Poster: TUDelft

PHOCUS: Target locusts from within

Authors:

Alicia Rodríguez Molina, Eline Doornenbal, Fiona Horne, Gabriela van Leersum, Iris Forkink, Javier Navarro Delgado, Maartje Spaans, Nick Bowring, Ramon van Valderen, Shikha Sebastian, Willem van Holthe

Abstract:

Since ancient history, locust plagues have been devastating crops and pastures, threatening food security across the globe and affecting the livelihood of ten percent of the world population. Current strategies to fight locust swarms rely on unspecific and dangerous chemical pesticides that harm other insects, or on biopesticides that are too slow to be used in an outbreak. Our mission is to provide a novel biopesticide against locusts that is fast-acting and safe. We introduce PHOCUS, a biopesticide based on engineered bacteriophages that infect the gut bacteria of the locust. After infection, the bacteria produce a crystal protein (Cry7Ca1) from Bacillus thuringiensis (Bt) that specifically harms locusts, and RNA interference (RNAi) precursors, all encoded within the phage genome. Cry7Ca1 punctures the gut lining, harming the locust and allowing the RNAi precursors to reach the hemolymph, where they mature and silence the expression of vital locust genes. With this unique complementary approach, PHOCUS kills the locusts from within.

Mission statement and vision:

Our mission is to provide a novel biopesticide against locusts that is fast-acting and safe bio-pesticide through responsible innovation and collaboration. By doing this, we aim for food security and economic stability across the globe to achieve a brighter future for all.

From left to right: Maartje Spaans, Shikha Sebastian, Willem van Holthe, Iris Forkink, Alicia Rodríguez Molina, Gabriela van Leersum, Nick Bowring, Fiona Horne, Javier Navarro Delgado, Eline Doornenbal, Ramon van Valderen

A worldwide crisis

Locust plagues have afflicted human civilisations throughout history. Unfortunately, this problem of biblical proportion has not been left in the past. Recently, humongous swarms of the desert locust, Schistocerca gregaria, have threatened the livelihood of communities, endangering food security and economic stability [3]. The Food and Agriculture Organization (FAO) of the United Nations predicts that a staggering 10% of the world population will be affected by the current plague[1].

Our target is the desert locust as this is the species that inflicts the most damage [4]. Normally, locusts live solitary lives. Why do they become problematic?

According to the Food and Agricultural Organisation (FAO) of the United Nations there is a need to produce a pesticide that is fast, easy to use and environmentally safe [2].

Locusts aggregate during periods of drought and consequent habitat decline follow periods of heavy rainfall, vegetation growth, and breeding at (sub)tropical temperatures [5].
As locusts turn gregarious, breed excessively, have a less restricted diet and become voracious [6]. A typical swarm consumes the same amount of food as 3.5 million people per day [3].
The gregarious locusts devour any crops and vegetation they find and migrate when there is nothing left, resulting in widespread destruction of farmland and pastures.

*Figure 1. Areas affected by desert locust plagues [5].*

*Figure 2. Phase polyphenism of the desert locust (Schistocerca gregaria). Alterations of the desert locusts from a solitary phage to a gregarious phase.*

Current solutions

Two control methods exist, both with serious drawbacks; chemical pesticides and biopesticides.

Chemical pesticides, although effective and fast, are not specific to the desert locust. As such, they are also harmful to other animals, humans and the environment [7].
The alternative, a bio-pesticide called Metarhizium anisopliae, is a fungus that is specific to locusts. However, it works slowly, with a killing time of 7 to 14 days [2]. This lack of speed prevents its use in emergency responses to sudden outbreaks.

In essence, the people fighting the desert locusts are in need of a safe and fast solution to end these troubles once and for all.

Our solution: PHOCUS

The solution: A bacteriophage based biopesticide

We designed PHOCUS, a biopesticide targeting the desert locust based on engineered phages that combines the safety of a biopesticide with the speed of a chemical pesticide. The biopesticide consists of a cocktail of engineered bacteriophages targeting the Enterobacter genus in the gut of the locusts [8].

