See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/346140843 Can CRISPRized crops save the global food supply? Chapter · November 2020 DOI: 10.1016/B978-0-12-821910-2.00006-0 CITATIONS READS 0 85 2 authors, including: Ki-Taek Lim Kangwon National University 108 PUBLICATIONS 974 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Lim's Group View project CRISPR and RNAi Systems View project All content following this page was uploaded by Ki-Taek Lim on 24 November 2020. The user has requested enhancement of the downloaded file. CHAPTER PR OO F Can CRISPRized crops save the global food supply? 1 Kamel A. Abd-Elsalam1, Ki-Taek Lim2 1 2 Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt, Department of Biosystems Engineering, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Republic of Korea EC TE D 1.1 Introduction UN CO RR The world’s population will rise from 9.1 billion to 9.6 billion, 34% higher than today by the year 2050; thus global food production is expected to increase by as much as 70%–100% to sustain the increasing world population (Tilman et al., 2011; Fróna et al., 2019). To take care of and sustain a quickly expanding populace confronted with a warming domain, reduced arable land, and a lack of available water supplies, progresses in plant breeding innovation are critically needed to increment rural efficiency and lift practical farming development (Chen et al., 2019). Cross-reproducing, transformation rearing, and transgenic rearing are the essential strategies for crop advancement in current horticulture. Such tedious, arduous, and untargeted reproducing procedures cannot satisfy the expanding worldwide need for food in near future (Wolter et al., 2019). To meet the challenge of feeding 10 billion people and increasing the efficiency of crop breeding systems, type of marker-assisted breeding methods and plant transgenic approaches have been employed to produce necessary characters through exogenous transformation into selected genotypes. (Hickey et al., 2019). Genome editing has already been successfully implemented for commercially important agricultural organisms, such as crops and farm animal husbandry, improving the productivity of plant and animal breeding, and providing new approaches for pest and disease control. The rapidly increasing use of genome editing has policy implications and boosts considerations concerning human health and environmental safety (Lassoued et al., 2019). Plant genome editing became conceivable when the principal designer nucleases were presented in the last part of the 1990s, but since 2012 after the development of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 method, the field has exploded in popularity (Jansing et al., 2019). CRISPR-associated protein (Cas) and RNA interference (RNAi) systems have recently gained growing attention in the Academic Press and Wider Media (Wada et al., 2020). The CRISPR/Cas9 system was applied successfully to plant breeding programs for different plant species, such as fruit, vegetables, ornamental crops, and grain crops (Jaganathan et al., 2018; Corte et al., 2019; Hussain et al., 2019). CRISPR/Cas systems have transformed plant genome engineering, due to their high performance, simple engineering and robustness, and democratized its application. The current situation with this innovation takes into account numerous applications that are CRISPR and RNAi Systems. https://doi.org/10.1016/B978-0-12-821910-2.00006-0 Copyright © 2021. 1 2 CHAPTER 1 Can CRISPRized crops save the global food supply? UN C OR RE C TE D PR OO F appropriate for improving crop yield, resistance for plant diseases, and environmental change transformation (Sedeek et al., 2019: Ansari et al., 2020). CRISPR/Cas9 would likely grow in popularity, mainly based on genome editing and speed breeding. It will be an important technique for obtaining plants with particularly desirable characteristics and helping to achieve our global zero-hunger targets (Eş et al., 2019). In the first volume of the Nanobiotechnology for Plant Protection Book Series, we review developments in RNAi and CRISPR systems and analyze their applications in plant genome editing and plant protection. We sum up RNAi and CRISPR–Cas applications as genome editing tools for disease-resistance development in field breeding and horticultural crops such as cereals, cotton, and wheat. Such new plant genomic devices would permit innovative development, for example, double-strand RNA (dsRNA) splashes, to build crop protection from pests, microbes, and virus infections; in some applications, such may even replace chemical pesticides. Subsequently, targeted genome engineering was reported for insect control and the relocation of crop pesticides. For example, the recent progress in