Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency

Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Biotechnology Journal This is a preprint and has not been peer reviewed. Data may be preliminary. 26 August 2024 V1 Latest version Share on Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency Authors : Pu Yuan , Muhammad Usman , Wenshan Liu , Ashna Adhikari , Chunquan Zhang , Victor Njiti , and ye xia 0000-0002-4749-3119 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.172471610.04317310/v1 Published Biotechnology Journal Version of record Peer review timeline 480 views 271 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the CRISPR-Cas9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to non-model plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress. Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency Pu Yuan 1 , Muhammad Usman 1,2 , Wenshan Liu 1 , Ashna Adhikari 1 , Chunquan Zhang 3 , Victor Njiti 3 , Ye Xia 1* 1 Department of Plant Pathology, College of Food, Agricultural and Environmental Sciences, The Ohio State University, Columbus, OH 43210, USA. 2 Department of Plant Pathology, University of Agriculture, Faisalabad, 38000, Pakistan 3 College of Agriculture and Applied Sciences, Alcorn State University, Lorman, MS 39096, USA *Corresponding author: Ye Xia, E-mail: [email protected] Abstract: Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the CRISPR-Cas9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to non-model plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress. KEYWORDS Plant gene editing; CRISPR-Cas9; construct design; transformation efficiency; non-model plant species 1. INTRODUCTION 1.1 Overview of plant gene editing technology The rapid increase in the world’s population and the competitive market for agricultural products are reducing agricultural productivity while increasing the demands for biofuels, food, and feed. [1] . By 2050, there is a prediction of increase in world’s population up to 9 billion, potentially doubling the demands for crop production [2, 3] . Therefore, there is a significant need to increase the production of staple crops (such as wheat, rice, maize, soybean, and cotton) by 38–67% [3] . To address these challenges and improve the production of essential crops, it is critical to enhance the process of developing high-yielding varieties through traditional/conventional breeding or genetic engineering. Traditional/conventional breeding has a complex set of limitations, including the potentials for undesirable mutations and time-consuming nature of the process, which can take several years from initial screening to commercial variety release [4, 5] . Genome editing tools have emerged as potent techniques for precisely altering crop genomes at targeted locations within the genome [6] . These tools offer the great potentials to enhance crop productivity, disease resistance, breeding efficiency, and the creation of novel plant and animal models [7] . In the mid-twentieth century, Watson and Crick published the molecular model of deoxyribonucleic acid (DNA), the fundamental carrier of genetic information. Approximately two decades later, in 1972, Paul Berg’s laboratory achieved a milestone by developing recombinant DNA technology, which was considered a significant achievement in the field of molecular biotechnology and genetics [8] . Since then, a complex array of research activities has been carried out in the fields of biochemistry and molecular genetics, with a special focus on viruses and bacteria. These efforts have led to the elucidation of numerous genetic and molecular mechanisms. These dedicated efforts have opened an innovative door in the field of molecular genetics, leading to the manipulation of DNA and the development of various vector systems for cellular uptake. These advancements have facilitated the successful generation of transgenic microorganisms and genetically modified crops. These insights in the field of genetic engineering have accelerated the development of new genome editing tools by scientists and researchers. For instance, at the end of the twentieth century, it was observed that specific protein domains known as zinc fingers can act as zinc finger nuclease (ZNFs) when coupled with a specified endonuclease domain Fokl [9] .This new insight initiated the editing of cultured cells in both model and non-model plants [10] . Continuous research efforts led to the development of most advanced genome editing techniques, including TALENs (transcription activator-like effector nucleases), multiplex automated genome engineering (MAGE), CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein), and RNA interference (RNAi). Traditionally, the process of designing TALENs requires the re-engineering of a novel protein for each target. Over time, advancements and modifications in this technique have streamlined the design process, resulting in a reduction of cloning required for each target. Usage, handling, and designing of CRISPR is much easier as compared to TALENs. MAGE is a genome editing technique that allows the rapid editing of an organism’s DNA to produce multiple changes across the genome. This technology can efficiently create a diverse set of genetic changes, such as mismatches, insertions, and deletions [11] . Although both CRISPR/Cas and MAGE can be used for multiplexing by simultaneously targeting multiple genomic sites, CRISP/Cas may require more optimization to achieve efficient multiplexing compared to MAGE. Additionally, RNA interference (RNAi) as important tools to suppress the expressions of the gene of interest have also gained a significant attention from the scientific community to achieve the desired traits in crop protection and improvement [12] . The mechanism of RNA interference is accelerated by low molecular weight interfering RNA molecules to silence the gene of interest and achieve desired traits [13] . The silencing mechanism starts with Dicer enzyme, which processes double-stranded RNA to transform the silencing signal into small interfering RNAs (~22-nucleotide). Currently, a variety of various genome-editing tools have been embraced to address challenges encountered in plants, aiming to meet the increased food requirements [14] . All these genome-editing techniques are currently used by scientists for the betterment of mankind, addressing complex challenges through the creation of mutants and transgenic plants [15-16] for improved plant health, quality, and yield. In summary, the most popular and commonly used gene-editing tools are EMNs ( endo/meganucleases ), ZFNs, TALENs, CRISPR, MAGE, and RNAi, as they facilitate the modification of important crops for improved traits and yields [17] . 