In short, PHOCUS works as follows:

The engineered bacteriophages contain the DNA coding for the locust specific Cry7Ca1 toxin and shRNAs.
The locusts ingest the engineered bacteriophages that are sprayed on crops, resulting in infection of the gut bacteria. The gut bacteria produce the locust specific toxins encoded within the phage genome. These molecules are released upon lysis.
The locusts are killed through the combined insecticidal action of the Cry7Ca1 toxin and gene silencing by shRNAs.

Figure 3. Project graphical overview. 1. Bacteriophages are engineered to contain DNA coding for our toxic molecules. 2. Engineered bacteriophages will be sprayed on vegetation in affected areas. 3. Locusts will eat the crops and ingest the bacteriophages. 4. Bacteriophages infect host bacteria and produce Cry7Ca1 and shRNA. Upon lysis, the cell bursts and the toxins and shRNAs are released. 5. Toxin and shRNA release causes locusts to die, swarms disappear.

Delivery: engineered bacteriophage

Phages engineered using BRED (Bacteriophage Recombineering of Electroporated DNA)

To engineer a phage to encode toxic molecules to locusts, we first investigated whether we could successfully engineer bacteriophages by replacing non-essential genes. For this reason, we performed proof of concept experiments using enhanced GFP del6(229) (eGFP) as a reporter,. The non-essential early/middle genes, 0.6, 1.1 and 4.3, were replaced separately by a double-stranded DNA (dsDNA) construct, coding for enhanced GFP, through Bacteriophage Recombineering of Electroporated DNA (BRED), [9]. As the first product of BRED results in a mixture of wild-type and engineered phages [9], a CRISPR/Cas9 system was designed and expressed in E.coli BL21 (DE3) as described by Kiro et al [10] to speed up the selection process for the engineered phage, as it results in the cleavage of the T7 wild-type phage DNA. Screening of positive plaques was performed by PCR.
Further PCR screening should be done to isolate the T7 phage engineered at position 4.3, using the constructed E.coli BL21 (DE3) host strain containing the CRISPR/Cas9 system targeting the 4.3 gene of the wild-type T7 phage.

Figure 4. BRED. dsDNA containing homologous regions to the bacteriophage are electroporated into cells containing bacteriophages. Recombination results in recombinant bacteriophages, containing the dsDNA fragment

Figure 5. T7 bacteriophage engineering replacing gene 4.3 by eGFP using BRED. A) Representation of the amplification of the engineered region of T7 with external primers marked as E, and one internal and one external primer marked as I. B) PCR screening of BRED samples for replacement of non-essential genes 0.6, 1.1 and 4.3 (lanes 2 through 7) using internal and external primers. Lane 8 (wt) shows amplification of the entire lysate of T7 wild-type obtained after electroporation without the addition of dsDNA substrate. C) PCR Screening of secondary plaques of engineered phages in the 4.3 gene position, with both internal and external primers. Numbers of each row correspond to each plaque screened. Each gel consisted of 1% agarose, the DNA ladder used is the Smartladder (Eurogentec)

Non-essential genes 0.6, 1.1 and 4.3 of the wild type T7 phage were replaced with eGFP using BRED. We successfully obtained positive engineered phages in each of the positions after screening by PCR.
A CRISPR/Cas9 system targeting the 4.3 gene of the wild type T7 phage was constructed to isolate the T7 phages engineered in the 4.3 genome.

Molecules toxic to locusts: Cry7Ca1 and shRNA

Figure 6: Cry7Ca1 protein mechanism (left side). The protoxin is then solubilised in the insect gut and processed by gut proteases, leading to their activation. The activated Cry toxin subsequently binds to specific receptors located on the insect gut epithelium, leading to the formation of a pore structure in the cell membrane. This leads to cell lysis, gut damage and eventually larvae death Schematic of the RNAi approach (right side). When shRNA enters the cell it breaks down mRNA, leading to gene silencing. shRNA (shRNA) is processed by Dicer into short interference RNA (siRNA). The RNA-induced silencing complex (RISC) is formed, hybridising with the target messenger RNA (mRNA), producing its cleavage and gene silencing. .

Toxic Molecules:

Our approach to kill the locusts is based on the production of Cry7Ca1, a toxin specifically harmful to locusts, from Bacillus thuringiensis, and silencing of vital genes by means of RNA interference (RNAi).