genetic engineering at oomycetes and Phytophthora, RNAi strategy has been used to manage phytopathogenic fungi. RNAi and CRISPR/Cas9 applications have been applied to control mycotoxins, and CRISPR has applications in plant bacteriology, too. CRISPR–Cas systems were used as antimicrobial agents in the agrifood field to prevent pathogenic biofilms. Prospects for integrating this innovative technology with phytoalexin biosynthesis through RNAi for disease resistance are also discussed, such as the role of the CRISPR/Cas system in altering plant-defense phenolics and carotenoid biosynthesis and the role of small RNA, RNAi, and CRISPR technology in improving abiotic stress tolerance. Until this point, numerous bioinformatics methods have been created to design and produce single guide RNA and are accessible on the web. Thus CRISPR/Cas and RNAi databases and bioinformatics methods for genome editing in plants are explored. Several novel cargo-vector systems have recently been implemented which demonstrate promising potential as efficient delivery systems, for example, the use of inorganic smart nanoparticles, polymers, and lipid-based nanoparticles to deliver RNAi and CRISPR systems to plant cells. We likewise assessed novel advancements that expand the capability of genome editing systems in crops and their commercialization prospects, such as patenting dynamics in CRISPR gene editing technologies. Agricultural applications for RNAi and CRISPR might face the same challenges as standard genetically modified organisms (GMO); thus the regulatory issues, risk evaluation, and toxicity associated with RNAi and CRISPR are discussed. Given population growth, global food security is becoming increasingly challenging; therefore the world needs more nutrient-rich, environmentally sustainable food production. The CRISPR system may be integrated soon with speed plant breeding programs to reshape the future of agrifood supply and safety. 1.2 Gene editing techniques Impressive advancement has been made in molecular plant breeding in the course of the most recent couple of years. The whole-modified DNA sequence of some genomes was elucidated and annotated (Michael and Jackson, 2013), whereas datasets were collected with data about a huge number of qualities and their gene expression (Wingender et al., 2000). Furthermore, a more profound comprehension of the elements of the structure and capacity of the genome, present-day biotechnology has made new methods that empower controlled adjustment of DNA modifications inside various plant 1.3 RNAi and CRISPR systems for plant breeding and protection: where are we 3 CO RR EC TE D PR OO F genomes. Currently, the likely significance of plant genome editing innovation for essential and applied plant science cannot be overvalued. The capacity to change hereditary data in an exact and precise manner, and to reestablish harmed plants, permits quality capacity and examines organic systems, yet additionally the advancement of innovative phenotypes, conceivably. As interest for farming yield develops with an expanding human populace, more modern methodologies will be expected to build more unpredictable characteristics in different plants, for example, improved yield and stress resilience (Petolino, 2016). In this way, we are trying to demonstrate the most common techniques for quality improvement in plants in the accompanying passages. The tool compartment for genome design has three primary stages: zinc-finger nucleases (ZFNs), CRISPR/Cas frameworks, and transcription activator-like actuator nucleases (TALENs). ZFNs and TALENs are protein-based frameworks that involve protein design for each user-defined sequence. CRISPR/Cas technique is in this manner an RNA-guided gadget and can be effectively arranged to tie to the targeted DNA (Belhaj et al., 2013; Osakabe and Osakabe, 2015; Quétier, 2016). There are five types of genome modification systems that can be utilizing the devices portrayed above. Each of the five sorts of modifications has been reported in crop plants, and a few instances of quality alterations related to significant characteristics, and results are examined underneath. Fig. 1.1 presents an outline of the different genome editing techniques, the possible consequences of genome modification for various examples from plant genotypes and instances of adjusted and developed yield characteristics (Jansing et al., 2019). RNAi technique is being created and used to improve plants by adjusting endogenous quality articulation just as focusing on both pests and plant pathogenic in addition to yield qualities. RNAi is an accepted mechanism that can be used to make a significant commitment to integrated pest management and practical horticultural methodologies that are required