Application Of RNA Interference Research Articles

Advancing RNAi-based Strategies for Eco-friendly and Targeted Insect Pest Management in Sustainable Agriculture

The RNA interference (RNAi) has emerged as a promising approach for targeted and eco-friendly insect pest management in sustainable agriculture. The RNAi involves the silencing of specific genes through the introduction of double-stranded RNA (dsRNA), leading to the degradation of complementary mRNA and subsequent reduction in the expression of targeted proteins. This chapter provides a comprehensive overview of the current state and future prospects of RNAi-based strategies for insect pest control. We discuss the molecular mechanisms underlying RNAi, delivery methods for dsRNA, and the design and selection of effective target genes. The application of RNAi in controlling various insect pests, including lepidopterans, coleopterans, and hemipterans is extensively reviewed. also highlight the potential challenges and limitations associated with RNAi-based pest management, such as off-target effects, variable efficacy across insect species, and the development of resistance. Strategies to overcome these challenges, including the use of nanoparticle-based delivery systems and the combination of RNAi with other pest control methods, are explored. Furthermore, discuss the environmental and ecological considerations surrounding the use of RNAi in agriculture, emphasizing the importance of assessing non-target effects and the need for appropriate risk assessment frameworks. The chapter concludes by outlining future research directions and the potential for RNAi to revolutionize insect pest management, contributing to the development of sustainable and resilient agricultural systems.

Biosafety aspects of RNAi-based pests control.

While the overuse of classical chemical pesticides has had a detrimental impact on the environment and human health, the discovery of RNA interference (RNAi) offered the opportunity to develop new and sustainable approaches for pest management. RNAi is a naturally occurring regulation and defense mechanism that can be exploited to effectively protect crops by silencing key genes affecting the growth, development, behavior or fecundity of pests. However, as with all technologies, there is a range of potential risks and challenges associated with the application of RNAi, such as dsRNA stability, the potential for off-target effects, the safety of non-target organisms, and other application challenges. A better understanding of the molecular mechanisms involved in RNAi and in-depth discussion and analysis of these associated safety risks, is required to limit or mitigate potential adverse effects. © 2024 Society of Chemical Industry.

Biotechnological Innovations in Sustainable Food Production

This review article explores the critical role of biotechnology in addressing the global food security crisis aggravated by the COVID-19 pandemic and Eastern European conflict. It starts by examining the profound impacts of these events on food supply chains and pricing, supported by relevant statistical evidence. The study then discusses agricultural biotechnology, comparing conventional and sustainable agriculture, with an emphasis on genome editing and modification techniques such as Zinc Finger Nucleases (ZFNs), Oligonucleotide-directed mutagenesis (ODM), and CRISPR. The application of RNA interference in agriculture and microbial biotechnology in promoting soil health and sustainable food production is discussed. Furthermore, the review shifts focus to the utilization of microbial biotechnology for soil health enhancement and sustainable food production, encompassing the development of pest-resistant and drought-tolerant crops and biotechnological methods for optimizing water use efficiency. Additionally, it examines biotechnology's applications in food processing, including enhancing nutritional content and improving shelf life and safety. The European Green Deal is analyzed, particularly its influence on agriculture through strategies like Farm to Fork, Biodiversity, and Circular Economy. Finally, the review concludes by addressing policy considerations, ethical challenges, and the necessity of international cooperation in biotechnology research, exemplified by projects like the Golden Rice and Heat-Tolerant Maize for Asia (HTMA), highlighting the multidisciplinary nature and global significance of biotechnological innovations in ensuring sustainable food production.