1.2 Importance of precise genetic modifications in agriculture and biotechnology Genome-edited crops offer a variety of advantages for consumers, with numerous success stories for the improved desired traits [18] . Examples include the modification of numerous oilseed crops to enhance their oil content, the improvement of flavor and color traits in tomato fruits, the extension of shelf life in apples and potatoes by inducing non-browning traits, the development of disease-resistant fruits and vegetables, and much more [18-19] . Similarly, several desired traits have been achieved in various genome editing crops, such as the knocking out of VInv gene and PPO gene in potatoes and mushrooms, respectively [20-21] . Moreover, extensive research is conducted worldwide in various oilseeds, staples, fruits, and vegetable crops to enhance their nutritional and functional traits (Table1). Over the last twenty years, the genetically modified (GM) crops have provided health, environmental, and economic benefits to the adopting countries worldwide [44] . These GM crops have significantly contributed to a healthier environment by reducing the indiscriminate use of harmful synthetic pesticides. Over the last two decades, it has been estimated that the consumption of 671.4 million kilograms of active ingredients has been reduced by using insect/disease-resistant GM crops on the same agricultural land area where conventional crops were cultivated [45] . Furthermore, compared to conventional/traditional farming, the cultivation of GM crops has reduced 2945 million kilograms of CO 2 emissions by eliminating the need for fuel usage in operating various machinery for spraying GM insect/disease-resistant crops [45] . In addition to nutritional improvement and economic and environmentally friendly benefits, significant work has been conducted in genetic engineering to enhance crop resistance against various invading fungal, bacterial, and viral pathogens [46] . Fungal pathogens are prominent in causing rusts, smuts, mildews, vascular wilts, and many other destructive diseases in economically-important crops [47] . Mildews are common fungal diseases hosting by a variety of crop plants [48] . The discovery of mildew resistance locus O ( Mlo ) is a well-known S (susceptibility) gene, which had been recognized for its robustness in durable pathogen-resistance programs through mutations [49] . Significant efforts have been made to enhance crop resistance against biotic and abiotic stresses using genetic engineering (GE) tools . For instance, improvements have been made in cotton resistance against Verticillium dahliae by targeting the Ghl4-3-3d gene [50] ; targeting the aLpx-1 gene from wheat to create resistance against Fusarium graminearum using CRISPR/Cas9 [51] ; modifications in the grape genome by targeting WRKY52 (a transcription factor involved in biotic stress) to establish resistance against the gray mold pathogen Botrytis cinerea [52] ; and targeting the Solyc08g075770 gene from the tomato genome against the fungal pathogen Fusarium oxysporum [53] . In addition to fungal pathogens, S genes have also been targeted to induce immunity against important bacterial pathogens. For instance, citrus canker is one of the most devastating diseases caused by bacterial pathogen Xanthomonas citri subsp. citri ( Xcc ), which affects the citrus production drastically worldwide [54] . CsLOBl, previously known as an S gene for Xcc , is a transcription factor targeted by using CRISPR/Cas to induce plant resistance against canker disease [55] . Other successful stories of genome editing against bacterial diseases include the induced resistance in tomato plants against Pseudomonas syringae by targeting SlJAZ2/cds gene through CRISPR/Cas9 [56] ; altering rice genome by targeting OsSWEET11 gene to create resistance against Xanthomonas oryzae pv. oryzae [57] ; and manipulating rice genome by targeting OsSWEET14 against Xanthomonas oryzae pv. oryzae causing bacterial blight of rice [58] . There are also many successful stories of gene editing to improve plant resistance against attacking pests, as discussed in Figure 1. All these clearly demonstrated the effectiveness of these GE tools in inducing resistance against invading pathogens and pests. 2. CRISPR-Cas9 SYSTEM 2.1 Principles of CRISPR-Cas9-Mediated Gene Editing CRISPR-Cas9 is a revolutionary gene editing tool adapted from a bacterial defense system. It allows for the introduction of precise genetic modifications in bacteria, plants, fungi, animals, humans, and other living organisms. Originally discovered in bacteria and archaea, the system harnesses a natural bacterial defense mechanism. Bacteria utilize CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) arrays and Cas9 (CRISPR-associated protein 9) enzyme to target and cleave the invading viral DNA [59] . By mimicking this natural process, scientists have targeted specific locations within the genome and edited DNA with remarkable precision [60] . This technology holds immense potential for researchers to modify genes, potentially correcting mutations that cause genetic disorders. The CRISPR-Cas9 system acts as molecular scalpel for editing DNA. It consists of two main components: Cas9 nuclease enzyme and the guide RNA molecule. The guide RNA is engineered to contain a spacer sequence complementary to the target DNA and a recognition sequence for the Cas9 enzyme. Cas9 binds to the guide RNA and locates the target DNA using the spacer sequence. Once the target is found, Cas9 makes a double-stranded break in the DNA at a specific location next to the spacer sequence. The cell’s natural DNA repair mechanism then takes over, and the break can be repaired either by inserting or deleting nucleotides (insertion/deletion or indel mutations), effectively editing the target DNA sequences [60] . CRISPR technology serves as a powerful toolkit for plant gene editing. It can perform various modifications with high precision, including knocking out unwanted genes, inserting entirely new ones, replacing existing ones with improved versions, or even fine-tuning gene regulation [61] . This versatility empowers researchers to tackle a multitude of agricultural challenges. For instance, CRISPR can be used to introduce disease resistance by disabling genes susceptible to pathogen infection or by inserting genes that bolster the plant’s immune system. Additionally, it can help develop crops with improved nutritional content or better tolerance to harsh environmental conditions. 2.2 Simultaneous Targeting of Multiple Genes Although CRISPR-Cas9 excels at targeting specific locations within the genome, editing can also be directed toward multiple genes simultaneously. This multiplex CRISPR approach offers advantages in various scenarios. One strategy involves designing multiple guide RNAs within a single vector, each targeting a different gene. This allows researchers to efficiently edit multiple genes in a single experiment, saving time and resources [62] . It’s particularly useful for investigating gene interactions or engineering complex genetic modifications. The other method utilizes libraries containing vast numbers of pre-designed guide RNAs targeting a large pool of genes. This approach is valuable for large-scale functional genomics studies, where researchers aim to identify genes involved in specific biological processes [63] . However, multiplex CRISPR editing also presents challenges. For instance, the off-target effects, where Cas9 can mistakenly cleaves in the unintended locations when targeting multiple genes. Additionally, delivering large numbers of guide RNAs into cells can be technically demanding [64] . Despite these hurdles, advancements are being made to improve the efficiency and accuracy of multiplex CRISPR editing. This holds immense promise for accelerating research in areas, such as deciphering complex genetic diseases caused by pathogens. By creating targeted mutations in specific genes, scientists can mimic the effects of various plant pathogens and diseases. This allows them to study the mechanisms of these disease initiation and development at a deeper level, leading to the development of more effective treatments and preventative measures of plant pathogens and diseases for improving plant health. 