worldwide to make sure about current and future food supply (Mezzetti et al., 2020). CRISPR/Cas9–Cas9 is a multipurpose hereditary designing innovation that relies upon the complementarity of the guide RNA to a specific nucleic acid sequence and the action of Cas9 endonuclease. Recently, the revelation of the CRISPR/Cas9 framework has changed genome editing and stood out as an incredible asset for an assortment of modern applications (Eş et al., 2019). Two primary methodologies have been utilized to bind the RNAi pathway for silencing gene expression: treatment with manufactured small interfering RNA (siRNA) molecules or expression of short hairpin RNAs that are intracellularly handled into dynamic siRNAs or microRNAs. In the present volume, we survey the improvement of these two impedance devices, portray the specialized difficulties yet to be tended to, and give some knowledge on how these two RNA-based innovations can be used in plant breeding and protection applications. UN 1.3 RNAi and CRISPR systems for plant breeding and protection: where are we now? Crop breeders work hard to grow crops that resistant to abiotic stress like climate-resilient and stress-tolerant, with better-quality for various parameters and improved plant production (Zaidi et al., 2016). Therefore the techniques of RNAi and CRISPR have various applications for plant gene functional genomic research that assume a critical function in the hereditary improvement of numerous significant agronomic attributes. Specifically, the knockout of specific qualities will advance unrivaled attributes which incorporate disease resistance, transformation to different abiotic stressors, utilization of supplements, and promotions in yields (Razzaq et al., 2019). Recent advances in genome editing CHAPTER 1 Can CRISPRized crops save the global food supply? OR FIGURE 1.1 RE C TE D PR OO F 4 Outline of genome editing methods, conceivable hereditary results for each case, and instances of yield qualities created utilizing described protocols. Colored arrows and boxes have connected yield trait examples to associated genome editing tools and their effects. - Data from Jansing, J., Schiermeyer, A., Schillberg, S., Fischer, R., Bortesi, L., 2019. Genome editing in agriculture: technical and practical UN C considerations. Int. J. Mol. Sci. 20 (12), 2888. This is an Open-Access article distributed under the terms of the Creative Commons CC-BY license. via RNAi and CRISPR/Cas allows successful targeted modification in most crops, thereby promising to accelerate crop development. The removal of negative elements is a positive method to improve genetics. Thus the simplest and most common application of CRISPR/Cas9 is the knocking out genes which confer undesirable traits. Traits that have been improved with CRISPR/Cas9 to date include yield, quality, and resistance to biotic and abiotic stress (Chen et al., 2019). The approaches of RNAi and CRISPR, and their contribution to achieving desirable traits by modifying genetic expression, have shown their potential for crop enhancement. 1.3 RNAi and CRISPR systems for plant breeding and protection: where are we 5 1.3.1 Improving yield and quality in crops UN CO RR EC TE D PR OO F Yield is the main target for crop enhancement gene editing to ensure improved food safety. It is a complex trait which depends on a lot of factors. There are additionally numerous utilizations of CRISPR/ Cas9 innovation for improving yield quality, for example, nutritional value, quality of storage, aroma, and starch content (Fig. 1.2). For example, the cooking and eating nature of rice has been improved by using CRISPR/Cas9 to mutate the Waxy gene (Zhang et al., 2018). The removal of negative regulators considered to influence yield-determining variables, for example, grain size, grain weight, grain number, panicle size, and tiller number has helped to establish the predicted some phenotypes in plants with loss-of-function mutations in these genes indicate that CRISPR/Cas9 is an important method for improving yield-linked characteristics (Razzaq et al., 2019). Develop unique functional components from naturally existing peripheral devices such as genes, promoters, cis-regulatory elements, small RNAs, and epigenetic modifications that would energize the making of regulatory pathways for assorted rice grain quality components (Fiaz et al., 2019). Based on the particular breeding requirements the quality traits differ. Up to now, improved yield quality through genome editing has affected crop starch content, aroma, nutritional value, and storage efficiency. Several examples of the development of quality traits via genome editing based on CRISPR-Cas9 include (1) removal of undesirable compounds (e.g., compounds that negatively impact nutrient absorption, have allergenic potential, or exhibit toxicity) and (2) increasing value compounds (e.g., oleic acid for the production of high thermal stability oils). FIGURE 1.2 Applications of RNAi and CRISPR systems to increase and improve crop yields. RNAi, RNA interference; CRISPR, clustered regularly interspaced short palindromic repeat. 