Genome Editing For Crop Improvement Research Articles

CRISPR/Cas9 mediated genome editing for crop improvement against Abiotic stresses: current trends and prospects.

Abiotic stresses associated with climate change, such as heat, cold, salinity, and drought, represent a serious threat to crop health. To mitigate the risks posed by these environmental challenges, both transgenic technology and conventional breeding methods have been extensively utilized. However, these methods have faced numerous limitations. The development of synthetic nucleases as precise genetic tools allows for the targeted alteration of stress-responsive genes in crop improvement. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas) genome-editing technique has transformed gene editing with its broad applicability, accessibility, adaptability, flexibility, and simplicity. Its application shows promise for the development of crop types that are more able to survive abiotic stress conditions. The present study presents recent scenario and application of CRISPR/Cas genome-editing technology in enhancing crop tolerance to a variety of abiotic stresses.

Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

CRISPR-Cas-Based Genome Editing for Crop Improvement: Progress, Challenges and Future Prospects

The discovery of the CRISPR-Cas genome editing technology has opened up new opportunities for crop improvement through precise genetic modifications. This new technology has shown great promise in improving crop yields, quality, and resilience to biotic and abiotic stresses. This review presents the recent advances in CRISPR-Cas technology, including new tools and techniques for precise genome editing, as well as the challenges associated with off-target effects and unintended consequences. It explores the applications of CRISPR-Cas-based genome editing in different crops, including maize, rice, wheat, and tomato, highlighting the progress achieved in improving important traits such as disease resistance, drought tolerance, and nutrient content. The regulatory concerns around CRISPR-Cas-based genome editing, as well as the ethical considerations associated with this technology are also adressed. Finally, insights into the potential impact of CRISPR-Cas-based genome editing on crop breeding and food security, and the challenges that need to be addressed to fully realize its potential are provided. This review thus highlights the potential of CRISPR-Cas-based genome editing in crop improvement and emphasizes the importance of continued research in this area for sustainable agricultural production.

Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis

The enhancement of CRISPR-Cas gene editing with robust nuclease activity promotes genetic modification of desirable agronomic traits, such as resistance to pathogens, drought tolerance, nutritional value, and yield-related traits in crops. The genetic diversity of food crops has reduced tremendously over the past twelve millennia due to plant domestication. This reduction presents significant challenges for the future especially considering the risks posed by global climate change to food production. While crops with improved phenotypes have been generated through crossbreeding, mutation breeding, and transgenic breeding over the years, improving phenotypic traits through precise genetic diversification has been challenging. The challenges are broadly associated with the randomness of genetic recombination and conventional mutagenesis. This review highlights how emerging gene-editing technologies reduce the burden and time necessary for developing desired traits in plants. Our focus is to provide readers with an overview of the advances in CRISPR-Cas-based genome editing for crop improvement. The use of CRISPR-Cas systems in generating genetic diversity to enhance the quality and nutritional value of staple food crops is discussed. We also outlined recent applications of CRISPR-Cas in developing pest-resistant crops and removing unwanted traits, such as allergenicity from crops. Genome editing tools continue to evolve and present unprecedented opportunities to enhance crop germplasm via precise mutations at the desired loci of the plant genome.

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum

Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio.

Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.

Impacts of the regulatory environment for gene editing on delivering beneficial products.

Various genome-editing technologies have been embraced by plant breeders across the world as promising tools for the improvement of different crops to deliver consumer benefits, improve agronomic performance, and increase sustainability. The uptake of genome-editing technologies in plant breeding greatly depends on how governments regulate its use. Some major agricultural production countries have already developed regulatory approaches that enable the application of genome editing for crop improvement, while other governments are in the early stages of formulating policy. Central to the discussion is the principle of “like products should be treated in like ways” and the subsequent utilization of exclusions and exemptions from the scope of GMO regulations for these products. In some countries, the outcomes of genome editing that could also have been achieved through conventional breeding have been defined as not needing GMO regulatory oversight. In this paper, we provide a short overview of plant breeding and the history of plant biotechnology policy development, the different classes of current regulatory systems and their use of exemptions and exclusions for genome-edited plants, and the potential benefits of such approaches as it relates to achieving societal goals.