Exogenous dsRNA sequence based on miR1917 downregulates its target gene related to ethylene signaling in tomato seedlings and fruit

The recent developments in application of RNA interference present a novel strategy for combating plant diseases by targeting pathogen genes. This study aimed to design, synthesize, and administer a double-stranded RNA (dsRNA) in tomato (var. Microtom) plants, drawing inspiration from the interaction between miR1917 and its target, the negative regulator of ethylene perception, Constitutive Triple Response 4 splicing variant 3 (CTR4sv3). An assessment of the potential endogenous effects stemming from the application of RNA molecules onto plant tissues was conducted, with a particular focus on observing resultant phenotypic changes. For seedling development analysis, seeds were soaked in a dsRNA solution for analysis, and they showed triple response phenotypic traits: reduction in root and hypocotyl length by up to 81 % and 60 % respectively, coupled with the noticeable development of hook. Furthermore, an 81 % decrease in CTR4sv3 expression was recorded, along with a 17-fold increase in the ethylene biosynthesis gene ACO1. Conversely, green-mature tomato fruit were subjected to dsRNA injections, resulting in a 43 % reduction in CTR4sv3 levels, accompanied by a more than 3-fold increase in ACO1 levels. Altogether, it is reported that the utilization of dsRNA based on miR1917 downregulates CTR4sv3, and the concomitant increase in ACO1 in both seedlings and fruit. With these findings, we propose that miRNA and target interactions can serve as a basis for RNA interference design and its application in plants to inhibit the plant's own mRNA can modify biosynthetic pathways, addressing both scientific inquiries and technological objectives.

Non-invasive dsRNA delivery via feeding for effective gene silencing in teleost fish: A novel approach in the study of gene function analysis

RNA interference (RNAi) is a powerful technique for gene silencing, involving post-transcriptional regulation of gene expression. Successful application of RNAi has been demonstrated in various organisms like nematodes, insects, and oysters by ingesting double-stranded RNA (dsRNA)-expressing bacteria. In this study, we attempted a non-invasive feeding method in the marine economy teleost fish, Sebastes schlegelii. To validate the feasibility of this approach in S. schlegelii, we utilized rotifers, Artemia nauplii, and commercial pellet feed as vectors to deliver Escherichia coli strain HT115 engineered to express dsRNA, targeting dead end (dnd) gene, known to be important for primordial germ cells (PGCs) migration and proliferation in fish. Our experimental results consistently showed that knockdown of the dnd gene effectively inhibited the development of PGCs in S. schlegelii, resulting in gonadal dysplasia and even sex reversal. This study marks a significant advancement in understanding gene function in teleost fish, laying the groundwork for future large-scale research in this field.

Improvement of economic and useful characters of wheat using RNA interference technology

Wheat is a strategic cereal crop of global importance and plays a leading role in the food supply of mankind. Despite the general trend to increase in its production, climatic changes leading to significant temperature changes, unpredictable precipitation or droughts and the appearance of new races of pathogens and pests significantly affect its yield. In order to prevent the negative impact of changes in climatic conditions on the productivity of this crop, it is necessary to develop innovative technologies for improving the resistance of wheat to environmental stresses. RNA interference (RNAi) represents a new potential tool for wheat breeding by introducing small non-coding RNA sequences with the ability to silence gene expression in a sequence-specific manner. A decrease in the expression of a certain gene determines the acquisition of a new characteristic through the elimination or accumulation of certain plant traits, which leads to biochemical or phenotypic changes that the original plants do not have. This literature review describes the progress achieved over the past decades in the application of RNAi to create wheat plants with improved economic and valuable traits. The main stages of the gene silencing mechanism mediated by short interfering RNA (siRNA) and microRNA (miRNA), features of their biogenesis, modes of action and distribution are presented. Examples of the use of various biotechnological approaches to wheat improvement using gene transformation, endogenous and exogenous double-stranded RNA molecules (dsRNA) are given. The possibility of using RNAi technology to increase the nutritional value and quality of grain, remove toxic compounds and allergens is highlighted. Considerable attention is paid to the practical results of various applications of RNAi to increase the resistance of wheat to biotic stress factors, in particular, viruses, bacteria, fungi, insect pests, and nematodes. Examples of the use of siRNA-mediated RNAi and the role of miRNA in improving wheat tolerance to abiotic stresses are summarized.

Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.

RNA-Based Therapeutics: Past, Present and Future Prospects, Challenges in Cancer Treatment.

