Keywords

1 Introduction

From the time of understanding of cropping, humans have continuously been focusing on the yield and quality of plant products via various traditional approaches until the green revolution . On the other hand, the human population is increasing continuously, but arable land is constant in dimensions. So, to feed the world population, it is imperative to make efficient use of non-cultivable lands such as pH imbalanced, salt-stressed, drought areas, and marshy lands (Singh et al. 2018; Mehta et al. 2019a). Gene revolution has been possible due to technological revolutions in the field of biotechnology. However, despite countless benefits and qualities in cereal plants, there are a few problems which need to be addressed such as cultivation issues , selective nutrient enrichment, overall nutritional content, and stress tolerance using genetic engineering of genes in the genome of affected cereal crops. GM crops can fight poverty and malnutrition by increasing yield, bio-fortification, reuse of salt-affected areas, overcome energy crisis, pest resistance, pesticide and herbicide tolerance, drought tolerance, efficient CO2 use, nitrogen use efficiency, and phytoremediation. ISAAA report March 2020 reported an increase in GM crops planting area. In 1996, 1.7 million hectares of land cover were utilized for GM plants, while in 2018, it was 191.7 million hectares. Genetic engineering techniques for cutting and introducing the desired gene sequence involves the use of DNA-based biomolecules such as transcription activator-like effector nuclease (TALEN), zinc finger domain nucleases (ZFNs), CRISPR/Cas9, CRISPR/Cpf1 most importantly, and non-DNA based biomolecules such as guide RNA-based gene editing (Mehta et al. 2020). These genome-editing tools have advantages as well as limitations (Mehta et al. 2020). Both TALENs and CRISPR/Cas9 allow precise alterations by target specificity. TALENs provides greater freedom in target site selection compared to CRISPR/Cas9. However, the popularity of CRISPR is due to its capability for modifying chromosomal targets at higher frequencies. Simplicity in design and usage makes CRISPR/Cas9 an attractive tool. However, TALENs designing gets streamlined by modules with repeated combinations, thus reducing the cloning time. TALENs is sensitive to cytosine methylation, expensive and targets one site at a time. ZFNs are another class of nucleases that can edit/modify any targeted genomic sequence. However, they have low efficacy and is time consuming. Here, we highlight various targets in cereals to fight biotic and abiotic stresses using gene-editing techniques to boost the productivity and nutrient content of cereals to fight world hunger in an eco-friendly manner.

2 Genetic Engineering Tools for Crop Resiliency

There are various DNA and non-DNA-based techniques for editing the crop’s genome, which includes insertion, deletion, or modification of a gene of interest. Many classical techniques are available for the transformation of the edited genes of interest into crops, such as electroporation, Agrobacterium-mediated , and nanoparticle-mediated transformation. The Agrobacterium-based technique is the most widely accepted because of its simplicity, and a higher integration probability even for low-copy DNA fragments with minimum problems to the host genome (Sindhu et al. 2019). The genetically modified crops created using gene-editing tools have desired characteristics (Griffiths et al. 2005). The transgene may have its origin from any organism having a beneficial trait. It can be from prokaryotes such as bacteria and viruses, or eukaryotes such as fungi and animals. Apart from this, there are other popular modern breeding techniques such as genome-wide association studies (GWAS), genome selection (GS), and marker-assisted selection (MAS) that are being employed by scientists for incorporating useful traits into the crops.

However, the current trend in life sciences is genome engineering using methods such as ZFN, TALEN, and CRISPR with variants such as CRISPR/Cas9, and CRISPR/Cpf1. These are well-known approaches which have been used to develop crops that can tolerate climate variabilities and biological stresses (Liu et al. 2013; Ali et al. 2015; Andolfo et al. 2016; Wang et al. 2016; Li et al. 2017; Kumar et al. 2018). There are other approaches such as RNA silencing using microRNA, trans-acting siRNA, hairpin RNA, and virus-induced gene suppression which reduce the translated product of marked genes (Bernstein et al. 2001). ZFNs, TALENs and CRISPR technologies create dsDNA cuts at particular genomic sequences which are further repaired using either NHEJ (non-homologous end-joining) or HR (homologous recombination) . ZFNs and TALENs use engineered proteins along with DNA binding regions, and DNA endonuclease enzymes, whereas CRISPR/Cas utilizes laboratory synthesized sgRNA (synthetic guide RNA) targeted to precise DNA sequences by base pairing. As far as ZFNs and TALENs are concerned, protein engineering is expensive, laborious, and time consuming, which limit their usage in high-throughput studies. On the other hand, CRISPR/Cas9 technique has circumvented these problems by being cheaper, versatile, less laborious, precise, and most importantly, efficient in genome-editing purposes at multiple levels (Mehta et al. 2020).