Cry7Ca1:

The Cry7Ca1 protein has insecticidal activity specifically against Locusta migratoria manilensis [11], unlike unspecific chemical pesticides. Upon ingestion, the toxin is activated by proteases in the gut of the targeted insect.

We expressed the Cry7Ca1 toxin from B. thuringiensis (BBa_K3407017) in E. coli BL21 (DE3) and successfully purified it.

RNAi:

RNA interference (RNAi) is a biological process where RNA is used to silence genes. Normally DNA is transcribed into messenger RNA (mRNA), this is then translated to a protein. However, when potentially hazardous double-stranded RNAs (dsRNA) are detected by the cell, the mRNA corresponding to that dsRNA is cleaved, thus degrading the mRNA.

Short-hairpin RNA

We produced short-hairpin RNA instead of long double-stranded RNA used in literature, to limit off-targets and ensure stability.

Transport

One of our main challenges was to design an approach to transport the shRNA from the gut, where it is produced, to the hemolymph, where it has been shown effective [12]. We developed a strategy with which we co-express transporting proteins together with our shRNA to make this possible.

We successfully produced shRNAs in vitro with viral T7 polymerase, which indicates viruses can be used for its production (BBa_K3407022).
We showed our shRNA can be processed by Dicer-2, an essential enzyme from the RNAi pathway in insects, showing it has the potential to trigger gene silencing inside locusts (Figure 7).
We demonstrated that the loop in our shRNA can bind to the Fox-1 RNA Binding Domain (RBD). This is a key protein that will participate in the transport of the shRNA to its target.
We successfully designed, cloned, expressed and purified Fox-1 RBD (BBa_K3407020) and Fox-1 RBD* (BBa_K3407024) (mutated, used as control).
We demonstrated that shRNAs binds to Fox-1 RBD (Figure 8)
We cloned and expressed Mini-3 and YmdB, additional proteins that are used to enhance the RNAi.

Modelling toxic molecules production

We chose to model the temporal evolution of the number of bacteria, phages, and produced toxin. With modelling we aimed to gain insight in the feasibility and speed of our biopesticide. To this end, we used two different models:

Model of phage infection and toxin production

The first model is a mechanistic model with the simplifying assumption that our system is always well-mixed, making it in effect 0-dimensional. Figure 9 shows the concentrations of susceptible bacteria, infected bacteria, phage and toxin concentrations of Cry7Ca1 and shRNA over time, as predicted with our model.

Figure 9. Phage infection and toxin production prediction in the desert locust gut. a) Susceptible bacteria concentration, b) infected bacteria concentration, c) bacteriophage concentration, d) Cry7Ca1 toxin concentration and e) shRNA concentration against time. This figure shows that lethal concentration of each toxin, 1.05 g/mL Cry7Ca1 and 1.2*10^12 shRNA molecules/mL, is produced within hours of infection[13 ][11 ].

With this model we demonstrated:

The capability of our biopesticide to produce lethal concentrations of each toxin (Cry7Ca1 and shRNA) within hours of infection. At these concentrations each toxin has the ability to kill the locust within 7 days.
That increasing the number of target bacteria, increasing the number of toxins produced per cell and increasing the stability of shRNA in the locust gut are the best design strategies to improve the effectiveness of PHOCUS.
That the rate at which bacteria acquire resistance is not a direct threat to our biopesticide.

2D biofilm simulation

Since the bacteria in the locust gut reside in microcolonies, which are small groups of cells packed close to each other, we modelled phage propagation and toxin production using a 2D biofilm simulation framework.

Figure 10. a) Heat map of a parameter sweep over impedance (x-axis) and initial phage concentration (y-axis). When impedance decreases, the phages can more easily diffuse through the biofilm. This increases the speed of phage propagation and results in a higher toxin production. Similarly, increasing the phage burst size speeds up phage propagation and results in a higher toxin production. b) Heat map of a parameter sweep over impedance (x-axis) and impedance (y-axis). This result demonstrates that the spatial organization of cells in biofilm structures promotes toxin production, as phages can easily spread when bacteria are packed closely. The values in each grid correspond with the mean maximum produced number of toxins toxins of 3 repeated simulations.