2.3 Advantages and Applications in Plant Genome Engineering CRISPR-Cas9 technology accelerates the process of developing new plant varieties compared to traditional breeding methods, reducing the time and resources required for crop health and improvement. Therefore, CRISPR-Cas9 offers significant advantages for plant genome engineering. It’s remarkable precision allows targeted modifications of genes that control desirable traits, such as increased crop yield, resistance to pests and diseases, tolerance against abiotic stresses, and improved nutritional content [65] . Researchers can use CRISPR system to correct genes responsible for genetic disorders in crops, potentially eliminating these disorders together. For instance, this paves the way for developing disease-resistant varieties that wouldn’t require harsh chemical treatments. Therefore, CRISPR’s ability to manipulate plant genomes with such precision offers immense promise for accelerating research in plant health and disease management in agriculture. CRISPR technology also revolutionizes agriculture by creating crops with enhanced nutritional profiles, such as sugars and beneficial secondary metabolites. Additionally, CRISPR-Cas9 can be used to introduce genes from different plant species or the other living organisms, enabling the development of plants with entirely new characteristics, like resistance to specific herbicides. However, careful consideration of potential unintended consequences and ethical regulations surrounding genetically modified organisms (GMOs) remains crucial [66] . 3. OPTIMIZATION OF CRISPR CONSTRUCTS IN PLANT GENOME EDITING CRISPR-based genome editors typically consist of Cas protein, guide RNAs (gRNAs), gene regulatory elements (GREs), and other essential components [67] . These elements play pivotal roles in facilitating the expression of Cas gene and gRNAs, thereby enabling efficient editing in plants. This section discusses the strategies for selecting the appropriate Cas protein and GREs, focusing on optimizing promoter and terminator elements for the robust expression of Cas gene and gRNAs, and designing effective gRNAs for specific gene targeting. 3.1 Selection of Cas Proteins in Plant Genome Editing Cas proteins commonly used for plant genome editing include SpCas9 ( Streptococcus pyogenes Cas9), SaCas9 ( Staphylococcus aureus Cas9), NmCas9 ( Neisseria meningitides ), FnCas12a ( Francisella novicida Cas12a, formerly Cpf1), and Cas12b (formerly C2c1) [65, 68] . SpCas9 stands out as the first and one of the most widely used Cas proteins, owing to its high efficiency and well-characterized properties [59] . The process involves loading the sgRNA (single guide RNA) onto Cas9, enabling it to guide the cleavage of target DNA sequences adjacent to 5′-NGG-3′ (N = A, T, C, G) protospacer-adjacent motifs (PAMs). SaCas9, being a smaller Cas9 ortholog compared to SpCas9, exhibits a distinct recognition pattern of 5′-NNGRRT [69] . Furthermore, NmCas9 is the other Cas9 ortholog that has been explored for its potential in plant genome editing, demonstrating efficient genome editing at target sites with N 4 GATT and N 4 CC PAMs using Nm1Cas9 and Nm2Cas9, respectively [70] . Additionally, Cas12 nucleases, among the most widely used Cas proteins in plants, encompass Cas12a and Cas12b, both of which recognize T-rich PAM, typically represented as 5’-TTTV-3’ [71] . 3.2 Designing Effective gRNAs for Precise Genome Editing in Plants Guide RNAs (gRNAs), the essential components of CRISPR-Cas systems, direct Cas proteins to target DNA sequences for accurate genome editing. They comprise a CRISPR RNA containing a spacer and a scaffold sequence referred to as trans-activating CRISPR RNA (tracrRNA) [67] . The design of effective gRNAs is crucial for achieving precise editing outcomes in plants, as it directly impacts editing efficiency specificity, and the occurrence of off-target effects. Websites designed especially for creating guide RNAs (gRNAs) for plant CRISPR-Cas genome editing applications are listed below: CRISPR-PLANT [72] , CRISPR-P [73] , sgRNA Scorer 2.0 [74] , CRISPR-GE [75] , CRISPR-PLANT2 [76] , PlantPAN 3.0 [77] , and PlantRegMap [78] . 3.3 Optimization of GREs Ensuring the effective expression of Cas proteins and sgRNA components is essential for successful CRISPR/Cas-mediated genome editing in plants. Promoters and terminators are pivotal in modulating the levels of Cas proteins and sgRNAs, thus ensuring precise editing outcomes. Here, we delve into the criteria for selecting suitable promoters to drive Cas gene or gRNA expression. For instance , the constitutive promoters, Cauliflower Mosaic Virus promoter (e.g., CaMV35S ) and UBIQUITIN (e.g., UBI10 ), are commonly utilized [79] . Furthermore, the maize DMC1 promoter has demonstrated the notable activity in callus tissue, resulting in a significant production of biallelic or homozygous mutants [80] . gRNAs are predominantly expressed through RNA polymerase III promoters like U6/U3, with the occasional use of RNA polymerase II promoters like CmYLCB [81] . Preferential selection of endogenous U6/U3 promoters is recommended for the optimal editing outcomes, considering their varied expression patterns. Attention to nucleotide composition, such as starting with ’G’ for U6 and ’A’ for U3 promoters, enhances transcription efficiency, which is often aided by design tools like CRISPR-P [67] . Terminators, along with promoters, play pivotal roles in modulating the stability of Cas and gRNA transcripts. For instance, the nopaline synthase (NOS) terminator, derived from Agrobacterium tumefaciens , serves as an effective terminator for Cas protein expression. Similarly, terminators such as the U6 small nuclear RNA (snRNA) terminator and sequences derived from transfer RNA (tRNA) genes are commonly utilized for guiding RNA (gRNA) expression in CRISPR/Cas systems [82] . These terminators significantly influence the editing efficiency of the CRISPR/Cas system in plants. It is crucial to emphasize the importance of utilizing robust terminators, such as rbcS-E9 from Pisum sativum , to optimize Cas9 expression [83] . 4. DELIVERY METHODS AND TRANSFORMATION TECHNIQUE A diverse array of delivery and transformation techniques have been devised to introduce Cas9 and sgRNA molecules into plant genomes, facilitating precise genetic modifications. These encompass both conventional methods like Agrobacterium -mediated transformation and biolistic particle delivery, as well as advanced techniques, such as protoplast transfection and nanotechnology-based approaches [84] . 4.1 Agrobacterium-Mediated Transformation Agrobacterium tumefaciens serves as a widely used vector for delivering CRISPR/Cas constructs into plant cells, facilitating the transfer of CRISPR/Cas components into the plant genome through infection, thereby offering a versatile and effective method suitable for various plant species and tissues, especially for stable transformation. The process typically involves the introduction of CRISPR/Cas constructs into Agrobacterium tumefaciens , either through plasmid transformation or direct integration into the bacterial genome. Subsequently, the engineered Agrobacterium strains are employed to infect plant tissues, facilitating the transfer of CRISPR/Cas constructs into the plant cells. Once inside the plant cells, the CRISPR/Cas system orchestrates targeted genetic modifications, such as gene knockouts, knock-ins, or point mutations [85] . Agrobacterium tumefaciens -mediated transformation not only provides high transformation efficiency but also exhibits broad host ranges, such as Arabidopsis thaliana , Rice ( Oryza sativa ), Wheat ( Triticum aestivum ), Potato ( Solanum tuberosum ) , Tomato ( Solanum lycopersicum ), Maize ( Zea mays ), Soybean ( Glycine max ), Apple ( Malus domestica ), Grapevine ( Vitis vinifera ), Strawberry ( Fragaria × ananassa ), Switchgrass ( Panicum virgatum ), and Artemisia annua . 