2.1 DNA-Based Genetic Engineering Approaches

2.1.1 Transcription Activator-Like Effector Nucleases (TALENs)

These are a variety of DNA-cutting proteinic enzymes especially designed to cleave at specific target DNA sequences. They are formed of the TAL effector DNA-binding domain with DNA cleaving sphere acting as nuclease which is taken from restriction endonuclease FokI. DNA-binding domain has various tandem DNA repeats ranging from 1.5 to 33.5 characteristically. Every repeat has 34 aa stretch with deviation at 12–13th amino acid named repeat variable residues, that is, RVD. These repeats recognize one nucleotide specifically. There are unique RVDs such as IN (Ile Asn), NN (Asn Asn), DH (Asp His) and GN (Gly Asn) which recognize A, G, C, and T, nucleotide bases respectively. Double-strand breaks after nicks are mended by HDR or NHEJ. These repairs result in deletions, insertions, duplications, inversions, transversions, translocations, or point mutations (Wei et al. 2013). The key features are explained in Table 3.1.

Table 3.1 Key features of different genetic engineering approaches used nowadays for enhancing crop resiliency
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2.1.2 Zinc Finger Nucleases (ZFNs)

ZFNs are a type of DNA-cutting enzymes used for genome-editing applications such as duplication, addition, deletion, or substitution of gene sequences, with more precision than conventional plant breeding techniques for improvement of various crop attributes. They also include a DNA-sticking domain (Zinc finger class) and DNA cleavage sphere. DNA binding part of a molecule usually has 3–4 zinc finger proteins conjugated with di-cysteine and di-histidine motifs. These motifs recognize nucleotide triplets based on the residues from the alpha helix. DNA cleaving domain comes from FoKI restriction endonuclease. It dimerizes for DNA cutting in a non-sequence-specific manner and induces dsDNA cuts. The DSBs (double-stranded breaks) are mended by two paths, namely (1) HDR using homologous DNA as a model sequence to reinstate the DSBs for accurate and controllable repairs and (2) NHEJ machinery leads to additions, deletions, or substitutions in the broken dsDNA of host genome (Miller et al. 2007). ZFNs’ key attributes are well-documented in Table 3.1.

2.1.3 CRISPR/Cas9 Approach

CRISPR usage in plants has promised precise and accurate gene editing for targeted crop trait improvements (Arora and Narula 2017). They have been used versatility in almost all model organisms of different origins including plants (Sander and Joung 2014). It has been showcased and used for various attributes in rice, tomato, maize, wheat, woody plants such as apple, poplar, etc., to extended level for alleviating biotic, abiotic, and other climatic stress issues (Osakabe and Osakabe 2017; Mehta et al. 2020). It involves induction of dsDNA breaks at selective sites in genomic sequences with the help of guided RNA complementary sequences that bind with DNA and Cas endonucleases of CRISPR/Cas system targets and cuts genomic loci of DNA and pair using Watson-Crick base pairing. The breaks are mended by the cell’s inner restoration mechanisms involving NHEJ and HR. Cas9 or Cpf1 plays molecular scissor role and RNA marks the address at genomic sequence level, thus guaranteeing precise and accurate cutting for further action. The entire process can be conceptualized as effective designing of Cas nucleases, assemblage of gRNA cassettes, Cas and RNA vector’s delivery, screening, selection, efficient gene-editing detection, and plant regeneration with selective traits. Thus, a characteristic trait is extensively utilized in a variety of model cells and organisms for targeted mutagenesis. Thus, it has been widely accessed for achieving functional annotation of various biotic and abiotic stress resilience in genetic elements. However, the efficient delivery of gene-editing tools and components via transformation is a key bottleneck in gene-editing techniques. There is low transformation efficiency in Agrobacterium-mediated and particle bombardment-mediated transformation. Alternative strategies such as ex vitro plant composite development can help in efficient functioning of CRISPR/Cas9 and thus help in the elucidation of gene function, generation of valuable traits for yield, and quality improvement which usually gets affected with biotic stresses such as pests and pathogens (bacterial, viral, and fungal) and abiotic stresses. Biological stresses are usually coded by solo gene and gene erasure using CRISPR/Cas9-targeted modification or inactivation of susceptible genes gives protection against pathogens and has proven to produce resistant plants against bacterial, fungal, and viral pathogens (Borrelli et al. 2018). Further, CRISPR/Cas has the ability to edit multiple genes simultaneously (Sasano et al. 2016). Abiotic stress responses are usually controlled by multiple genes because of their complex nature. CRISPR/Cas-based unique allele variants for a non-biological stress-related gene (s) (Shi et al. 2017; Osakabe and Osakabe 2017) or clubbing multiples genes via HDR-induced gene aiming (Devkota 2018) has been used to develop resistance against abiotic stresses. The mutants failed to develop nodules when inoculated with Sinorhizobium sp. strain NGR234. Moreover, clubbing CRISPR/Cas with the expression of morphogenic regulators such as BBM/WUS may improve gene-edited plant’s regeneration capacity with reduced tissue and genotype dependency.