It is becoming more and harder in today's climate to disregard the impact of cancer on social health. Even though a significant amount of money is spent annually on cancer research, it still ranks as the second leading cause of death worldwide. Additionally, only about half of the patients suffering from complex forms of cancer survive a year after receiving traditional cancer therapies. A method for silencing genes is called RNA interference (RNAi). Such a method is very effective in focusing on genes linked to cancer. Most gene products implicated in cancer have recently been used as RNA interference (RNAi) therapeutic targets. According to the findings from this research, RNAi application is necessary for today's cancer treatment to target functioning carcinogenic molecules and tumor resistance to chemotherapy and radiation. Proapoptotic and antiproliferative activity has been reported from previous research studies on cell culture systems, animal models, and clinical trials through the knockdown of gene products from RNAi technology. Numerous novel RNAi-based medications are now in the clinical trial stages thanks to the discovery of the RNAi mechanism and advancements in the area. In the future, genomic-based personalized medicines can be developed through this RNAi therapy. Hopefully, cancer sufferers will find this sort of therapy to be one of the most effective ones. Various kinds of RNA-based treatments, such as aptamers, small interfering RNAs, microRNAs, antisense oligonucleotides, and messenger RNA, are covered in broad terms in this study. We also present an overview of the RNA-based therapies that have received regulatory approval in the past or are now undergoing clinical studies.

Recent Advances in Engineering Carriers for siRNA Delivery.

RNA interference (RNAi) technology has been a promising treatment strategy for combating intractable diseases. However, the applications of RNAi in clinical are hampered by extracellular and intracellular barriers. To overcome these barriers, various siRNA delivery systems have been developed in the past two decades. The first approved RNAi therapeutic, Patisiran (ONPATTRO) using lipids as the carrier, for the treatment of amyloidosis is one of the most important milestones. This has greatly encouraged researchers to work on creating new functional siRNA carriers. In this review, the recent advances in siRNA carriers consisting of lipids, polymers, and polymer-modified inorganic particles for cancer therapy are summarized. Representative examples are presented to show the structural design of the carriers in order to overcome the delivery hurdles associated with RNAi therapies. Finally, the existing challenges and future perspective for developing RNAi as a clinical modality will be discussed and proposed. It is believed that the addressed contributions in this review will promote the development of siRNA delivery systems for future clinical applications.

RNA Interference in Agriculture: Methods, Applications, and Governance

RNA interference (RNAi) is a naturally occurring gene silencing mechanism conserved across organisms with a clearly defined cell nucleus (eukaryotes). Gene silencing by RNAi through the degradation of a target messenger RNA (mRNA) has historically been used as a research tool to study the function of genes. Over the past two decades, silencing of vital genes through RNAi has been explored for agricultural applications, including managing plant insect pests and pathogens, improving plant agronomic traits, and increasing consumer desirability of food. Using RNAi for crop protection is especially attractive because of its high specificity, which minimizes unintended effects on non-target organisms and improves the safety profile of RNAi products. This paper describes how RNAi functions, its current applications in agriculture, the current regulatory views of RNAi-based pesticides, and concludes with a discussion of current challenges for the commercial application of RNAi in agriculture. The content presented is intended to serve as a resource for regulatory agencies, policy and lawmakers, private and public institutions, and the general public to inform regulatory assessments and consumer choice decisions.

Bacteria-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

Since the discovery of the RNA interference (RNAi) mechanism, it has been widely used in the fields of gene function, biomedicine, and crop pest control. In the direction of agricultural application, this technology is highly expected, especially in the field of pest control, which is called "the third revolution in the history of pesticides". Currently, RNA biopesticides are developing rapidly all over the world. A genetically modified product (MON87411) has been approved for marketing, and a large number of agricultural companies are developing products based on direct spraying RNA biopesticides and submitting them for regulatory approval. The biggest problem that has emerged for spray RNA biopesticides is the technology for large-scale and low-cost production of dsRNA. At present, the bacterial fermentation production technology can realize large-scale dsRNA production with a yield of 4.23~182mg/L bacterial solution. This article describes the experimental protocol for dsRNA production technology based on bacterial fermentation.

