MOVE

Understanding the Problem

Humans have invented various inventions for agriculture, including fertilizers, pesticides, agricultural equipment, and irrigation systems. Pesticides, among others, have had a major impact on increasing human crop production. Pesticides have contributed to increased yields in many crops, while at the same time contributing significantly to the economic interests of farmers [1]. Without the use of pesticides, it is estimated that fruit production would decrease by 78%, vegetable production by 54%, and grain production by 32%[2]. Although pesticides continue to be indispensable for food production in modern society, they are facing the need for new technological innovations. There are three reasons.

Current Solutions and Their Drawbacks

Need for IPM

One promising approach to solving the above modern pesticide challenges is the concept of Integrated Pest Management (IPM). IPM is the careful consideration of all available pest control technologies and the integration of appropriate measures to control pest population growth. It is an attempt to combine biological, chemical, physical, and crop-specific (cultivation) management strategies and practices to grow healthy crops, minimize pesticide use, and reduce or minimize the risks pesticides pose to human health and the environment for sustainable pest management[4]. Since the 1960s, IPM has been discussed in many countries around the world, and the need for IPM is becoming increasingly important.

Options in IPM: RNA Pesticides

One of the tools that is now receiving the most interest in conducting IPM is RNA pesticides. RNA pesticides are pesticides that utilize RNA interference (RNAi) technology, which suppresses gene expression in a sequence-specific manner through transcriptional repression against plants and pests. By utilizing this technology, it is possible to target specific genes of pests and pathogens and prevent their growth and survival, and implementation is currently underway. The main features and advantages of RNA pesticides are as follows:

1. Specificity : RNA pesticides pinpoint specific genes in targeted pests and pathogens, thus having less impact on non-target organisms.
2. Low environmental impact : RNA pesticides degrade faster in the environment and are less persistent than conventional chemical pesticides.
3. Reduction of resistance problems : Easier to avoid resistance in pests and pathogens, and can be expected to have long-term effects.
4. Wide range of application : Enables control of soil-borne bacteria and viruses that conventional chemical pesticides could not handle.

Problems with RNA Pesticides

Despite this promising new technology, RNA pesticides have not yet been widely implemented in the real world. Despite the effort put into RNA pesticides by many companies and researchers, including other iGEM teams, there are currently only four or five RNA pesticides approved worldwide. In our meetings with pharmaceutical companies, we found that almost all companies face one main challenge when introducing a new pesticide: Cost, building a mass production line . Interestingly, however, according to Greenlight, one of the companies currently implementing RNA pesticides in the US, the company offers 10,000 kg/year of RNA pesticides for less than $10/ha, a level less expensive than current chemical pesticides. In other words, in terms of RNA mass production and cost, RNA pesticides are already (for some companies) competitive with chemical pesticides. So, why have they not yet been widely implemented in society? It is because the real challenge lies in the duration of efficacy of RNA pesticides.

Duration of Effectiveness: The Biggest Barrier to Practical Application

While typical chemical pesticides remain effective for several weeks, RNA pesticides remain effective for only about 4 days. This short duration of effect is the biggest barrier to the practical application of RNA pesticides. Shorter durations require more frequent application, which increases labor costs, increases fuel consumption for spraying equipment, and damages farmland due to soil trampling. These factors make the overall cost of using RNA pesticides higher than that of conventional chemical pesticides, even if production costs are lower. Previous studies have attempted to protect RNA using artificial lipid membranes to increase the longevity of their effects. However, these approaches had fatal inconsistencies and drawbacks:

1. High cost : The high cost of producing artificial lipid membranes has been a major obstacle to their practical application[5].
2. Difficulty in control : it was difficult to precisely control the timing and amount of RNA release.
3. Scale-up issues : The manufacturing process was not suitable for large-scale production.

Due to these factors, existing approaches had not been implemented in society and remained a theoretical possibility.