4.2 Biolistic Particle Delivery Particle bombardment-mediated transformation involves introducing tiny gold or tungsten particles coated with CRISPR constructs into plant cells by bombarding embryos. This method enables the transformation of a wide range of plant species and tissues, such as rice [86] , wheat [87] , and maize [88] , and facilitates both stable transformation and transient expression experiments. Unlike Agrobacterium -mediated, particle bombardment does not require bacterial infection, simplifying experimental procedures, reducing potential risks of bacterial contamination, and accelerating the experimental process. However, particle bombardment may lead to the random integration, genomic damage, and lower transformation efficiency, while the need for specialized equipment incurs additional costs [89, 90] . 4.3 Protoplast Transfection Protoplast-based CRISPR/Cas editing typically involves the prior removal of the cell wall using cellulase and pectinase enzymes to enhance the susceptibility to transformation. CRISPR constructs are then introduced into protoplasts through techniques, such as PEG (polyethylene glycol)-mediated or electroporation, making protoplast transfection a valuable method for transient expression experiments in various plant species [67] . It offers several advantages, including the ability to study gene function and regulatory networks in a cell-type-specific manner, as well as the potential for the rapid screening of CRISPR-induced mutations. Additionally, protoplasts derived from recalcitrant plant species or tissues can be used for CRISPR/Cas editing, bypassing the challenges associated with traditional plant transformation methods. However, the transient nature of protoplasts may restrict the duration of CRISPR activity and the ability to generate stable transgenic plants directly from edited protoplasts. Additionally, optimizing transfection conditions for different plant species and cell types can be challenging, potentially impacting the efficiency of CRISPR/Cas editing under varying experimental conditions. 4.4 Other Transformation Methods In addition to the aforementioned methods, novel approaches have recently emerged, such as the delivery of DNA-free Cas9/gRNA into tobacco BY2 protoplasts through lipofection-mediated transfection [91] . Furthermore, plant viruses like potato virus X (PVX), tobacco mosaic virus (TMV), and tobacco rattle virus (TRV) can be engineered to transport CRISPR/Cas components into plant cells [92] . Another technique, agroinfiltration, enables the transient expression of CRISPR components in planta and is particularly useful for rapid screening of gene editing events, utilizing both syringe and vacuum infiltration methods applicable to a variety of plant species [93] . 5. GENE EDITING IN NON-MODEL PLANT SPECIES Expanding gene editing capabilities across diverse plant species is paramount for advancing agricultural biotechnology and tackling global food security challenges. Although CRISPR/Cas9 has revolutionized gene editing in model plant species, such as Arabidopsis thaliana and rice, its application in non-model plant species presents distinct challenges due to variations in genetic backgrounds, tissue culture requirements, and transformation efficiencies [94] . Non-model plant species encompass a wide array of economically significant, environmentally important, or research-valuable plants. For instance, in crops like wheat ( Triticum aestivum ) [95] and maize ( Zea mays ) [96] , CRISPR/Cas9 is employed to engineer traits, including herbicide resistance, nutritional enhancement, and disease resistance. Soybean ( Glycine max ) has been targeted for improving traits like oil content and protein quality [97] . Potato ( Solanum tuberosum ) has been manipulated to enhance traits, such as tuber quality, disease resistance, and reduced acrylamide formation [98] . Similarly, in horticultural plants, such as apple and tomato ( Solanum lycopersicum ), CRISPR/Cas9 has been utilized to augment traits like fruit ripening, shelf life, and disease resistance [99] . Additionally, Grapevine ( Vitis vinifera ) and Strawberry ( Fragaria × ananassa ) are subjected to gene editing to improve traits, such as fruit quality, disease resistance, and yield [100] . In energy plants like switchgrass ( Panicum virgatum ), CRISPR/Cas9 has been applied to boost traits, such as biomass yield, stress tolerance, and lignin content [101] . Additionally, medicinal herbs like Artemisia annua have been targeted for genetic modifications aimed at enhancing their medicinal properties [102] . In summary, extending CRISPR/Cas9 technology to a diverse range of plant species holds immense promises for revolutionizing agriculture, ensuring food security, and addressing various agricultural and environmental challenges. 6. CONCLUSION AND FUTURE PERSPECTIVES 6.1 Summary of advancements and challenges in plant gene editing technology Gene editing technology, such as TALENs, Zinc finger nuclease, RNAi, MAGE, and CRISPR/Cas, hold tremendous potential to revolutionize various fields, including healthcare and agriculture, with a forward-looking perspective. Plant gene editing techniques have been widely used in various fields in recent years, characterized by their increased speed, simplicity, precision, and potentially reduced costs compared to traditional approaches (Figure 2.) [103] . For instance, CRISPR/Cas-based genome engineering has expedited the process of developing beneficial crop traits via conventional breeding [104, 105] . RNAi exhibits a high degree of specificity, enabling accurate and focused targeting of individual genes or gene isoforms. This specificity enables researchers to analyze intricate biological pathways and discover new targets with great accuracy [106] . However, exogenous RNA molecules, especially long double-stranded RNA (dsRNA), may trigger the innate immune responses, resulting in inflammation, cytotoxicity, and off target effects, which have raised safety concerns in many studies [107] . A large-scale technology called multiplex automated genome engineering (MAGE) is known for employing a simultaneous targeting approach to modify many loci on the chromosome within a single cell or throughout a population of cells, resulting in the generation of diverse combinations of genetic modifications [108] . This approach enabled a high level of efficiency and the ability to generate varied genetic modifications. However, it also introduces increased complexity and reduced genetic stability. Importantly, continuous progress in CRISPR/Cas gene editing methodologies has led to a significant expansion in the potential applications for curing plant genetic diseases, enhancing crop quality and resilience, and mitigating climate change. However, there are still challenges to overcome. For instance, the precise mechanism by which Cas9 dissociates from its designed sgRNA and recycles thereafter remains unknown and requires additional investigation [109] . Additionally, the prevalent occurrence of off-target effects in most gene editing technologies can greatly reduce the efficacy of the process. These result in cellular signaling and physiological effects that are either unreported or unknown, difficult to detect, and very challenging to accurately assess [110] . To minimize these, various strategies have been reported, including improving target specificity by altering the sgRNA sequence, controlling the Cas9-sgRNA complex, and using a mutant Cas9 that can enhance cleavage efficacy [111-113] . Meanwhile, the complexity of multigenic traits also brings difficulties in the gene editing procedure. Many desirable agricultural features, including but not limited to yield production, resilience to climate change, disease resistance, and abiotic stress tolerance, are controlled by multiple genes and influenced by different environmental factors. This complexity of mechanisms presents technical challenges and requires a deeper understanding of plant genetics and physiology [114] . Nevertheless, the transition of genetically modified crops from research laboratories to agricultural fields requires cautious deliberation and poses challenges due to constraints, such as legal frameworks, public acceptance, regulatory hurdles, and potential ecological repercussions. Overall, the precision, efficiency, and versatility of different gene editing technologies have revolutionized plant gene editing efficiency and accuracy, enabling the selective targeting of individual and multiple genes and the precise alteration of features, such as eliminating undesired features but introducing advantageous ones for the plants. However, challenges also exist as mentioned. 6.2 Potential applications of gene editing technology in agriculture, food security, and plant biotechnology The utilization of plant gene editing technology has resulted in the development of enhanced new crop traits, some of which would have been unattainable through traditional breeding methods. Genetically modified (GM) crops, possessing novel traits and improved attributes, have been further developed and made available for commercial use worldwide [115] . For instance, research has demonstrated the development of unique varieties of plants with enhanced stress tolerance/resistance or nutrient contents with gene editing technologies, which cause significantly increased crop yield and quality leading to less economic losses [116,117] . Furthermore, gene editing can further alter plants’ traits for the purpose of biofuel generation, phytoremediation, and the creation of useful secondary metabolites for agriculture and human health. In summary, plant gene editing technology has revolutionized agriculture and plant traits, improved food security, and advanced biotechnological applications. However, it is very critical to develop the related approaches and applications properly and precisely for creating desired GM plants, to ensure the safety, ethical considerations, and regulatory compliance. AUTHOR CONTRIBUTIONS Y.X., P.Y., Z.C., and N.V. conceptualize the manuscript’s contents; P.Y.; U.M.; W.S.L.; and A.A helped in data collection and preparation of the draft of the manuscript. Y.X., Z.C., and N.V did the final editing of the manuscript. All authors have read and agreed to the published version of the manuscript. ACKNOWLEDGEMENTS We would like to thank Dr. Yiping Qi from the University of Maryland for the scientific support. No conflict of interest declared. FUNDING This work was supported by grants from the USDA-NIFA AWD-115812 and USDA Hatch Project OHO01511. CONFLICT OF INTEREST STATEMENT All authors are aware of this communication. This is an original review article and there is no conflict of interest among the authors with each other. DATA AVAILABILITY STATEMENT This study is a review article and contents no supporting data; all the literatures were cited within the manuscript from where the details were taken. REFERENCES 1. Hemathilake, D. M. K. S., Gunathilake, D. M. C. C. (2022). Agricultural productivity and food supply to meet increased demands. Future Foods , 539–553.2. Tian, X., Engel, B. A., Qian, H., Hua, E., Sun, S., Wang, Y. (2021). Will reaching the maximum achievable yield potential meet future global food demand? Journal of Cleaner Production , 294, 126285.3. Röös, E., Bajželj, B., Smith, P., Patel, M., Little, D., Garnett, T. (2017). Greedy or needy? Land use and climate impacts of food in 2050 under different livestock futures. Global Environmental Change , 47, 1–12.4. Yadav, R. K., Tripathi, M. K., Tiwari, S., Tripathi, N., Asati, R., Patel, V., Sikarwar, R. S., Payasi, D. K. (2023). Breeding and genomic approaches towards development of fusarium wilt resistance in chickpea. Life ,13, 988.5. Yadava, D. K., Dikshit, H. K., Mishra, G. P., Tripathi, S. (2022 ) . Fundamentals of field crop breeding. Springer Nature Singapore .6. Nerkar, G., Devarumath, S., Purankar, M., Kumar, A., Valarmathi, R., Devarumath, R., Appunu, C. (2022). Advances in crop breeding through precision genome editing. Frontiers in genetics, 13, 880195.7. Proudfoot, C., Carlson, D. F., Huddart, R., Long, C. R, Pryor, J. H., King, T. J., Lillico, S. G. (2015). Genome edited sheep and cattle. Transgenic research , 24, 147–153.8. Singer, M. F. (1979). Introduction and historical background. Genetic Engineering , 1–13.9. Kim, Y.-G., Cha, J., Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proceedings of the National Academy of Sciences of the United States of America , 1156–1160, 10. Weeks, D. P., Spalding, M. H., & Yang, B. (2016). Use of designer nucleases for targeted gene and genome editing in plants. Plant biotechnology journal, 14 (2), 483–495. 11. Wang H., Wu Y., Zhang Y., Yang J., Fan W., Zhang H., Zhao, S., Yuan, L., Zhang, P. (2019). CRISPR/Cas9-based mutagenesis of starch biosynthetic genes in sweet potato (Ipomoea Batatas) for the improvement of starch quality. International journal of molecular sciences, 20 (19), 4702. 12. Koeppe, S.; Kawchuk, L; Kalischuk, M. (2023). RNA interference past and future applications in plants. International journal of molecular sciences, 24 (11), 9755. 13. Zand Karimi, H. and Innes, R.W. (2022). Molecular mechanisms underlying host-induced gene silencing. The Plant cell, 34 (9), 3183-3199.14. Zhang, H., Zhang, J., Lang, Z., Botella, J. R., Zhu, J. K. (2017). Genome editing-principles and applications for functional genomics research and crop improvement. Critical Reviews in Plant Sciences , 36, 291–309.15. Noman, A., Aqeel, M., He, S. (2016). CRISPR-Cas9: tool for qualitative and quantitative plant genome editing. Frontiers in plant science , 7 , 1740. 16. Zhang, F., Wen, Y., Guo, X. (2014). CRISPR/Cas9 for genome editing: progress, implications and challenges. Human molecular genetics , 23 (R1), R40–R46.17. Mudziwapasi, R., Chekera, R., Ncube, C. Z., Shoko, I., Ncube, B., Moyo, T., Chimbo, J. G., Dube, J., Mashiri, F. F., Mubani, M. A., Maruta, D. (2021). Genome editing tools and genedrives: a brief overview. CRC Press. 1 st edition, 150.18. Lobato-Gómez, M., Hewitt, S., Capell, T., Christou, P., Dhingra, A., Girón-Calva, P. S. (2021). Transgenic and genome-edited fruits: background, constraints, benefits, and commercial opportunities. Horticulture research, 8 (1), 166.19. Ishii, T., Araki, M. (2016). Consumer acceptance of food crops developed by genome editing. Plant cell reports , 35 (7), 1507–1518.20. Clasen, B. M., Stoddard, T. J., Luo, S., Demorest, Z. L., Li, J., Cedrone, F., Tibebu, R., Davison, S., Ray, E. E., Daulhac, A., Coffman, A., Yabandith, A., Retterath, A., Haun, W., Baltes, N. J., Mathis, L., Voytas, D. F., Zhang, F. (2016). Improving cold storage and processing traits in potato through targeted gene knockout. Plant biotechnology journal , 14 (1), 169–176.21. Waltz E. (2016). CRISPR-edited crops free to enter market, skip regulation. Nature biotechnology , 34 (6), 582.22. Shan, Q., Zhang, Y., Chen, K., Zhang, K., Gao, C. (2015). Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant biotechnology journal , 13 (6), 791–800.23 Sun, Y., Jiao, G., Liu, Z., Zhang, X., Li, J., Guo, X., Du, W., Du, J., Francis, F., Zhao, Y., Xia, L. (2017). Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Frontiers in plant science, 8 , 298.24. Zhang, J., Zhang, H., Botella, J. R., Zhu, J. K. (2018). Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties. Journal of integrative plant biology , 60 (5), 369–375.25. Sánchez-León S., Gil-Humanes J., Ozuna C. V., Giménez M. J., Sousa C., Voytas D. F., Barro, F. (2018). Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9. Plant biotechnology journal , 16 (4), 902–910.26. Zhang, Y., Li, D., Zhang, D., Zhao, X., Cao, X., Dong, L., Liu, J., Chen, K., Zhang, H., Gao, C., Wang, D. (2018). Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits. The Plant journal, 94 (5), 857–866.27. Shukla, V. K., Doyon, Y., Miller, J. C., DeKelver, R. C., Moehle, E. A., Worden, S. E., Mitchell, J. C., Arnold, N. L., Gopalan, S., Meng, X., Choi, V. M., Rock, J. M., Wu, Y. Y., Katibah, G. E., Zhifang, G., McCaskill, D., Simpson, M. A., Blakeslee, B., Greenwalt, S. A., Butler, H. J., … Urnov, F. D. (2009). Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 459 (7245), 437–441.28 Liang, Z., Zhang, K., Chen, K., Gao, C. (2014). Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. Journal of genetics and genomics = Yi chuan xue bao , 41 (2), 63–68. 29. Li, A., Jia, S., Yobi, A., Ge, Z., Sato, S. J., Zhang, C., Angelovici, R., Clemente, T. E., Holding, D. R. (2018). Editing of an Alpha-Kafirin gene family increases, digestibility and protein quality in sorghum. Plant physiology , 177 (4), 1425–1438. 30. Andersson, M., Turesson, H., Nicolia, A., Fält, A. S., Samuelsson, M., & Hofvander, P. (2017). Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant cell reports , 36 (1), 117–128. 31. Kusano, H., Ohnuma, M., Mutsuro-Aoki, H., Asahi, T., Ichinosawa, D., Onodera, H., Asano, K., Noda, T., Horie, T., Fukumoto, K., Kihira, M., Teramura, H., Yazaki, K., Umemoto, N., Muranaka, T., & Shimada, H. (2018). Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato. Scientific reports , 8 (1), 13753.32. González, M. N., Massa, G. A., Andersson, M., Turesson, H., Olsson, N., Fält, A. S., Storani, L., Décima Oneto, C. A., Hofvander, P., & Feingold, S. E. (2020). Reduced enzymatic browning in potato tubers by specific editing of a polyphenol oxidase gene via ribonucleoprotein complexes delivery of the CRISPR/Cas9 System. Frontiers in plant science , 10 , 1649. 33. Yasumoto, S., Umemoto, N., Lee, H. J., Nakayasu, M., Sawai, S., Sakuma, T., Yamamoto, T., Mizutani, M., Saito, K., & Muranaka, T. (2019). Efficient genome engineering using Platinum TALEN in potato. Plant biotechnology (Tokyo, Japan) , 36 (3), 167–173. 34. Okuzaki, A., Ogawa, T., Koizuka, C., Kaneko, K., Inaba, M., Imamura, J., & Koizuka, N. (2018). CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus . Plant physiology and biochemistry: PPB , 131 , 63–69. 35. Haun W., Coffman, A., Clasen, B. M., Demorest, Z. L., Lowy, A., Ray, E., Retterath, A., Stoddard, T., Juillerat, A., Cedrone, F., Mathis, L., Voytas, D. F., & Zhang, F. (2014). Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant biotechnology journal , 12 (7), 934–940. 36. Wen, S., Liu, H., Li, X., Chen, X., Hong, Y., Li, H., Lu, Q., & Liang, X. (2018). TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut ( Arachis hypogaea L.) promotes the accumulation of oleic acid. Plant molecular biology , 97 (1-2), 177–185. 37. Ren, C., Liu, X., Zhang, Z., Wang, Y., Duan, W., Li, S., & Liang, Z. (2016). CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay ( Vitis vinifera L.). Scientific reports , 6 , 32289. 38. Ito, Y., Nishizawa-Yokoi, A., Endo, M., Mikami, M., & Toki, S. (2015). CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochemical and biophysical research communications , 467 (1), 76–82. 39. Čermák, T., Baltes, N. J., Čegan, R., Zhang, Y., & Voytas, D. F. (2015). High-frequency, precise modification of the tomato genome. Genome biology , 16 , 232. 40. Deng, L., Wang, H., Sun, C., Li, Q., Jiang, H., Du, M., Li, C. B., & Li, C. (2018). Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. Journal of genetics and genomics = Yi chuan xue bao , 45 (1), 51–54. 41. Li, R., Fu, D., Zhu, B., Luo, Y., & Zhu, H. (2018). CRISPR/Cas9-mediated mutagenesis of lncRNA1459 alters tomato fruit ripening. The Plant journal: for cell and molecular biology , 94 (3), 513–524. 42. Maioli, A., Gianoglio, S., Moglia, A., Acquadro, A., Valentino, D., Milani, A. M., Prohens, J., Orzaez, D., Granell, A., Lanteri, S., & Comino, C. (2020). Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L. Frontiers in plant science , 11 , 607161. 43. Jiang, M., Zhan, Z., Li, H., Dong, X., Cheng, F., & Piao, Z. (2020). Brassica rapa orphan genes largely affect soluble sugar metabolism. Horticulture research, 7 (1), 181.44. Turnbull, C., Lillemo, M., & Hvoslef-Eide, T. A. K. (2021). Global Regulation of Genetically Modified Crops Amid the Gene Edited Crop Boom - A Review. Frontiers in plant science , 12 , 630396. 45. Brookes, G., & Barfoot, P. (2018). Environmental impacts of genetically modified (GM) crop use 1996-2016: Impacts on pesticide use and carbon emissions. GM crops & food , 9 (3), 109–139. 46. Bishnoi, R., Kaur, S., Sandhu, J. S., & Singla, D. (2023). Genome engineering of disease susceptibility genes for enhancing resistance in plants. Functional & integrative genomics , 23 (3), 207.47. Muhammad, U., Muhammad, A., Nasir, A. R., Shahbaz, T. S., Mohsin, S., Nian, L., Shahid, I., Asif, M. A., Usama, A., Khurram, S. K., Muhammad, A., Fasih, U. H. (2024). Efficacy of green synthesized silver-based nanomaterials against early blight of tomato caused by Alternaria solani . Journal of Crop Health , 76 (1), 105-115.48.Sabir H., K., Mubeen, I., Abdul, J., Hafiz MF, S., Kiran, Fatima., Muhammad, U., Naveed, A., Hafiz MR, M. (2021). Impact of downy mildew disease caused by Pseudoperonospora cubensis on cucumber germplasm and its horticultural attributes. Pakistan Journal of Phytopathology, 33 (1): 117-123.49. Büschges, R., Hollricher, K., Panstruga, R., Simons, G., Wolter, M., Frijters, A., van Daelen, R., van der Lee, T., Diergaarde, P., Groenendijk, J., Töpsch, S., Vos, P., Salamini, F., & Schulze-Lefert, P. (1997). The barley Mlo gene: a novel control element of plant pathogen resistance. Cell , 88 (5), 695–705. 50. Zhang, Z., Ge, X., Luo, X., Wang, P., Fan, Q., Hu, G., Xiao, J., Li, F., & Wu, J. (2018). Simultaneous Editing of Two Copies of Gh14-3-3d Confers Enhanced Transgene-Clean Plant Defense Against Verticillium dahliae in Allotetraploid Upland Cotton. Frontiers in plant science , 9 , 842. 51. Wang, W., Pan, Q., He, F., Akhunova, A., Chao, S., Trick, H., & Akhunov, E. (2018). Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat. The CRISPR journal , 1 (1), 65–74. 52. Wang, X., Tu, M., Wang, D., Liu, J., Li, Y., Li, Z., Wang, Y., & Wang, X. (2018). CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant biotechnology journal , 16 (4), 844–855. 53. Prihatna, C., Barbetti, M. J., & Barker, S. J. (2018). A Novel Tomato Fusarium Wilt Tolerance Gene. Frontiers in microbiology , 9 , 1226. 54. Muhammad, A., Kiran, F., Nasir, A. R., Shahbaz, T. S., Akhtar, H., Muhammad, U., Usama, A., Adeel, S., Shahid, I., Ahmad, N. (2023). Estimation of biocidal potential of desert phytopowders for the management of citrus canker. Sains Malaysiana , 52 (3), 757-770.55. Peng, A., Chen, S., Lei, T., Xu, L., He, Y., Wu, L., Yao, L., & Zou, X. (2017). Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant biotechnology journal , 15 (12), 1509–1519. 56. Ortigosa, A., Gimenez-Ibanez, S., Leonhardt, N., & Solano, R. (2019). Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2. Plant biotechnology journal , 17 (3), 665–673. 57. Oliva, R., Ji, C., Atienza-Grande, G., Huguet-Tapia, J. C., Perez-Quintero, A., Li, T., Eom, J. S., Li, C., Nguyen, H., Liu, B., Auguy, F., Sciallano, C., Luu, V. T., Dossa, G. S., Cunnac, S., Schmidt, S. M., Slamet-Loedin, I. H., Vera Cruz, C., Szurek, B., Frommer, W. B., … Yang, B. (2019). Broad-spectrum resistance to bacterial blight in rice using genome editing. Nature biotechnology , 37 (11), 1344–1350. 58. Zafar, K., Khan, M. Z., Amin, I., Mukhtar, Z., Yasmin, S., Arif, M., Ejaz, K., & Mansoor, S. (2020). Precise CRISPR-Cas9 Mediated Genome Editing in Super Basmati Rice for Resistance Against Bacterial Blight by Targeting the Major Susceptibility Gene. Frontiers in plant science , 11 , 575. 59. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, N.Y.) , 337 (6096), 816–821.60. Gaj, T., Sirk, S. J., Shui, S. L., & Liu, J. (2016). Genome-Editing Technologies: Principles and Applications. Cold Spring Harbor perspectives in biology , 8 (12), a023754. 61. Saini, H., Thakur, R., Gill, R., Tyagi, K., & Goswami, M. (2023). CRISPR/Cas9-gene editing approaches in plant breeding. GM crops & food , 14 (1), 1–17. 62. Soste, M., Hrabakova, R., Wanka, S., Melnik, A., Boersema, P., Maiolica, A., Wernas, T., Tognetti, M., von Mering, C., & Picotti, P. (2014). A sentinel protein assay for simultaneously quantifying cellular processes. Nature methods , 11 (10), 1045–1048.63. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science (New York, N.Y.) , 339 (6121), 819–823.64. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature protocols , 8 (11), 2281–2308.65. Chen, K., Wang, Y., Zhang, R., Zhang, H., & Gao, C. (2019). CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. Annual review of plant biology , 70 , 667–697. 66. Zhu, H., Li, C., & Gao, C. (2020). Applications of CRISPR-Cas in agriculture and plant biotechnology. Nature reviews. Molecular cell biology , 21 (11), 661–677.67. Hassan, M. M., Zhang, Y., Yuan, G., De, K., Chen, J. G., Muchero, W., Tuskan, G. A., Qi, Y., & Yang, X. (2021). Construct design for CRISPR/Cas-based genome editing in plants. Trends in plant science , 26 (11), 1133–1152. 68. Gürel, F., Zhang, Y., Sretenovic, S., & Qi, Y. (2019). CRISPR-Cas nucleases and base editors for plant genome editing. aBIOTECH , 1 (1), 74–87.69. Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., Koonin, E. V., Sharp, P. A., & Zhang, F. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature , 520 (7546), 186–191.70. Xu, R., Qin, R., Xie, H., Li, J., Liu, X., Zhu, M., Sun, Y., Yu, Y., Lu, P., & Wei, P. (2022). Genome editing with type II-C CRISPR-Cas9 systems from Neisseria meningitidis in rice. Plant biotechnology journal , 20 (2), 350–359. 71. Riaz, A., Kanwal, F., Ahmad, I., Ahmad, S., Farooq, A., Madsen, C. K., Brinch-Pedersen, H., Bekalu, Z. E., Dai, F., Zhang, G., & Alqudah, A. M. (2022). New Hope for Genome Editing in Cultivated Grasses: CRISPR Variants and Application. Frontiers in genetics , 13 , 866121. 72. Xie, K., Zhang, J., & Yang, Y. (2014). Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. Molecular plant , 7 (5), 923–926. 73. Liu, H., Ding, Y., Zhou, Y., Jin, W., Xie, K., & Chen, L. L. (2017). CRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants. Molecular plant , 10 (3), 530–532. 74. Chari, R., Yeo, N. C., Chavez, A., & Church, G. M. (2017). sgRNA Scorer 2.0: A species-independent model to predict CRISPR/Cas9 Activity. ACS synthetic biology , 6 (5), 902–904.75. Xie, X., Ma, X., Zhu, Q., Zeng, D., Li, G., & Liu, Y. G. (2017). CRISPR-GE: A convenient software toolkit for CRISPR-based genome editing. Molecular plant , 10 (9), 1246–1249.76. Minkenberg, B., Zhang, J., Xie, K., & Yang, Y. (2019). CRISPR-PLANT v2: an online resource for highly specific guide RNA spacers based on improved off-target analysis. Plant biotechnology journal , 17 (1), 5–8. 77. Chow, C. N., Lee, T. Y., Hung, Y. C., Li, G. Z., Tseng, K. C., Liu, Y. H., Kuo, P. L., Zheng, H. Q., & Chang, W. C. (2019). PlantPAN3.0: a new and updated resource for reconstructing transcriptional regulatory networks from ChIP-seq experiments in plants. Nucleic acids research , 47 (D1), D1155–D1163.78. Tian, F., Yang, D. C., Meng, Y. Q., Jin, J., & Gao, G. (2020). PlantRegMap: charting functional regulatory maps in plants. Nucleic acids research , 48 (D1), D1104–D1113.79. Ahmar, S., Hensel, G., & Gruszka, D. (2023). CRISPR/Cas9-mediated genome editing techniques and new breeding strategies in cereals - current status, improvements, and perspectives. Biotechnology advances , 69 , 108248.80. Feng, C., Su, H., Bai, H., Wang, R., Liu, Y., Guo, X., Liu, C., Zhang, J., Yuan, J., Birchler, J. A., & Han, F. (2018). High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize. Plant biotechnology journal , 16 (11), 1848–1857.81. Čermák, T., Curtin, S. J., Gil-Humanes, J., Čegan, R., Kono, T. J. Y., Konečná, E., Belanto, J. J., Starker, C. G., Mathre, J. W., Greenstein, R. L., & Voytas, D. F. (2017). A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants. The Plant cell , 29 (6), 1196–1217. 82. Shan, S., Soltis, P. S., Soltis, D. E., & Yang, B. (2020). Considerations in adapting CRISPR/Cas9 in nongenetic model plant systems. Applications in plant sciences , 8 (1), e11314.83. Ordon, J., Bressan, M., Kretschmer, C., Dall’Osto, L., Marillonnet, S., Bassi, R., & Stuttmann, J. (2020). Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation. Functional & integrative genomics , 20 (1), 151–162.84. Nadakuduti, S. S., & Enciso-Rodríguez, F. (2021). Advances in Genome Editing with CRISPR Systems and Transformation Technologies for Plant DNA Manipulation. Frontiers in plant science , 11 , 637159. 85. Kumar, S., Barone, P., Smith, M. (2019). Transgenic plants: methods and protocols. Springer New York, 1864.86. Banakar, R., Schubert, M., Collingwood, M., Vakulskas, C., Eggenberger, A. L., & Wang, K. (2020). Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase ( OsPDS ) Gene. Rice (New York, N.Y.) , 13 (1), 4.87. Liang, Z., Chen, K., Li, T., Zhang, Y., Wang, Y., Zhao, Q., Liu, J., Zhang, H., Liu, C., Ran, Y., & Gao, C. (2017). Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature communications , 8 , 14261. 88. Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K., & Mark Cigan, A. (2016). Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nature communications , 7 , 13274. 89. Zhang, Y., Iaffaldano, B., & Qi, Y. (2021). CRISPR ribonucleoprotein-mediated genetic engineering in plants. Plant communications , 2 (2), 100168. 90. Liu, J., Nannas, N. J., Fu, F. F., Shi, J., Aspinwall, B., Parrott, W. A., & Dawe, R. K. (2019). Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize. The Plant cell , 31 (2), 368–383. 91. Liu, W., Rudis, M. R., Cheplick, M. H., Millwood, R. J., Yang, J. P., Ondzighi-Assoume, C. A., Montgomery, G. A., Burris, K. P., Mazarei, M., Chesnut, J. D., & Stewart, C. N., Jr (2020). Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant cell reports , 39 (2), 245–257.92. Laforest, L. C., & Nadakuduti, S. S. (2022). Advances in Delivery Mechanisms of CRISPR Gene-Editing Reagents in Plants. Frontiers in genome editing , 4 , 830178. 93. Kaur, M., Manchanda, P., Kalia, A., Ahmed, F. K., Nepovimova, E., Kuca, K., & Abd-Elsalam, K. A. (2021). Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants. International journal of molecular sciences , 22 (19), 10882. 94. Bao, A., Burritt, D. J., Chen, H., Zhou, X., Cao, D., & Tran, L. P. (2019). The CRISPR/Cas9 system and its applications in crop genome editing. Critical reviews in biotechnology , 39 (3), 321–336. 