Mesoporous Silica Nanoparticles Mediate SiRNA Delivery for Long-Term Multi-Gene Silencing in Intact Plants.

RNA interference (RNAi) is a powerful tool for understanding and manipulating signaling pathways in plant science, potentially facilitating the accelerated development of novel plant traits and crop yield improvement. The common strategy for delivering siRNA into intact plants using agrobacterium or viruses is complicated and time-consuming, limiting the application of RNAi in plant research. Here, a novel delivery method based on mesoporous silica nanoparticles (MSNs) is reported, which allows for the efficient delivery of siRNA into mature plant leaves via topical application without the aid of mechanical forces, achieving transient gene knockdown with up to 98% silencing efficiency at the molecular level. In addition, this method is nontoxic to plant leaves, enabling the repeated delivery of siRNA for long-term silencing. White spots and yellowing phenotypes are observed after spraying the MSN-siRNA complex targeted at phytoene desaturase and magnesium chelatase genes. After high light treatment, photobleaching phenotypes are also observed by spraying MSNs-siRNA targeted at genes into the Photosystem II repair cycle. Furthermore, the study demonstrated that MSNs can simultaneously silence multiple genes. The results suggest that MSN-mediated siRNA delivery is an effective tool for long-term multi-gene silencing, with great potential for application in plant functional genomic analyses and crop improvement.

Transcript Level and Sequence Matching Are Key Determinants of Off-Target Effects in RNAi.

Double-stranded RNA (dsRNA) pesticides, those based on RNA interference (RNAi) technology utilizing dsRNA, have shown potential for pest control. However, the off-target effects of dsRNA pose limitations to the widespread application of RNAi and raise concerns regarding potential side effects on other beneficial organisms. The precise impact and underlying factors of these off-target effects are still not well understood. Here, we found that the transcript level and sequence matching jointly regulate off-target effects of dsRNA. The much lower expressed target genes were knocked down to a lesser extent than genes with higher expression levels, and the critical sequence identity of off-target effects is approximately 80%. Moreover, off-target effects could be triggered by a contiguous matching sequence length exceeding 15 nt as well as nearly perfectly matching sequences with one or two base mismatches exceeding 19 nt. Increasing the dosage of dsRNA leads to more severe off-target effects. However, the length of mismatched dsRNA, the choice of different RNAi targets, and the location of target sites within the same gene do not affect the severity of off-target effects. These parameters can be used to guide the design of possibly selective sequences for RNAi, optimize the specificity and efficiency of dsRNA, and facilitate practical applications of RNAi for pest control.

RNAi turns 25:contributions and challenges in insect science.

Since its discovery in 1998, RNA interference (RNAi), a Nobel prize-winning technology, made significant contributions to advances in biology because of its ability to mediate the knockdown of specific target genes. RNAi applications in medicine and agriculture have been explored with mixed success. The past 25 years of research on RNAi resulted in advances in our understanding of the mechanisms of its action, target specificity, and differential efficiency among animals and plants. RNAi played a major role in advances in insect biology. Did RNAi technology fully meet insect pest and disease vector management expectations? This review will discuss recent advances in the mechanisms of RNAi and its contributions to insect science. The remaining challenges, including delivery to the target site, differential efficiency, potential resistance development and possible solutions for the widespread use of this technology in insect management.

Effective Agrobacterium-mediated genetic transformation of okra (Abelmoschus esculentus L.) and generation of RNAi plants resistant to Begomovirus infecting okra