6 CHAPTER 1 Can CRISPRized crops save the global food supply? 1.3.2 Biotic and abiotic stress resistance RE C TE D PR OO F Plant pathogens and plant insects such as viruses, bacteria, fungi, and nematodes are the main causative agents that cause biotic stress and reduce crop yield. Besides, the ongoing upsurge of many new strains of lethal pests makes the fight against these pathogens extremely difficult; worldwide losses associated with 137 pathogens, and pests associated with wheat, rice, maize, potato, and soybean have been reported (Savary et al., 2019). Therefore understanding plant–pathogen interactions are very important in protecting agriculture from the destructive effects of biotic stressors (Kettles and Kanyuka, 2016). Strategies for RNAi and CRISPR gradually expanded to the study of plant–pathogen interactions and processes underlying the plant responses to pathogenic attacks. Stress is a major factor that influences crop yield and quality. Numerous plants with increased resistance to biotic stress, including various plant pathogens and pests were obtained through CRISPR/Cas9 knockout. Another obstacle to the successful implementation of RNAi-based plant protection is the development of effective methods to deliver dsRNA at the correct dose and to the appropriate location (i.e., plant surface vs vasculature) (Liu et al., 2020). Chemically synthesized dsRNA can be delivered to the plant either by direct application to the leaf or through different other strategies as shown in Fig. 1.3. Ingestion of dsRNA/small RNA present on the surface of the plant and/or inner tissues by an insect predator results in the presence of this RNA in the lumen of the insect gut. Additionally, RNAi and CRISPR technology has been extensively applied to cope with various abiotic stressors in major crop plants such as rice, wheat, maize, soybean, cotton, tomato, and potato. The Cas9 system was applied as a reliable, effective, and practical approach to the production of climate tolerant crop varieties. Moreover, the possibility of creating new quantitative trait loci for abiotic stress tolerance has been investigated through CRISPR/Cas-mediated promoter targeting (Zafar et al., 2020). The current book outlines many recent studies showing significant progress of the RNAi and CRISPR system against different plant pests, diseases, and the growth of abiotic stress-tolerant crops (Fig. 1.4). OR 1.3.3 Speed breeding programs in plants UN C Overall, plant scientists use innovative methods such as speed breeding, genome editing programming, and high-performance phenotyping to improve the efficiency of plant breeding. A male-sterile maternal line is a prerequisite for producing a high-quality hybrid variety. To meet the needs of breeders, genome editing is also a successful approach to enhancing many other traits, such as enhancing haploid breeding (Dong et al., 2018; Yao et al., 2018), shortening growth times (Li et al., 2017), increasing silica shatter resistance (Braatz et al., 2017), and overcoming diploid potato self-incompatibility (Ye et al., 2018). Several crops are currently developing speed breeding protocols (Watson et al., 2018; Ghosh et al., 2018). Unlike double haploid technology in which haploid embryos are developed to produce fully homozygous genotypes, speed of breeding is sufficient for diverse germplasms and does not require advanced in vitro equipment (Slama-Ayed et al., 2019). Using speed breeding, successive generations of improved crops can be developed for field examination utilizing single-seed descent, which is cheaper than the production of doubled haploids (Hickey et al., 2017; Wolter et al., 2019; Ahmar et al., 2020). It will also help to achieve food security by creating crops with better-quality food through genome editing techniques (Narayanan et al., 2019; El- 7 EC TE D PR OO F 1.3 RNAi and CRISPR systems for plant breeding and protection: where are we FIGURE 1.3 RR dsRNA delivery methods for RNAi-mediated insect control. Each of these methods, depending on the conditions involved, provides certain benefits. Dashed circles show methods of delivery, which are widely used for research purposes but are inappropriate for the safety of large-scale crops. dsRNA, double-strand RNA; CRISPR, clustered regularly interspaced short palindromic repeat. - Data from Liu, S., Jaouannet, M., Dempsey, D.M.A., Imani, J., Coustau, C., Kogel, K.H., 2020. RNA-based technologies