Our Innovative Solution

We present a solution to this challenge, “Modules for Optimized Viability and Efficacy of RNA pesticides (MOVE) .

MOVE is a project to formulate RNA pesticides with the following features and advantages.

1. Envelopment of RNA in membrane vesicles : Encapsulating RNA in membrane vesicles (MVs), which are cell membrane vesicles, enhances the persistence of RNA’s effects in the outdoor environment.
2. Add functionality by surface presentation : Add functionality to enhance the efficacy of pesticides by presenting adhering proteins or proteins that break the membrane of pathogens on the surface layer of MVs, rather than just wrapping them in a membrane.
3. Predictive model for RNA pesticide implementation : Create an economic model surrounding pesticides to predict how much RNA pesticides can be implemented at what price.
4. RNA pesticide creation support platform : Create a platform where RNA sequences that are suitable as targets for RNA pesticides can be submitted to support the creation of RNA pesticides.

Through these projects, we will move RNA pesticides (MOVE), move pest control (MOVE), and move agriculture (MOVE).

Specifically, we do the following.

1. Encapsulating RNA in Membrane Vesicles: Fusion of shRNA and PIA-MVP

To solve the problem of short persistence of RNA pesticides in the field environment, we propose the world’s first innovative approach: encapsulation of shRNA (short hairpin RNA) with PIA-MVP (Polymer Intracellular Accumulation-triggered system for Membrane Vesicle Production). PIA-MVP is a mechanism proposed by Dr. Taguchi of Kobe University [4], which promotes highly efficient MV production through a good correlation between the activity of the glucose concentration-dependent PHB accumulation pathway and m-MV (multi-layer membrane vesicle) production. We have devised a novel application of this in combination with shRNA. Please check Design for more details. This unique combination has the following innovative features:

1. Dramatic improvement in cost efficiency through continuous production : PIA-MVP can produce MV without killing cells and at a significantly lower cost than conventional artificial lipid membranes. This removes the greatest barrier to the practical application of RNA pesticides.
2. Increased shRNA stability : Encapsulation with PIA-MVP can greatly reduce degradation of shRNA in the environment.
3. Scalability : PIA-MVP is suitable for large-scale production of MV. This enables a smooth transition from RNA-encapsulated MV production at the laboratory level to actual pesticide production.
4. High biocompatibility : PIA-MVP is expected to have a low environmental impact due to the use of biogenic materials. This is very important in terms of sustainable agricultural practices.

With this innovative approach, wet-lab aims to extend the effect duration of RNA pesticides from the current 4 days or so to at least 2 weeks.

2. Adding functionality through surface presentation: “Formulation” of RNA pesticides

In general, pesticides undergo a process called formulation in order to increase their effectiveness or to compensate for their weaknesses. In addition to compensating for the weakness of pesticides that are easily degraded as described in section 1, we are also trying to solve the weakness of pesticides that are sprayed and washed away by rain, or that RNA does not reach the target by surface presentation of several proteins on the MV. We decided to use the SpyCatcher-SpyTag system to present multiple proteins on the same membrane surface, Spontaneously forms an isopeptide bond between SpyTag, a short peptide, and SpyCatcher, a small protein. For more information on the SpyCatcher-SpyTag system, please check Design.

3. Predictive model for RNA pesticide implementation: economic model

Reducing the cost of RNA pesticides is essential for real-world implementation of RNA pesticides. Does the RNA pesticide module we are creating meet the constraints woven into the agricultural economy? By modeling the economic gains and losses for farmers, crop markets, and indirect environmental impacts of pesticides, we show that the implementation of MOVE will have a logically positive benefit to society. This is also a roadmap that iGEMers working on RNA pesticides in the future can refer to.