ABSTRACT Okra is an important vegetable crop of the Malvaceae family and is infected by varying numbers of viruses of the genus Begomovirus. Regardless of the importance of the crop, very little consideration has been given to its genetic improvement. RNA interference (RNAi), a potent biotechnological tool, is known to control Begomovirus in many crops. For the implementation of successful RNAi, there is a need for an efficient genetic transformation system in okra. In the present study, we developed a procedure for Agrobacterium-mediated tissue culture–dependent regeneration of okra plants for the application of RNAi. Eleven transgenic okra RNAi plants were regenerated by utilising hypocotyls as explants. Transformed plants were screened with hygromycin at the regeneration stage and the presence of transgenes (AC1, AC2 & AC4 codes for replication-associated protein, transcriptional activator protein and suppressor of PTGS) in putative transformed plants was confirmed by polymerase chain reaction (PCR). Wild and transgenic lines were challenged with a dimeric Begomovirus clone or viruliferous whiteflies and the level of resistance was estimated with quantitative real-time reverse transcriptase PCR (qRT-PCR) by utilising viral gene–specific primers. The resistant transgenic lines accumulated very low titres of viral gene products according to the qRT-PCR assays compared to the control plants. This is the first report of tissue culture–mediated RNAi-derived resistance in okra against Begomovirus infection.

Prospects for deploying microbes against tree-killing beetles (Coleoptera) in Anthropocene

Forests form rich biodiversity hubs that act as large reservoirs of natural carbon. The spatial and temporal heterogeneity of these complex habitats of forest floors provides ecological services of immense socio-economic importance. However, these socio-economic ecological hotspots are incessantly exposed to multifarious abiotic, biotic, and anthropogenic disturbances, amongst which unpredictable forest pest (i.e., bark beetle) outbreak account for the loss of vegetation and microbiome of measurable quantum. The importance of the microbiome in forming an inseparable functional unit of every host and shaping its interaction with other partners has been well realized. Interestingly, forest pests, including bark beetles, are also reported to rely on their endosymbiotic microbial partners to manipulate tree defense machinery. In contrast, the microbiome forming the holobiont of trees also regulates the overall function and fitness of the host and significantly contributes to tackling these challenging situations. Nevertheless, how the holobiont of trees directly or indirectly influence beetle holobiont is still an enigma. The present review shall elaborate on the role of microbial tools in enhancing tree performance and fitness, which helps counter beetle damage. Besides, it shall also emphasize exploiting the role of microorganisms in acting as biocontrol agents in shielding the trees against beetle destruction. The application of endosymbiont-mediated RNA interference (RNAi) in working with two-tier specificity for controlling beetle devastations shall be discussed as new-age technological advances. All explanations are expected to put forth the potential of the microbial toolbox in offering better and more sustainable beetle management strategies in the future.

Lab-scale method for plant-mediated delivery of dsRNAs to phloem-feeding leafhoppers

RNA interference (RNAi) regulates gene expression in eukaryotes, and it is an emerging tool in crop protection by exogenous applications of double-stranded RNAs (dsRNAs) to silence the expression of essential pest genes. Nevertheless, delivery of dsRNAs to sap-sucking insects is a major challenge for RNAi applications. The present work aimed at verifying whether in leafhopper species, RNAi can be triggered by plant-mediated delivery, and providing a proof of concept towards field applications. Two phytoplasma vectors species, Euscelidius variegatus and Scaphoideus titanus (Hemiptera: Cicadellidae), were used as case study. Gene silencing can be achieved efficiently in both species through microinjection of dsRNAs, despite the technique being time consuming and inapplicable on large scale. This protocol was set as gold standard for the development of a higher throughput approach. Soaking of nymphs in a solution with co-adjuvant and dsRNAs as well as insect feeding on whole plants or detached leaves immersed in a dsRNA solution were assayed as alternative delivery strategies. Nymph soaking did not induce specific gene silencing, while plant absorption proved to be suitable to deliver both a coloured solution and control dsRNAs targeting green fluorescent protein gene. Insect feeding on detached leaves immersed in dsRNA solution was selected to test silencing of two gut-specific (legumain and natterin) and one ubiquitous (ATP synthase β) genes. The expression of the three genes significantly decreased in E. variegatus insects fed on dsRNA-treated plants. Similarly, a significant reduction of ATP synthase β transcript was measured in S. titanus fed on dsRNA-treated plants.