Programming of CRISPR/Cas9 can be changed using sgRNA sequences and more than one sgRNA can work concurrently using similar Cas9 protein for diverse targets (Wu et al. 2014). However, there is a problem of off-target effects of CRISPR/Cas9, which must be minimized so that new mutants for abiotic and biotic stress response would be generated. One approach is to use optimized sgRNA designing (Montagne et al. 2014). CRISPR-P web software has been exploited for optimized designing of sgRNA as used for more than 20 plant species including cereals (Jain 2015). Numerous vectors and toolkits were also developed to facilitate plant genome editing using CRISPR/Cas9 (Xing et al. 2014). Thus, CRISPR/Cas9 usage in genetic engineering, genomic screening, and transcriptional intonation for dichotomizing molecular origin behind biotic and abiotic strain response and generating stress-tolerant cereal crop can be used by accessibility of the aforementioned attributes and knowledge. Key features of CRISPR/Cas9 are explained in Table 3.2.

Table 3.2 Review of literature highlighting the key examples of genome editing in improving resistance to various biotic stresses in cereals
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2.1.4 CRISPR/Cpf1

One other budding variant of CRISPR is CRISPR/Cpf1 which is CRISPR (Prevotella and Francisella 1) and was recently found to be a unique RNA-mediated site-specific class 11 type V nuclease. It was first used to engineer a mammalian cell (Mahfouz 2017). Cpf1 endonuclease has a RuvC-type nuclease domain but it does not have the HNH domain as in Cas9. Cpf1 produces double-stranded breaks with sticky or staggered ends, not blunt ends as in Cas9. Thus, it enables gene expurgation, inclusions, or substitution via HDR-homology-directed repair (Zetsche et al. 2015; Mehta et al. 2020). Cpf1 provides multiple rounds of cutting as opposite to Cas9 where a single cleavage occurs, and it cuts DNA at a specific distance from protospacer adjacent motif (PAM). Cpf requires shorter CRISPR RNA and utilizes a T-rich PAM instead of G-rich PAM as in the Cas9 system. So, Cpf1 can explore better and increase the number of plant genome editions (Stella et al. 2017). Cpf1 has increased target specificity than Cas9 in plants compared to animals (Tang et al. 2017).

2.1.5 CRISPRi

This variant of CRISPR utilizes catalytically inactive Cas9 (dCas9) which disrupts the gene’s function via gene intercession (Qi et al. 2013). CRISPRi technology essays the role of a perturbation tool for sequence-specific suppression of transcription in small prokaryotic organisms as well as complex eukaryotic organisms (Huang et al. 2016). Earlier, it was invented for transcriptional interference to silent gene expression, but nowadays, it is being exploited for transcriptional activation and epigenetic modifications such as exploiting the functionality of gene methylation or chromatin modifications for abiotic stress responses. It is highly specific and precise in nature along with a little off-target effects (Domingue et al. 2016). It was found that when dCas9 gets merged with Kruppel associated box (KRAB), the gene expression level was reduced equal to approximately 99% in human cells (Gilbert et al. 2013). Hence, this technique can be well applied in plants for stress-responsive genes using CRISPR-mediated and synthetically driven transcriptional activator or repressor molecules (Piatek et al. 2015). Transcriptional activation got enhanced if dCas9 was fused with a transcriptional activator (Gilbert et al. 2013). CRISPRi can regulate a particular target gene’s efficiency along with enhancing its effect (Lo and Qi 2017).