With this model we demonstrated:

That limitations in phage mobility (impedance) limits the ability of the phage to propagate which results in a decrease in produced toxins. With this, we showed that selecting high burst size phages is a good strategy to overcome this limitation.
That tight spatial organization of bacteria in such biofilm structures promotes phage propagation and toxin production.

Safety of PHOCUS

*Figure 11. Schematic of all the identified risks we came up with and for which we designed mitigation strategies*

Safe-by-Design

We want to develop our project according to Responsible Research and Innovation principles. Therefore we conducted a Value-Sensitive-Design assessment. From this we learned that the value of Safety is most important to our stakeholders. To best adhere to this value we executed the Safe-by-Design framework, in which risk assessment and management is integrated in the project from the start [14]. To increase safety of PHOCUS we considered the following:

Our delivery method phages: Our delivery method is safe because phages only infect bacteria. In addition, lytic phages are generally safe to humans and the human microbiome [20]

We use lytic phages: We use lytic phages minimizes the risk of horizontal gene transfer. Due to lysis, risk of creating a genetically modified (GM) phage is low [15].

How we engineer phages: We engineer phages for a range of hosts to limit propagation risk [16]. We insert sequences safe to the environment, humans or other animals.

We use the Cry7Ca1 toxin: This toxin is specific, non-pathogenic, non-toxic for humans and other animals [17,18].

We use double-stranded RNA: Double-stranded RNA is specific, non-pathogenic, non-toxic for humans and other animals [19].

Our choice of target bacteria: We target the Enterobacter genus. This species is also present in the human microbiome but unintended phage exposure will not negatively impact human health [20]. The effect of our biopesticide on the microbiome of other organisms should be studied.

We propose encapsulation to protect the phages from UV and temperature that is related to degradation and to limit the release of our bacteriophages in the locust gut.

Chances of resistance formation for toxin/shRNA is low: There is a chance that the locust can gain resistance to our shRNA or Cry7Ca1 toxin. In case of the RNAi resistance, the target sequences will be altered to target essential genes. The locust can gain resistance against Cry however our complimentary approach minimizes the chance of resistance propagation.

Chances of bacterial resistance towards phages is low: From our model we concluded that the bacteria are killed before they become resistant to phages. In addition to decreasing the chance of resistance formation, our cocktail can consist of phages targeting different receptors of the same bacteria to ensure susceptibility for our target bacteria.

Kill-switch is unnecessary: PHOCUS loses the competition as it most likely, has a reduced fitness [21], this will result in deletion of our insert. We use lytic phages, if the hosts die our phages cannot survive.

Field trials

The best approach for PHOCUS to overcome unfavorable legislation is by performing a risk assessment to show it is safe [22]. We investigated which data had to be gathered, and studied corresponding legislation. Before PHOCUS can be used, we concluded that:

PHOCUS has to comply with GM regulations, insecticide regulations and food regulations
PHOCUS needs to be tested for:
- Toxicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
- Pathogenicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
- Stability and potential to accumulate (GM phage, Cry7Ca1, RNAi)
- Uniqueness of sequence targeted (RNAi)
- Potential gene flow of insert (GM phage)
- Specificity to locusts (CryCa1, RNAi)
Environmental risk assessment [23] on PHOCUS needs to be done
Results have to be submitted to the Pesticide Referee Group

Integrated Human Practices

*Figure 12. Through talking to stakeholders from, among others, civil society, science, industry and end-users we designed PHOCUS to fit their needs.*

Throughout the development of our project we implemented feedback loops with the input from our stakeholders to create the best possible implementation. We started with understanding the problem in combination with the values and needs of our stakeholders and end-users through a Value-Sensitive Design assessment. This gives a primary understanding of the context in which PHOCUS should fit. To create further depth in our understanding we reached out to:

Affected farmers
The public at large
The scientific community
End-users, specifically the Food and Agriculture Organization (FAO) of the United Nations and national governments.

We did this by adhering to the principles of ethical research as described by Vanclay et al.[22]. This enabled us to also identify the desired solution, from the different perspectives of our stakeholders.