4. RNA Pesticide Creation Support Platform:

MOVE is a formulation module for social implementation of RNA pesticides. RNA pesticides can be created by determining the sequence of the required RNA, and there are already many examples of RNA sequences in the world. We have created an RNA pesticide sequencing platform that, when combined with this module (or others), will enable more people around the world to benefit from RNA pesticides. While chemical pesticides were characterized by their active ingredient chemicals, the sequences that can be inhibited by RNA interference are very limited and more universal. In implementing an RNA pesticide, the characteristics of the pesticide would not appear in its sequence, but rather in the way it is formulated. We wanted to develop RNA pesticides as quickly as possible by treating the sequences for RNA interference as open source.

Key Effects

MOVE is an attempt to implement RNA pesticides. The major impacts of MOVE are as follows.

1. Promoting the practical application of RNA pesticides : The technology of encapsulating RNA in membrane vesicles extends the duration of efficacy of RNA pesticides, and the added functionality of surface presentation reduces the need for frequent spraying, making the practical application of RNA pesticides feasible.
2. Comprehensive cost reduction due to significantly reduced application frequency : Extended duration of efficacy will reduce the number of applications, which will reduce labor costs, fuel consumption of equipment, and damage to farmland. In addition, manufacturing costs will be kept low, resulting in overall cost reductions for the market as a whole.

MOVE, which implements RNA pesticides, will also indirectly bring about the impact that RNA pesticides will ultimately have on humanity.

1. Providing effective countermeasures to the problem of resistant bacteria : Since RNA pesticides target specific genes, they can easily circumvent resistance in pests and pathogens and are expected to have long-term effects. This will reduce the occurrence of resistant fungi and resistant pests, and enable sustainable pest management.
2. Effective solution to difficult-to-control pests : RNA pesticides are effective against soil-borne pests and other diseases that have been difficult to control with conventional chemical pesticides. They provide a means of controlling pests that have been difficult to control, and can be used in combination with existing pesticides to achieve IPM.
3. Reduction of environmental impact : Compared to conventional chemical pesticides, RNA pesticides degrade faster in the environment and are less persistent. They also have minimal impact on non-target organisms, thus contributing to the protection of biodiversity.

Bigger Picture

Our goal is expressed in the following mottoes.

The three MOVEs are to build a delivery system for RNA pesticides and implement RNA pesticides (MOVE RNA Pesticides), to further advance current pest control with RNA pesticides (MOVE Pest Control), and to change agriculture for the better (MOVE Agriculture), and to change agriculture for the better (MOVE Pest Control). MOVE does not target specific pests like other agrochemical projects in the iGEM team. MOVE, which aims to implement and raise the bar in the formulation of RNA pesticides, does not target just one RNA, but wants to achieve the transport of as many RNAs as possible. Based on iGEM’s Give & Share Philosophy, MOVE will then aggregate information on RNA pesticides for various pests and diseases to expand the range of RNA pesticide applications with comprehensive knowledge. Ultimately, the project will contribute to comprehensive pest control that is not limited to existing pesticides alone, and will advance agriculture by improving production and ensuring stable livelihoods for farmers.

Reference

[1] Jerry Cooper, Hans Dobson, The benefits of pesticides to mankind and the environment. Crop Protection 26(9) , 1337-1348 (2007).

[2] Tudi M, Daniel Ruan H, Wang L, Lyu J, Sadler R, Connell D, Chu C, Phung DT, Agriculture Development, Pesticide Application and Its Impact on the Environment. Int J Environ Res Public Health 18(3) , 1112(2021).

[3] Bernardes, M. F. F., Pazin, M., Pereira, L. C., Dorta, D. J. , Impact of Pesticides on Environmental and Human Health in Toxicology Studies - Cells, Drugs and Environment (Publisher 2015).

[4] FAO, “Integrated Pest Management” in Pest and Pesticide Management, https://www.fao.org/pest-and-pesticide-management/ipm/integrated-pest-management/en/

[5] Yan, S., Ren, B.-Y. and Shen, J. , Nanoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management. Insect Science 28 , 21-34 (2021).