2.2 DNA-Free Genome Engineering

DNA based and non-DNA based CRISPR/Cas approaches have been implemented in recent scenarios. DNA-based approach usually involves Agrobacterium-based transformation , whereas non-DNA-based approach involves PEG-mediated protoplast fusion method. In the classical CRISPR/Cas method, T-plasmid is constructed along with the required sgRNA and Cas9/Cpf1/Cas variant coding sequence. During transformation, RNA and Cas sequences get incorporated into the host genome. Guide RNA and Cas9 get translated inside the host cell and in vivo gRNA-Cas9 RNP (ribonucleic protein complex) is formed. As the target DNA sequence is detected, double-strand DNA breaks are induced and mutations are generated by the cell’s internal DNA repair mechanisms. Here, the CRISPR/Cas complex is continuously getting expressed and actively produces desired mutations in host cells.

DNA-free CRISPR/Cas9 approach is getting wider acceptance, as it can create safer and ethically accepted GMO products. In one approach, in vitro-synthesized Cas protein and in vitro-translated gRNA are used to generate the RNP complex in vitro and delivered inside the host cell protoplast using the PEG-mediated fusion method. Since the complex is already formed in a tube, it is active and once inside the cells, it detects the target sequence and induces dsDNA breaks/nicks. The cellular repair mechanisms lead to the generation of mutations in the host genome at desired target sequences and do not add any foreign DNA in the host genome. With time, the CRISPR/Cas9 complex gets degraded inside the cell and their availability is decreased over time. A suitable screening process is required to identify stable cells for further growth (Metje-Sprink et al. 2019). Other DNA-free approaches include momentarily expressed CRISPR/Cas9 plasmid DNA (TECCDNA), and CRISPR/Cas9 in vitro transcripts (IVTs). All these techniques do not allow integration of foreign DNA into the host genomic DNA and hence, they diminish off-target effects. These techniques are comparatively faster and less costly than the CRISPR/Cas cassettes already available in the global market. The most suitable methods for the delivery of these CRISPR cargos are the polyethylene glycol (PEG), electroporation, biolistic bombardment, or cationic lipid-based method (Zhang et al. 2016; Yin et al. 2017).

The most advanced CRISPR/Cas variant in the market is CRISPR base editors which create single-nucleotide changes at the target loci. The dsDNA breaks or template DNA sequences are not required here. This approach for the generation of a single-nucleotide mutant is widely used in monocot and dicot plants. This variant utilizes dCas9 (dead/inactive cas9), nCas9 (Cas9 nickase), and adenine or cytidine deaminase enzymes. Deaminases convert cytosine to thymine via uracil and adenine to guanine via inosine (Monsur et al. 2020). They are more advanced, effective, and efficient in editing. SpCas9, Sacas9, SpVQR-Cas9, SpEQR-Cas9, SaKKH-Cas9, and SpVRER-Cas9 are a few variants which are available in the market having differential specificity toward PAM (protospacer-adjacent motif) . They provide product purity at a high level and have low off-target editing efficiency.

3 Role of Genome Editing Tools in Biotic Stress Management

The factors eliciting biological stress in plants are a major threat to cereal crops causing substantial yield losses annually on a global scale (Langner et al. 2018). Cereal crops are attacked by numerous pathogens and pests, including myriad bacteria, virus, fungal entities, insects, and parasites (Rahman et al. 2019; Singh et al. 2019; Mehta et al. 2019b). These biotic stressors generally harm the host plant by directly or indirectly depriving its host of its vital nutrients and become a major cause of pre- and post-harvest crop damage. The native defense mechanisms of plants protecting against biotic stresses are diverse and dynamic, which are genetically governed by resistance genes encoded within the plant genome (Diaz 2018). Numerous methods such as traditional breeding, molecular breeding, and genetic modulation tools have been deployed with the ultimate goal of enhanced food security by enabling the crops to combat pathogenic infections. In recent years, gene-editing technology has appeared to be an opportunistic strategy that serves to improve resistance in crops toward biotic stress-inducing agents through targeted gene manipulation. Numerous pioneer studies reporting the application of gene-editing tools to improve resistance in various cereal crops towards bacterial and fungal diseases are presented in Table 3.2.