With the FAO, the global responsible authority on locust control, we have had contact throughout the project to best learn the ins and outs of the field. For example we learned that the scale of locust operations are so big, that sole farmers cannot make an impact. Therefore, there are specially trained professionals who perform the control operations.

We learned that our initial idea of “de-swarming” locusts was not a good approach. Both Em. Prof. Arnold van Huis and Harold van der Valk told us that this would only worsen the problem by dispersing the locusts and they advised to focus our efforts on a approach that kills locusts. Therefore, we changed our experimental design from de-swarming to killing locusts.

To further work towards the best possible implementation, we expanded our stakeholder scope to also include:

Regulators
Environmental organisations
The agrochemical industry

With these and the previous stakeholders we discussed ethical, safety, security and legal concerns.

From Simonis B.V., a preselected supplier of the FAO, we learned that all locust pesticide application is done in a standardized manner, using Ultra-Low Volumes (ULV), and it is be important that PHOCUS also complies with these standards to obtain successful implementation. Our models showed that with a low initial amount of phages, PHOCUS is could still be effective and could potentially be applied using ULV methods.

We combined all stakeholder insights to create an integrated and complete overview of the problem, and to figure out how PHOCUS can best contribute to a solution.

Entrepreneurship

Before PHOCUS can be used on desert locust swarms, it has to be further developed, manufactured and exported. Locust control organizations rely on agrochemical companies for these activities and thus, it is important that PHOCUS is commercialized to be translated from the lab to the real world. Therefore, we analysed the feasibility of commercializing PHOCUS and developed a business plan to attract investors:

We showed that our bacteriophage-based biopesticide PHOCUS is good for the world and developed it in close collaboration with our potential customer: the United Nations Food and Agriculture Organization (FAO).
We showed the feasibility of commercializing PHOCUS through our extensive analyses of the customers and the market. We concluded a.o. that PHOCUS meets unmet customer needs in the market and that the demand for a fast and safe biopesticide is large.
We were guided by one of FAO’s locust pesticide suppliers on developing ideas about how we would distribute PHOCUS, what regulations it has to comply with and how we can obtain funding.
Based on their advice and the promising results from the analyses, we wrote a business plan, which contains a company description, multiple market analyses (assessing current methods, its competitiveness and external threats), marketing strategies, manufacturing plans (including a bioprocess design and life cycle analysis), and a financial analysis.
We pitched our business plan to a potential investor, after which he showed great interest in funding us.

Here, you can see a Gantt chart that touches on our planning of the further development and commercialization of PHOCUS for the coming five years:

*Figure 13. Gantt chart showing the planning of PHOCUS for the coming 5 years*.

Science Communication

Novelty in science brings along challenges in the matter of regulation, social acceptance and ethics. The best way to overcome such barriers within synthetic biology (SynBio) is to involve a broader range of people than scientists alone in the discussion regarding the discipline and its implications to achieve social acceptance of it. This is why we reached out to a wide demographic, particularly the very young and the elderly. The young represent the future of SynBio, and the elderly may have diverging opinions due to their experiences. Involving them leads to the consideration of well grounded concerns. Furthermore, we reached out to a wider audience through cooking shows and national television to introduce them to SynBio and our project.

*Figure 14. Outreach activities for children*.

*Figure 15. Outreach activity for the elderly*.

*Figure 16. Outreach activities for the broader audience*.

*Figure 17. Outreach activities for the broader audience through media*.

We created a children's book which was translated to Dutch, English, French, and German, and shared it with iGEM teams worldwide and (inter)national schools to seed interest in SynBio.
We engaged with elderly people through a series of lectures on the locust problem, biotechnology, and our project, followed by an open discussion.
We reached out to a broader audience through playful cooking shows, blogs, podcasts and videos.
We also reached out to a broader audience through news fragments on TV, radio, and newspapers.

Acknowledgements

Supervisors

Dr. Timon Idema
Dr. Christophe Danelon
Ana Maria Restrepo Sierra
Britte Bouchaut
Charlotte Koster
Elisa Godino
Esengül Yildirim
Martin Holub

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Team:TUDelft/Poster