopectin content (Waltz 2016). Consumption of cereals with enriched amylose or resistant starch benefits human health and reduces the risks of non-infectious serious diseases such as diabetes (Regina et al. 2006). Using the CRISPR/Cas tool, mutagenesis was implemented in the starch branching enzyme gene (SBEIIb), leading to an increased proportion of amylose and resistant starch in rice grains (Sun et al. 2017).

Modifications of the fatty acid composition of plant oils have also been carried out by the CRISPR genome editing tool. Targeting the Fad2 (Morineau et al. 2016; Jiang et al. 2017) and CsDGAT1 and CsPDAT1 (Aznar-Moreno and Durrett 2017) genes in Camelina sativa altered the fatty acid composition of the seed oil. CRISPR/Cas9 tool was used to interfere with the ripening process of tomato and enhanced the fruit shelf-life by targeting RIN, SLALC, and lncRNA1459 genes (Ito et al. 2015; Yu et al. 2017; Li et al. 2018). Precise mutations of RIN, SLALC and lncRNA1459 genes of tomato interfered with the ripening process and enhanced the fruit shelf-life (Ito et al. 2015; Yu et al. 2017; Li et al. 2018). Other studies carried out with the aim of improving quality, yield, and breeding-related attempts has been summarized in Table 16.3.

16.7 Conclusion

CRISPR/Cas is a simple, versatile, and robust tool to induce target-specific mutations in different plant species. The employment of the CRISPR/Cas system has indicated an unprecedented contribution to the genome editing of crops. This tool has paved the way for conferring desired characteristics to various plant species and eliminating foreign genes in subsequent generations. Using this technology along with the high throughput system, quantitative traits with polygenic inheritance can be improved by simultaneously targeting the gene loci in crops. CRISPR/Cas system is also useful in the identification of the roles of genetic elements involved in different metabolic pathways. Despite the advances achieved to date, the existence of impediments such as off-target effects, delivery of the CRISPR reagents to host cells, and plant regeneration are still a limiting factor for some species. Unlike the commonly reported knockout-based mutation studies, knock-in researches are less reported and have been carried out on a limited scale. The recent use of the prime editing method in human genome editing has raised the hope for more efficient and precise base editing attempts without relying upon double-strand breaks or donor DNA (Anzalone et al. 2019). The prime editing technology is also expected to be used for plant cells, which will lead to new directions in the precise engineering of the plant genome (Xu et al. 2020).