3.1 Resistance Against Fungal Diseases

Shan et al. 2013 was the first to report the successful application CRISPR/Cas9 technology to enhance resistance against Blumeria graminis F. sp. tritici which causes powdery mildew disease in wheat plants leading to significant yield losses. CRISPR/Cas9-mediated knock-out of TaMLO (MILDEW-RESISTANCE LOCUS) in wheat, imparted disease resistance, wherein 28.5% mutational frequency was observed in TaMLO for protoplasts (Shan et al. 2013). Further, Wang and his team demonstrated the use of CRISPR/Cas9 and TALENs systems for the concurrent expurgation of three homoalleles of TaMLO gene in hexaploid bread wheat and reported similar mutation frequency (5.6%) by both the editing methods (Wang et al. 2014). More recently, the feasibility of CRISPR/Cas9 in wheat for achieving fungal resistance was confirmed by Zhang and his colleagues, who targeted the TaEDR1 gene which negatively regulates powdery mildew resistance (Zhang et al. 2017). They were able to simultaneously knock-down the three homologs of EDR1 generating Taedr1 wheat lines tolerant to mildew-induced cell death. Similarly, a group of researchers targeted lipoxygenase genes (TaLpx1 and TaLox2) for enhancing wheat resistance to fusarium, the causative agent of one of the most destructive fungal diseases. Lipoxygenases stimulate jasmonic acid-mediated defense mechanism in plants by hydrolyzing polyunsaturated fatty acids and initiating oxylipin biosynthesis. TaLpx-1 gene silencing has been reported to render wheat plant resistant towards Fusarium graminearum (Nalam et al. 2015). The mutation frequency of 9% and 45% was achieved for TaLpx1 and TaLox2 genes, respectively (Shan et al. 2014; Wang et al. 2018b). Hexaploid wheat plants carrying mutated TaLOX2 were developed with a frequency of 9.5%, accounting for 44.7% homozygous mutants (Zhang et al. 2016). Similarly, three genes earlier reported to be linked with disease susceptibility (TaNFXL1, TaABCC6) and resistance (TansLTP9.4) toward fusarium head blight (FHB) (Ouellet et al. 2013; Balcerzak et al. 2016) were also targeted in wheat to confer resistance against FHB (Cui 2017) (Table 3.3).

Table 3.3 Review of literature summarizing the reports of genome editing for enhancing tolerance to abiotic stresses
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Fungal blast has been associated with extensive losses in rice yields worldwide. Therefore, various techniques are being explored to develop blast-resistant cultivars using advanced gene-editing techniques. In an attempt to develop resistance in japonica rice, Wang et al. performed CRISPR/Cas9-targeted knockout of OsERF922, a negative regulator of fungal blast resistance (Wang et al. 2016). Additionally, Cas9/sgRNAs-mediated multiplex targeting of two or three sites within the OsERF922 gene was also shown to increase mutagenic frequency (Wang et al. 2016). Agronomic traits of the mutant lines were significantly consistent with those of wild-type plants, indicating no negative effect on plant growth and sustainability. Likewise, blast resistance in rice plants was achieved through the disruption of genes OsERF922 and OsSEC3A using the CRISPR/Cas9 system (Ma et al. 2018). OsSEC3A mutation was found to be associated with enhanced resistance against Magnaporthe oryzae, elevated concentrations of salicylic acid, as well as upregulation of defense responsive genes and salicylic acid synthesis (Ma et al. 2018). CRISPR/Cas9-based knock-out of a stress-responsive gene “mitogen-activated Protein Kinase5” (OsMPK5) using three sgRNAs, reportedly enhanced fungal as well as bacterial disease resistance in rice plants by showing constitutive expression of pathogenesis-related (PR) genes. The mutation frequency ranging from 3% to 8% was observed in Nipponbare rice protoplasts (Xie and Yang 2013). DuPont Pioneer is exploring the CRISPR/Cas9-mediated approach to generate improved disease-resistant maize cultivar by targeting the NLB-sensitive allele.

The development of multiplex genome-editing methods further increases the application of CRISPR system in cereal crops to confront more challenging attributes encompassing multiple genes by utilizing a single CRISPR construct (Wang et al. 2018a). A study adapting CRISPR/Cas9 technology to disrupt bE1 and bW2 genes in Ustilaginoidea maydis with efficiency of 70% and 100% in progeny from a single transformant provided proof of concept for developing resistance against corn smut. In more recent reports, the use of CRISPR-based multiplexed genome-editing method has been attempted in hexaploid wheat by employing three sgRNA to target three genes viz. TaGW2, TaLpx-1, and TaMLO to achieve resistance toward FHB and powdery mildew (Wang et al. 2018b). Further, Liang et al. successfully demonstrated an approach which employed CRISPR/Cas9 for efficient gene replacement or editing in Ustilaginoidea virens for imparting resistance against rice false smut, one of the major fungal diseases of rice (Liang et al. 2018). Additionally, a novel co-editing and counter-selection strategy presented by Foster and his colleagues allows precise editing in fungal strains to generate completely isogenic lines with no foreign DNA. The study demonstrated rapid plasmid-free CRISPR/Cas9-mediated editing in Magnaporthe oryzae with improved precision and speed of genetic manipulation which are likely to be applicable to a range of fungal species (Foster et al. 2018).