Genome editing for crop improvement: A perspective from India

Human population is expected to reach to about 10 billion by 2050. Climate change affects crop production, thus posing food security challenges. Conventional breeding alone will not bridge the gap between current level of crop production and expected levels in the decades to come in the food production systems. Rate of genetic gain with time has remained narrow considerably. Biotechnology-enabled crops developed through genome editing will have a part to play in improving crop productivity, meeting food, nutrition security besides catering to regional preferences and fetching valuable foreign exchange. Political, social, economical proposition, scientific will, retailer and consumer acceptance are a must for genome editing (GE) to succeed and add value in the food value chain. This will also help to make agriculture a lucrative profession and attract youth. Therefore, the present review looks into existing regulations governing crops developed using biotechnology in India, institutes involved in genome editing, prospects of new tools developed in this sphere such as DNA-free editing systems, nanotechnology, their applicability in crop improvement efforts, social and future prospects taking cue from recent global developments. This will make GE more appealing to stakeholders and defray any safety concerns.

CRISPR/Cas9 gene editing in legume crops: Opportunities and challenges

AbstractLegumes are an excellent source of proteins and health‐promoting phytochemicals. Recognizing their importance in human nutrition and sustainable agricultural production, significant efforts are currently being made to accelerate genetic gain related to yield, stress resilience, and nutritional quality. Recent increases in genomic resources for multiple legume crops have laid a solid foundation for application of transformative breeding technologies such as genomic selection and genome editing for crop improvement. In this review, we focus on the recent plant‐specific advances in CRISPR/Cas9‐based gene editing technology and discuss the challenges and opportunities to harnessing this innovative technology for targeted improvement of traits in legume crops. Gene‐editing methods have been successfully established for soybean, cowpea, chickpea, and model legumes such as Medicago truncatula and Lotus japonicus. However, the recalcitrance of other legumes to in vitro gene transfer and regeneration has posed a serious challenge to application of gene editing. We discuss various modifications to in vitro culture methods, in terms of the choice of explant, media composition, and DNA delivery and gene‐editing detection methods that can potentially improve the rate of transformation and regeneration of whole plant in legume crops. Although gene‐editing technology can bring enormous benefits to legume breeding, regulatory hurdles are a cause for serious concern. We compare the regulatory environments existing in the European Union and the United States of America. A favorable regulatory framework and public acceptance are important factors in realizing CRISPR's potential benefits to global food security.

Precise gene replacement in plants through CRISPR/Cas genome editing technology: current status and future perspectives.

CRISPR/Cas, as a simple, versatile, robust and cost-effective system for genome manipulation, has dominated the genome editing field over the past few years. The application of CRISPR/Cas in crop improvement is particularly important in the context of global climate change, as well as diverse agricultural, environmental and ecological challenges. Various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci, transcriptome regulation and epigenome editing, base editing, and precise targeted gene/allele replacement or tagging in plants. In particular, precise replacement of an existing allele with an elite allele in a commercial variety through homology-directed repair (HDR) is a holy grail in genome editing for crop improvement as it has been very difficult, laborious and time-consuming to introgress the elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop breeding practice. However, it still remains very challenging in crop plants. This review intends to provide an informative summary of the latest development and breakthroughs in gene replacement using CRISPR/Cas technology, with a focus on achievements, potential mechanisms and future perspectives in plant biological science as well as crop improvement.

Current status, challenges, and future prospects of plant genome editing in China

Genome editing (GE) is the most powerful tool for creating genetic variation in plants. This approach is valuable for studying the mechanism of gene function and regulation as well as to improve desirable traits using sequence-specific endonucleases. It is typically performed with diverse molecular scissors that cleave a particular gene at a defined position. The advent of sequence-specific nucleases such as ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), and CRISPR (clustered regularly interspaced short palindromic repeats), in particular, have allowed for the precise and efficient introduction of genetic variation into the genome. The newly developed CRISPR-associated protein 9 (Cas9) variants, base-editing systems, novel RNA-directed nucleases, and DNA-free CRISPR/Cas9 delivery methods offer great opportunities for plant genome engineering. China has made tremendous progress in the field of GE for crop improvement to meet the demand of growing population. Herein, we reviewed the recent progress in GE of different crops in China, highlighting advanced GE tools/methods, and also discussed the specific challenges and prospects of plant GE.