Biomimetic hypoxia-triggered RNAi nanomedicine for synergistically mediating chemo/radiotherapy of glioblastoma

Although RNA interference (RNAi) therapy has emerged as a potential tool in cancer therapeutics, the application of RNAi to glioblastoma (GBM) remains a hurdle. Herein, to improve the therapeutic effect of RNAi on GBM, a cancer cell membrane (CCM)-disguised hypoxia-triggered RNAi nanomedicine was developed for short interfering RNA (siRNA) delivery to sensitize cells to chemotherapy and radiotherapy. Our synthesized CCM-disguised RNAi nanomedicine showed prolonged blood circulation, high BBB transcytosis and specific accumulation in GBM sites via homotypic recognition. Disruption and effective anti-GBM agents were triggered in the hypoxic region, leading to efficient tumor suppression by using phosphoglycerate kinase 1 (PGK1) silencing to enhance paclitaxel-induced chemotherapy and sensitize hypoxic GBM cells to ionizing radiation. In summary, a biomimetic intelligent RNAi nanomedicine has been developed for siRNA delivery to synergistically mediate a combined chemo/radiotherapy that presents immune-free and hypoxia-triggered properties with high survival rates for orthotopic GBM treatment.Graphical

Inhibition of PD-L1 and tumor growth in triple-negative breast cancer using a magnetic nanovector with microRNA34a

BackgroundClinical applications of RNA interference for cancer treatment and immune therapy require the development of simultaneous therapy and imaging systems for microRNA. This research was performed to fabricate the miRNA34a-loaded magnetic nanoparticles and investigate its anticancer effects against triple-negative breast cancer (TNBC) in mice model.ResultsUsing two types of polymers to improve their water dispersibility and gene delivery, iron oxide magnetic nanoparticles were prepared for delivery of miRNA34a. The iron oxide magnetic nanoparticles were delivered to TNBC cells, and their efficacy was evaluated in vitro and in vivo. Delivery of miRNA34a reduced TNBC cell migration and decreased the expression of PD-L1 at the mRNA and protein levels. In animal experiments, delivery of miRNA34a reduced tumor growth, and immunostaining and algorithmic analysis confirmed the decrease in PD-L1 expression.ConclusionThis study is the first to modulate PD-L1 by delivering miRNA34a with magnetic nanoparticles, and the results suggest that miRNA34a can be delivered effectively using magnetic nanoparticles and has potential as a molecular imaging contrast agent.

Delivery of Methoprene-Tolerant dsRNA to Improve RNAi Efficiency by Modified Liposomes for Pest Control

The application of RNA interference (RNAi) technology for pest control is environmentally friendly and accurate. However, the efficiency of RNAi is often inconsistent and unreliable, and finding a suitable carrier element is considered critical to success in overcoming biotic and abiotic barriers to reach the target site. The fall armyworm, Spodoptera frugiperda (FAW), which is one of most important global agricultural pests, has recently spread rapidly to other parts of the world. In this study, a method to improve the stability and RNAi efficiency of the dsRNA carrier complex was reported. Methoprene-tolerant gene (Met) was selected as a target, a gene which is critical to the growth and development of FAW. Biomaterials nanoliposomes (LNPs) were modified with polyethylenimine (PEI) to deliver the dsRNA of Met. The synthesized Met3@PEI@LNPs reached a size of 385 nm and were found to load dsRNA effectively. Through stability and protection assays, it was found that LNPs provided reliable protection. In addition, the release curve also demonstrated that LNPs were able to prevent premature release under alkaline condition of the insect midgut but accelerate the release after entering the acidic environment of the target cells. The cell transfection efficiency of the prepared LNPs reached 96.4%. Toxicity tests showed that the use of LNPs could significantly improve the interference efficiency, with 91.7% interference efficiency achieved when the concentration of dsRNA in LNPs was only 25% of that of the control. Successful interference of Met demonstrated it could significantly shorten the larval period and make the larvae pupate earlier, thus achieving the purpose of control. In this study, we have demonstrated the use of nanotechnology to provide a novel RNAi delivery method for pest control.