3.2 Resistance Against Bacterial Diseases

Bacterial diseases such as bacterial leaf blight (BLB) and bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively, are two of the most devastating diseases of rice (Verdier et al. 2012; Mehta et al. 2019b). Moreover, the advent of novel virulent pathotypes of Xanthomonas oryzae (Xoo) (Gonzalez et al. 2007; Mehta et al. 2019b) has rendered conventional breeding programs and resistant cultivars ineffective and further intensifying the utilization of advanced gene-editing tools to combat such diseases. The transcription activator-like effectors (TALEs) are key determinants of Xanthomonas pathogenicity since the activation of host target genes by TALEs is associated with susceptibility and/ or resistance in rice (Bogdanove et al. 2010; Bogdanove and Voytas 2011; Boch et al. 2014). This provides the opportunity to target several TALEs to enhance resistance towards Xoo and Xoc diseases. The sucrose-efflux transporter (SWEET) gene family in rice is the best-studied class of TALE virulence targets which include OsSWEET11, OsSWEET13, and OsSWEET14 genes. The activation of OsSWEET14 gene by TAL effectors AvrXa7 or PthXo3 of Xoo is known to facilitate the export of sugars from plant cell to pathogen which requires it for growth and virulence (Antony et al. 2010). The TALEN-based system was implemented for disrupting the gene associated with bacterial blight defense OsSWEET14 (Os11N3) to confer resistance in rice lines toward AvrXa7- and PthXo3-based Xoo strains in rice lines (Li et al. 2012). Thereafter, some investigations employing TALEN and CRISPR/Cas9-targeting susceptible genes have been conducted to impart resistance toward blight disease in rice (Jiang et al. 2013; Hutin et al. 2015; Blanvillain-Baufum et al. 2017; Cai et al. 2017). In an attempt to demonstrate the CRISPR/Cas9-mediated targeted gene alterations, Jiang et al. 2013 designed CRISPR/Cas9-sgRNA constructs capable of inducing site-specific disruption in the promoter sequence of bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11 for enhanced resistance (Jiang et al. 2013). CRISPR/Cas9 system was also deployed to construct OsSWEET13 null mutant in indica rice, IR24 to prevent TAL effector gene pthXo2-mediated neutralization, thereby improving the resistance toward bacterial blight disease (Zhou et al. 2015). Genetic modification through TALEN of EBEtal7 binding site in promoter sequence of Os09g29100 gene by removal of its Tal7-binding sequence can reduce the severity of bacterial disease through avrXa7-Xa7 defense in rice (Cai et al. 2017). Some other TAL effectors such as AvrXa7, TalC, and Tal5 contributing to Xoo susceptibility in rice can also be exploited to target OsSWEET14 to improve blight resistance (Blanvillain-Baufum et al. 2017). The potential of rice disease resistance genes Xa10-Ni or Xa23-Ni to impart broad-spectrum resistance to Xanthomonas oryzae pv. Oryzae has also been explored through TALEN and CRISPR/Cas9 technology (Wang et al. 2017).

Recently, a Japonica rice cultivar with an improved resistance to Xanthomonas oryzae pv. oryzae was developed by CRISPR/Cas9-based gene mutagenesis of Os8N3/OsSWEET11. The mutant lines were observed to possess equivalent agronomic characteristics such as plant height, leaf length/width, the number/length of panicles, and pollen development (Kim et al. 2019). Furthermore, a research group engineered a broad-spectrum resistance to bacterial blight in rice through CRISPR–Cas9 gene editing. They systematically targeted multiple sites in SWEET promoters to confer resistance in Kitaake, and elite varieties IR64 and Ciherang-Sub1 exhibiting normal agronomic features (Oliva et al. 2019). A multitude of such studies ascertain the prospects of advanced genome-editing strategies for engineering important cereal cultivars with reduced susceptibility to bacterial diseases. In 2019, Oliva et al. showed the role of CRISPR/Cas9 in curing bacterial blight using genes SWEET11, 13, and 14 in rice cultivar Kitaake. They used ZmUbi promoter and mutations in promoter regions were observed.

3.3 Resistance to Viral Diseases

Rice tungro disease (RTD) is caused by the concurrence of two different viruses , namely rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (RTBV), which severely affect rice production across tropical Asia (Macovei et al. 2018; Mehta et al. 2019b). Macovei and his team attempted to engineer resistance in RTSV-susceptible IR4 rice through CRISPR/Cas9-mediated changes in the eIF4G gene (Macovei et al. 2018), which is known to control RTSV resistance. These resistant plants carrying mutant eIF4G alleles can further be exploited as a source for additional RTSV-resistant rice varieties. Kis et al. (2019) showed the role of DsRED protein for reporting purposes by indicating that the role of CRISPR/Cas9 under ZmUbi promoter via the Agrobacterium-mediated transfection method led to resistance against wheat dwarf virus.