CRISPR/Cas-mediated genome editing for crop improvement: current applications and future prospects

Conventional breeding techniques for crop improvement are based on hybridization and selection. However, due to the long breeding cycles of crops and the potentially unpredictable effects of traditional breeding, these techniques are not sufficient to meet market demands for crops with a variety of traits or to address the emerging food crisis we could face in the near future. In the past decade, advanced technologies such as next-generation sequencing have been used to rapidly produce massive amounts of genome sequence information in many crop species. These techniques, together with targeted genome editing tools such as Zinc Finger Nuclease (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Sequences (CRISPR)/CRISPR-associated protein (Cas) have increased the possibilities for crop improvement via targeted genome editing. The use of these technologies in crop biology has opened up a new era of genome editing-mediated crop breeding. In this review, we summarize the current techniques used for site-directed genome editing in plants, focusing on the CRISPR/Cas system, and discuss their current and future applications for crop biology.

Genome Editing for Crop Improvement - Applications in Clonally Propagated Polyploids With a Focus on Potato (Solanum tuberosum L.).

Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato, Solanum tuberosum Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop.

Policy and Governance Perspectives for Regulation of Genome Edited Crops in the United States.

Genome editing for crop improvement lies at the leading edge of disruptive bioengineering technologies that will challenge existing regulatory paradigms for products of biotechnology and which will elicit widespread public interest. Regulation of products of biotechnology through the US Coordinated Framework for Biotechnology is predicated on requiring burden of proof that regulation is warranted. Although driven by considerations of newly emerging processes for product development, regulation has, for the most part, focused on characteristics of the biotechnology product itself and not the process used for its development per se. This standard of evidence and product focus has been maintained to date in regulatory considerations of genome edited crops. Those genome edited crops lacking recombinant DNA (rDNA) in the product intended for environmental release, lacking plant pest or pesticidal activity, or showing no food safety attributes different from those of traditionally bred crops are not deemed subject to regulatory evaluation. Regardless, societal uncertainties regarding genome editing are leading regulators to seek ways whereby these uncertainties may be addressed through redefinition of those products of biotechnology that may be subject to regulatory assessments. Within US law prior statutory history, language and regulatory action have significant influence on decision making; therefore, the administrative law and jurisprudence underlying the current Coordinated Framework strongly inform policy and governance when considering new plant breeding technologies such as genome editing.

Recent advances in CRISPR/Cas mediated genome editing for crop improvement

Plant breeding is one of the oldest sustainable agriculture methods used to increase the yield, quality and other biomaterial for human use. Many crops like fruits, vegetables, ornamental flowers, shrubs and trees, have been long cultivated to satisfy human food and aesthetical needs. Conventional breeding technologies like selection, hybridization, mutation through physical and chemical methods, and modern transgenic approaches are often used to improve the desired traits without inducing the pleiotropic effects. But these breeding methods are highly laborious and complicated to enhance crop production. Recently, targeted genome editing through engineered nuclease including zinc finger nuclease, transcription activator like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPRs) have been used to improve various traits in plants. Genome editing has emerged as a novel alternative approach to classical breeding with higher mutagenic efficiency. Here, we briefly cover the strengths of CRISPRs in comparison with other genome editing techniques. We also discuss its potential applications in genetic improvement of various crops and future prospective.

Challenges in wide implementation of genome editing for crop improvement

Genome editing technologies using customizable sequence specific nucleases (SSNs) enables precise genome modification in plants, which has revolutionized functional genomics research as well as crop improvement. Applicability of SSN-mediated genome editing ranges from targeted gene knockout and single base modification to multiple gene stacking into desired genomic sites. However, there are still considerable challenges in implementing genome editing technologies for practical crop improvement. Here, we briefly discuss the technological challenges, especially those associated with the delivery of SSNs and with homologous recombination-mediated genome editing, and address some promising solutions to overcome the issues.