for insect control in CO plant production. Biotechnol. Adv. 39, 107463. This is an open-access article distributed under the terms of the Creative Commons CC-BY license. UN Mounadi et al., 2020). With the aid of speed breeding, current genome editing techniques can be enhanced (e.g., genes responsible for late flowering could be knocked out using CRISPR/Cas9). When Cas9 has been effectively inserted into the plant, the transgenic plant would then be able to develop under-speed growing conditions instead of the typical conditions in the greenhouse to create transgenic seeds that were ahead of schedule, as could reasonably be expected. Through using that method, stable homozygous phenotypes can be obtained in less than a year. Moreover, this approach often reductions time needed for breeding, as the production of a GMO crop normally takes many years. CHAPTER 1 Can CRISPRized crops save the global food supply? TE D PR OO F 8 FIGURE 1.4 Applications of RNAi and CRISPR strategies for biotic and abiotic stress tolerance. RNAi, RNA interference; CRISPR, clustered regularly interspaced short palindromic repeat. RE C 1.4 What are future perspectives? UN C OR Both RNAi and CRISPR/Cas systems have enormous potential to improve plant modifictions and engineered science. For instance, artificial DNA sequences, including promoters, chromosomes, genome congregations, and transcriptional regulatory elements, might be embedded into plant genomes to adjust cell or plant conduct to produce novel capacities and functions (Chen et al., 2019). Engineered neo-domesticates can be used directly as crops or crossed by elite lines to add new features without the time lag associated with wild germplasm use. In the coming years, new domesticated crops will be more tolerant to a variety of difficult conditions, including deserts, coastal areas, low-nutrient soils, and cold climates, it is likely to enhance crop diversity and help to solve many of the concerns related to sustainable agriculture. Producing valuable gene transmission systems and developing new modification systems will be crucial to reducing challenges to inexpensive plant gene editing applications. Delivery systems based on nanomaterials can deliver functional genes or siRNA to intact plant cells and produce genetically engineered transgene-free plants. This system enables highly efficient and organ-specific delivery that can transcend the limitations of the host range. This approach would have a wide range of applications in plant biotechnology and plant biology (Wang et al., 2019). CRISPR and gene-controlled systems based on RNAi will prove to be beneficial to humans in time. For example, they can eliminate disease epidemics, improve farming production, and control the transmission of pathogens, such as plant cultivars that are known to be resistant to insect pests and herbicide-sensitive pathogens. For using this technology effectively and durably in crop improvement, the numerous biosafety and societal concerns regarding this technology need to be resolved by the scientific community. There is also a need to reconsider the drawbacks of genome edit Conclusion 9 PR OO F ing plants and to notify the general public of their resources. However, sustainable crop production to ensure global food security can only be done by the joint efforts of the private industry, extension staff, and the government sector. We should expect to be creating a wide variety of strategies in the future using interdisciplinary concepts to maximize their advantages (Fig. 1.5). Crop production techniques, breeding methods, field test approaches, genotyping technologies, even equipment and facilities across crop species need to be applied to keep our fiber, food, and biobased economy diverse. Conclusion CO RR EC TE D A strong breeding strategy, such as speed breeding, that saves time is required to feed the world’s population, which is expected to be 9–10 billion in 2050 (Hickey et al., 2017). The speed breeding method is also being developed with the help of the RNAi and CRISPR techniques to decrease the life cycle of the crop. In a very short time, a variety can be developed by decreasing the crop cycle. These modern techniques will maximize yield, adaptability, and biotic and abiotic resistance in a very short time as compared to traditional breeding methods. By using both techniques sufficient, food can be produced to meet the growing population’s food requirements. FIGURE 1.5 UN Future applications of RNA interference systems in crop improvement. 10 CHAPTER 1 Can CRISPRized crops save the global food supply? References UN C OR RE C TE D PR OO F Ahmar, S., Gill, R.A., Jung, K.H., Faheem, A., Qasim, M.U., Mubeen, M., et al., 2020. 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