4 Engineered Abiotic Stress Tolerance

Plants experience unfavorable environmental conditions very often called abiotic stress, for example, excess of sunlight, excess CO2, decreased availability of soil minerals, decreased availability of water or excess of water, very extreme temperatures (hyperthermia or hypothermia), presence of toxic ions in the soil, etc. (Compant et al. 2010; Hirayama and Shinozaki 2010; Lal et al. 2018). These abiotic stresses influence various attributes of the plant affecting development and output (Rejeb et al. 2014; Lal et al. 2018). Abiotic stress is responsible for up to 50% losses in crop yields (Rodziewicz et al. 2014; Sharma et al. 2020). According to IPCC (intergovernmental panel on climate change), plants experience abiotic stress due to ever-changing climatic circumstances (Mittler 2006). which result in huge loss of food security and environmental sustainability in developing countries (Andy 2016). This has attracted the attention of researchers to develop advanced adaptation strategies for plants under stress and make them adaptable under changing environmental conditions (Wheeler and Von Braun 2013). The major challenge is to identify how these plants respond to different stresses by activating different pathways and switching on/off of responsible genes (Wallace et al. 2003; Andy 2016; Anamika et al. 2019). The capability of plants to overcome or to develop resilience against all these factors solely depends upon photosynthesis in addition to various other physiological or genetic processes. A well-known feature of plants to combat abiotic stresses is the involvement of several genes, and activation and deactivation of several interlinked molecular pathways. Abruptly changing abiotic stresses such as temperature, air humidity, or light evokes multiple intracellular processes at the molecular, biochemical, cellular, and physiological levels (Vahisalu et al. 2010; Mittler et al. 2012; Vainonen and Kangasjärvi 2015; Suzuki et al. 2015; Dietz 2015; Hulsmans et al. 2016; Pommerrenig et al. 2018). CRISPR/Cas9 is used extensively for inducing site-specific mutations in many grass plant species such as rice, sorghum, wheat, and switchgrass.

4.1 Drought Resistance

Drought stress is one of the outcomes of climate change that has an adverse impact on crop growth, and yield by affecting biochemical and physiological processes (Husen et al. 2014, 2017; Getnet et al. 2015; Embiale et al. 2016; Siddiqi and Husen 2017, 2019). It is due to unavailability of water to plants which may cause shoot biomass reduction and grain yield losses. Yield losses are maximum due to water stress as compared to other stresses (Farooq et al. 2009). Water depletion in the rooting area causes increased vapor pressure deficit, which multiplies drought stress (Ahanger et al. 2014). This stress leads to increased crop yield losses when compared to other abiotic stresses. Thus, the intensity and duration of drought stress coupled with other environmental factors play a key role in determining crop yields. The reductions in yields depends on plant type, growth stage, severity, and longevity of the drought conditions. Lou et al. (2017) showed amelioration of drought stress by employing CRISPR/Cas9 approach on SAPK gene under SAPK promoter via PEG-mediated transformation. ROS detoxification was observed in the mutant plants.

4.2 Cold and Heat Resistance Stress

Every crop requires optimal temperature for its optimal growth. Below optimum temperatures result in cold stress while temperatures above optimal temperature results in heat stress. The temperature of 0–10 °C results in chilling stress which is very common in temperate and subtropical species such as cereals. Cold shocks in early reproduction stages lead to flower abortion, pollen and ovule infertility, as well as low seed sets in cereals which hamper metabolic rates and hence, grain yields (Thakur et al. 2010). The low-temperature shock causes enormous changes in membrane permeability, free proline content, and malonic dialdehyde (MDA) (Nesterova et al. 2019). Under frost conditions, photosynthesis gets hampered due to low temperature and internal injuries occur, resulting in ROS production (Sharma et al. 2020). Different physiological processes are greatly affected due to temperatures above the ambient temperature such as rate of photosynthesis and respiration, production of ROS, etc. Since flowering has low threshold value, it gets affected the most at high-temperature stress and thus, there is less seed formation and loss in grain yield (Prasad et al. 2017). Ann3 gene was edited by CRISPR/Cas9 for curing cold response using the Agrobacterium-mediated approach where survival of knockout plants was observed. There was an improvement in cold stress tolerance as well as reduced electrical conductivity as observed by Shen et al. (2017).