95. Kumar, R., Kaur, A., Pandey, A., Mamrutha, H. M., & Singh, G. P. (2019). CRISPR-based genome editing in wheat: a comprehensive review and future prospects. Molecular biology reports , 46 (3), 3557–3569. 96. Shi, J., Gao, H., Wang, H., Lafitte, H. R., Archibald, R. L., Yang, M., Hakimi, S. M., Mo, H., & Habben, J. E. (2017). ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant biotechnology journal , 15 (2), 207–216.97. Gan, W. C., & Ling, A. P. K. (2022). CRISPR/Cas9 in plant biotechnology: applications and challenges. Biotechnologia , 103 (1), 81–93.98. Tussipkan, D., & Manabayeva, S. A. (2021). Employing CRISPR/Cas Technology for the Improvement of Potato and Other Tuber Crops. Frontiers in plant science , 12 , 747476.99. Xia, X., Cheng, X., Li, R., Yao, J., Li, Z., & Cheng, Y. (2021). Advances in application of genome editing in tomato and recent development of genome editing technology. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik , 134 (9), 2727–2747.100. Zhou, J., Li, D., Wang, G., Wang, F., Kunjal, M., Joldersma, D., & Liu, Z. (2020). Application and future perspective of CRISPR/Cas9 genome editing in fruit crops. Journal of integrative plant biology , 62 (3), 269–286. 101. Ul Haq, S. I., Zheng, D., Feng, N., Jiang, X., Qiao, F., He, J. S., & Qiu, Q. S. (2022). Progresses of CRISPR/Cas9 genome editing in forage crops. Journal of plant physiology , 279 , 153860. 102. Senthilnathan, R., Ilangovan, I., Kunale, M., Easwaran, N., Ramamoorthy, S., Veeramuthu, A., & Kodiveri Muthukaliannan, G. (2023). An update on CRISPR-Cas12 as a versatile tool in genome editing. Molecular biology reports , 50 (3), 2865–2881. 103. Eş, I., Gavahian, M., Marti-Quijal, F. J., Lorenzo, J. M., Mousavi Khaneghah, A., Tsatsanis, C., Kampranis, S. C., & Barba, F. J. (2019). The application of the CRISPR-Cas9 genome editing machinery in food and agricultural science: Current status, future perspectives, and associated challenges. Biotechnology advances , 37 (3), 410–421 104. Whitworth, K. M., Rowland, R. R., Ewen, C. L., Trible, B. R., Kerrigan, M. A., Cino-Ozuna, A. G., Samuel, M. S., Lightner, J. E., McLaren, D. G., Mileham, A. J., Wells, K. D., & Prather, R. S. (2016). Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nature biotechnology , 34 (1), 20–22. 105. Svitashev, S., Young, J. K., Schwartz, C., Gao, H., Falco, S. C., & Cigan, A. M. (2015). Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA. Plant physiology , 169 (2), 931–945. 106. Agrawal, N., Dasaradhi, P. V., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA interference: biology, mechanism, and applications. Microbiology and molecular biology reviews : MMBR , 67 (4), 657–685. 107. Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Burchard, J., Mao, M., Li, B., Cavet, G., & Linsley, P. S. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nature biotechnology , 21 (6), 635–637. 108. Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M. (2009). Programming cells by multiplex genome engineering and accelerated evolution. Nature , 460 (7257), 894–898. 109. Zischewski, J., Fischer, R., & Bortesi, L. (2017). Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnology advances , 35 (1), 95–104. 110. Kadam, U. S., Shelake, R. M., Chavhan, R. L., & Suprasanna, P. (2018). Concerns regarding ’off-target’ activity of genome editing endonucleases. Plant physiology and biochemistry: PPB , 131 , 22–30. 111. Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., Cradick, T. J., Marraffini, L. A., Bao, G., & Zhang, F. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology , 31 (9), 827–832. 112. Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R. (2013). High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature biotechnology, 31(9), 839–843. 113. Cho, S. W., Kim, S., Kim, Y., Kweon, J., Kim, H. S., Bae, S., & Kim, J. S. (2014). Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome research , 24 (1), 132–141. 114. Arora, L., & Narula, A. (2017). Gene Editing and Crop Improvement Using CRISPR-Cas9 System. Frontiers in plant science , 8 , 1932. 115. Georges F., & Ray, H. (2017). Genome editing of crops: A renewed opportunity for food security. GM crops & food , 8 (1), 1–12. 116. Brooks, C., Nekrasov, V., Lippman, Z. B., & Van Eck, J. (2014). Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant physiology , 166 (3), 1292–1297. 117. Miao, J., Guo, D., Zhang, J., Huang, Q., Qin, G., Zhang, X., Wan, J., Gu, H., & Qu, L. J. (2013). Targeted mutagenesis in rice using CRISPR-Cas system. Cell research , 23 (10), 1233–1236. Figure legends: Figure 1 . Schematic description of a successful case study of genome editing in cotton plants for enhancing their resistance against pest invading . Previously, insects with chewing types were vulnerable challenges for cotton industry. These insects feed on plant leaves & balls and destroy the whole crop by making holes in cotton balls. Genome editing can create successful transgenic cotton plants by inserting Vip gene from bacteria Bacillus thuringiensis (BT). Vip gene can produce vegetative insecticidal protein during the vegetative growth of cotton plants. It is a binary toxin that destroys the epithelial cells of the midgut in feeding insects by binding to receptors, displaying its ADP-ribosyltransferase activity against actin, and preventing microfilament formation. Figure 2 . This figure illustrates various techniques employed in plant gene modification and their applications. The techniques depicted include mutation technique, which involves inducing random mutations in plant genomes through chemical or radiation treatments, leading to the discovery of novel traits or the alteration of existing ones; transgenic technique, which entails introducing foreign genes to confer traits like pest resistance or enhanced nutrition; and genome editing, which utilizes precise molecular tools, such as zinc finger nucleases (ZFNs), TALENs, and CRISPR-Cas9 to make targeted modifications in plant DNA, facilitating traits like disease resistance, improved yield, and altered growth patterns. Applications encompass crop improvement; nutritional enhancement; environmental remediation; and biofuel production. Supplementary Material File (figures.pptx) Download 642.82 KB Information & Authors Information Version history V1 Version 1 26 August 2024 Peer review timeline Published Biotechnology Journal Version of Record 18 Dec 2024 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Biotechnology Journal Keywords construct design genetic engineering plant biotechnology plant gene editing transformation efficiency Authors Affiliations Pu Yuan The Ohio State University View all articles by this author Muhammad Usman The Ohio State University View all articles by this author Wenshan Liu The Ohio State University View all articles by this author Ashna Adhikari The Ohio State University View all articles by this author Chunquan Zhang Alcorn State University View all articles by this author Victor Njiti Alcorn State University View all articles by this author ye xia 0000-0002-4749-3119 [email protected] The Ohio State University View all articles by this author Metrics & Citations Metrics Article Usage 480 views 271 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Pu Yuan, Muhammad Usman, Wenshan Liu, et al. Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency. Authorea . 26 August 2024. DOI: https://doi.org/10.22541/au.172471610.04317310/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.172471610.04317310/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a028bebc4c034193',t:'MTc3OTkyMzk3Mg=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();