Regulatory aspects of genome-edited crops

Genome editing with engineered nucleases (GEEN) is a highly efficient means of generating useful traits in crops. The application of GEEN for multiple traits and crops is resulting in products that are already entering the marketplace. Genome editing represents diverse and robust techniques targeting highly specific genome locations to cause double-strand breaks and subsequent repair by homologous and non-homologous mechanisms. Genome edits range from simple random insertions or deletions to template additions to stacking of multiple transgenes at a specific locus. Consequently, it is not easy to generalize the regulatory view of genome editing for crop improvement since a case-specific consideration is necessary which, in many regulatory regimes, encompasses the process used and the genome edit achieved in addition to the phenotype derived. Key process considerations for regulators of a GEEN-derived product are the nature of the edit, specificity, the source of the editing machinery, and post-editing trait segregation. These considerations along with the nature of the derived trait and performance of the crop phenotype inform the safety and benefit of a given GEEN product. The view of regulators toward a particular genome-edited crop is driven by the degree that regulatory statutes are product-versus process-focused and will be shaped by emerging scientific and public views of genome-editing technology. In order that the public will not confuse genome-edited crops with genetically modified organisms (GMOs), scientists and regulators alike need to carefully frame and communicate GEEN techniques in terms of the specific process used and the benefits derived through development of useful crop traits.

Targeted Genome Editing for Crop Improvement

Crop improvement is essential to attaining world food security and enhancing nutrition for human beings. Both conventional breeding and modern molecular breeding have contributed to increased crop production and quality. However, the time and resources for breeding practices have been limited. It takes a long time to bring a novel improved crop to the market, and the genetic sources from wild species cannot be always available for crops of our interests. Genome editing technology implemented molecular breeding can overcome those limitations of time and resource by facilitating the specific editing of plant genomes. However, there is a long-lasting argument about the safety of genetically modified organisms (GMOs). In this review, we briefly summarize the principle of genome editing tools, focusing on the CRISPR/Cas9 system and the application of these tools to plants in the service of crop engineering.

Genome editing for crop improvement: Challenges and opportunities

ABSTRACTGenome or gene editing includes several new techniques to help scientists precisely modify genome sequences. The techniques also enables us to alter the regulation of gene expression patterns in a pre-determined region and facilitates novel insights into the functional genomics of an organism.Emergence of genome editing has brought considerable excitement especially among agricultural scientists because of its simplicity, precision and power as it offers new opportunities to develop improved crop varieties with clear-cut addition of valuable traits or removal of undesirable traits. Research is underway to improve crop varieties with higher yields, strengthen stress tolerance, disease and pest resistance, decrease input costs, and increase nutritional value.Genome editing encompasses a wide variety of tools using either a site-specific recombinase (SSR) or a site-specific nuclease (SSN) system. Both systems require recognition of a known sequence. The SSN system generates single or double strand DNA breaks and activates endogenous DNA repair pathways. SSR technology, such as Cre/loxP and Flp/FRT mediated systems, are able to knockdown or knock-in genes in the genome of eukaryotes, depending on the orientation of the specific sites (loxP, FLP, etc.) flanking the target site. There are 4 main classes of SSN developed to cleave genomic sequences, mega-nucleases (homing endonuclease), zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the CRISPR/Cas nuclease system (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein). The recombinase mediated genome engineering depends on recombinase (sub-) family and target-site and induces high frequencies of homologous recombination.Improving crops with gene editing provides a range of options: by altering only a few nucleotides from billions found in the genomes of living cells, altering the full allele or by inserting a new gene in a targeted region of the genome. Due to its precision, gene editing is more precise than either conventional crop breeding methods or standard genetic engineering methods. Thus this technology is a very powerful tool that can be used toward securing the world's food supply. In addition to improving the nutritional value of crops, it is the most effective way to produce crops that can resist pests and thrive in tough climates.There are 3 types of modifications produced by genome editing; Type I includes altering a few nucleotides, Type II involves replacing an allele with a pre-existing one and Type III allows for the insertion of new gene(s) in predetermined regions in the genome. Because most genome-editing techniques can leave behind traces of DNA alterations evident in a small number of nucleotides, crops created through gene editing could avoid the stringent regulation procedures commonly associated with GM crop development. For this reason many scientists believe plants improved with the more precise gene editing techniques will be more acceptable to the public than transgenic plants. With genome editing comes the promise of new crops being developed more rapidly with a very low risk of off-target effects. It can be performed in any laboratory with any crop, even those that have complex genomes and are not easily bred using conventional methods.