4.3 Salinity and Submergence Stress

High salt concentration above a certain threshold concentration is considered as salt stress. Soil salinity is one of the most damaging abiotic stresses. This stress damages plant crops and yield loss has been reported in several investigations (Husen et al. 2016, 2018, 2019; Hussein et al. 2017; Siddiqi and Husen 2017, 2019). It has been reported that 7% of total cultivable land area and 20% from irrigated arable land are stressed due to excessive salt concentrations (Li et al. 2014), thus leading to decrease in crop yields via overpowering the crop performance due to non-availability of nutrients to plants. India has a 6.74 million ha area under salt stress. Submergence stress is due to waterlogging, Water-deficient conditions result in drought and excess of water results in waterlogging which further affects biological processes in crop and results in yield loss. During the early flowering, crop metabolism is very sensitive. Waterlogging gives rise to several fungal diseases, and under these conditions, requirement of metabolic energy shoots up. Production of metabolic energy decreases due to anaerobic respiration and growth of the crop is arrested. RR22 gene was mutated using the CRISPR/Cas9 approach for salinity tolerance using the Agrobacterium-mediated transformation method. Similarly, the successful application of CRISPR resulted in improved agronomic traits as well as salt tolerance (Zhang et al. 2019a, b).

4.4 Adaptation Stress

Plants face different kinds of biotic or abiotic stress, and each of them has their genetic makeup to fight against these stresses and come up with a solution for adaptation in the changing environment. “Survival of the fittest” or the natural law of selection is applicable everywhere regarding the survival of the fittest. Adaptation stress is the stress faced by different crops to adjust themselves according to the evolving environmental conditions. Physiological changes, molecular changes, or biochemical changes are constitutive processes undergoing within cellular structures and switching ON/OFF of different genes allows plants to survive in the evolving environmental conditions. Zhang et al. (2020) recently published a study where CRISPR/Cas9-based base editing in maize inbred line resulted in transgene-free semi-dwarf maize, which is more efficient in withstanding environmental stress.

5 Conclusion

Cereal crops are a prime component of the human diet accounting for a significant proportion of nutrition consumed worldwide. The trends of the progressively growing population and predicted climate changes are expected to boost the global crop demand. Genome engineering techniques are powerful tools that are likely to contribute significantly to the redressal of these anticipated challenges. Advanced gene engineering techniques such as CRISPR/Cas9 have superseded the limitations of conventional genomics-based breeding approaches for crop improvement by eliminating the obstacle of genotypic limitation. Moreover, genome-editing methods surmount the imprecision associated with the use of markers by engineering innovative alternatives affecting yield and stress tolerance. Genome engineering facilitates the production of cereal crops with superior agronomical traits such as better yield, enhanced resistance to biological as well as non-biological stress, by targeting a suite of genes controlling these factors. The applications of genome-editing techniques have broadened in the field of cereal research, as it permits the biofortification of cereals in terms of favoring human health. The cereals of the future can now be developed with a specified composition and quality and offering desired nutritional performance and end-use applicability in foodstuffs. With the ability of genomic rearrangement combined with its high potential to simultaneously edit multiple genes associated with plant characteristics and the regulatory elements, the modern gene-editing tools enable crop improvement by targeting complex traits. This multiplexing approach further allows a useful combination of edits to stack multiple traits in a new variety. These techniques enable the production of transgene-free crop varieties. However, the regulation of genome-edited cereals and public acceptance puts significant constraints in the commercialization of these crops. Therefore, these issues must be addressed to accurately differentiate between transgenic and non-transgenic genome-edited crops because, unlike transgenic varieties, genetically engineered crops can be indistinguishable from crops formed by conservative breeding methods. The challenges associated with off-target alterations and changes in cleavage effectiveness remain to be overcome for establishing efficient genome-editing methods for crop improvement. The systematic analysis of target sites using efficient genomics, the upgraded delivery systems, and the availability of high throughput screening methods needs to be taken into account to modify essential cereal crops. Further, the capability of the CRISPR/Cas9 system to deploy multiple gRNAs and the availability of NGS-next-generation sequencing technologies will provide adequate data for the comparison of gene-editing systems in a diverse range of crop species. The progressive research being carried to develop and improve gene-editing methods is expected to bring a revolution by addressing the agricultural issues related to yield, quality, and biotic/abiotic stress management. Overall, the genome engineering system offers numerous opportunities to improve cereal crops by overcoming the antagonistic effects of climate change and may support global food security.