2022 GreenLight work published in peer-reviewed academic journals

We will continue to update this list as more research is published throughout the year. Our team has at least three papers in the pipeline for publication this year.

Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays – American Chemical Society, 7/26/22

Editorial: New Applications of Insecticidal RNAi – Sec. Pest Management, 5/27/22

Toxicity of a novel dsRNA-based insecticide to the Colorado potato beetle in laboratory and field trials – Pest Management Science, 2/15/22

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International crop network validates ledprona as a new Mode of Action group

An effective and novel pest-management solution with low-to-no residues aimed at the Colorado potato beetle (CPB) has received validation as a new mode of action (MoA) by the Insecticide Resistance Action Committee.

Ledprona, the active ingredient in GreenLight’s CalanthaTM product, was approved as a new mode of action in the recent winter meeting of IRAC, an international association of crop-protection companies that focuses on resistance management and sustainable agriculture.

The first foliar-applied, dsRNA-based bioinsecticide that provides effective control of CPB, CalanthaTM is expected to be registered in the United States this year. The Colorado potato beetle (Leptinotarsa decemlineata) ravages plants like potatoes and eggplant and accounts for more than $500 million in annual crop loss worldwide. 

For decades, insect resistance to pesticides has challenged growers. Using integrated pest management, which includes rotating insecticides, farmers and agronomists can prolong the useful lifespan of crop treatments. GreenLight’s product is designed to work well with standard growers’ programs to control first- and second-generation Colorado potato beetle infestations.

CalanthaTM has a unique mode of action among chemical and biological insecticides, which will provide farmers with a new tool aimed at protecting potato and eggplant fields from the Colorado potato beetle and support their efforts at resistance management. 

Ledprona, expected to be classified as IRAC MoA group 35 (RNAi-mediated targeted suppressors), specifically targets only CPB, causing the beetle to stop eating and expire from accumulation of its own metabolic waste. Because it is based on double-stranded ribonucleic acid, CalanthaTM degrades quickly in the environment, supports biodiversity, and is an example of the next generation of eco-friendly crop-protection products. 

The Insecticide Resistance Action Committee helps growers around the world by developing mode-of-action classification schemes; identifying new technologies for insect, mite, and tick control products; and  implementing insecticide resistance management strategies for crop protection, plant biotechnology, and public health.

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GreenLight research recognized in four top tier peer-reviewed papers in 2021

Journal of Chromatography B

Versatile separation of nucleotides from bacterial cell lysates using strong anion exchange chromatography

ACS: Crop Protection Products for Sustainable Agriculture

Development of dsRNA as a Sustainable Bioinsecticide: From Laboratory to Field

Frontiers in Plant Science

First Sprayable Double-Stranded RNA-Based Biopesticide Product Targets Proteasome Subunit Beta Type-5 in Colorado Potato Beetle (Leptinotarsa decemlineata)

Frontiers in Agronomy

Knockdown of Genes Involved in Transcription and Splicing Reveals Novel RNAi Targets for Pest Control

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A tractor harvests in a potato field with farmers working alongside it

ACS book chapter details GreenLight’s progress in the development of dsRNA solutions

Agricultural pests are responsible for more than $100 billion in global crop losses each year. Meanwhile, the amount of arable land for farming is shrinking and traditional pesticides currently in the market are losing their efficacy. 

To secure a sustainable food supply for future generations, GreenLight is working on RNA-based agricultural solutions that are designed to affect the target pest and limit harm to any non-targeted organisms. In Crop Protection Products for Sustainable Agriculture, a new American Chemical Society book about crop protection innovation, a GreenLight team, led by Ken Narva and Thais Rodrigues,  shares the science behind sprayable dsRNA as a new mode of action plant health product that has shown efficacy comparable to market standards and fits integrated pest management systems. 

This product is intended for large-acre control of the Colorado potato beetle and has been shown to be effective at extremely low use rates. A challenge to wide-scale use of dsRNA is the cost-effective production of large quantities for field applications, which GreenLight has overcome with its cell-free process to manufacture high-quality dsRNA faster, on a larger scale, and much more inexpensively than traditional methods.

Successful registration of this solution will pave the way for additional dsRNA products for agricultural pest control, providing growers with biological alternatives to synthetic insecticides.

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two people attend to a vat in a large clean factory space

How to scale RNA production

The art and science of taking the RNA scale-up recipe from the lab to a manufacturing plant

“There’s a difference between baking a normal loaf of bread and a gigantic loaf 10,000 times bigger,” says Adam Shanebrook, a biotechnologist and pilot plant director at GreenLight Biosciences. “You’ll burn the outside and leave the inside uncooked.” His point: You can’t simply scale up chemical and biological processes. You have to be clever about it.

That is very much on the mind of researchers at GreenLight as they scale up their manufacturing from laboratory to commercial production for their RNA-based solutions targeted at Colorado potato beetles, a crop pest, and varroa mites, which decimate bee colonies. The company’s innovative cell-free RNA synthesis was shown to work at laboratory scale some years ago in Medford, Massachusetts, but GreenLight has opened a pilot plant at an old Kodak factory in Rochester, New York to make it work at a far greater scale.

Producing RNA in a lab or a factory typically involves gene-editing a cell, often a harmless bacterium. But the process doesn’t just manufacture RNA, it also makes the rest of the bacterium. That means that it is wasting a lot of energy and raw materials in making unwanted byproducts. What’s more, those byproducts are mixed in with the RNA you actually want, so you have to either separate them—an expensive and difficult process—or deal with an impure final product, full of debris left over from the cells.

The cell-free process

The cell-free process works rather differently. It uses harmless strains of E. coli—long used in the pharmaceutical industry to make things like human insulin—to make the starting materials for the cell-free reaction. To prepare for that, short rings of DNA called “plasmids” are made to provide assembly instructions for the cell-free RNA production step. Additionally, E. coli are used to make the enzymes that catalyze the production and assembly of the RNA molecule.

Then those products are put into a large vat containing a “soup” of nucleosides, the building blocks of RNA and DNA, and the enzymes pluck the floating nucleosides out of the soup, energize them so that they snap together like magnets, and assemble them in a line according to the DNA plasmids’ instructions. This has the advantages that it is faster than bacterial-cell production—perhaps 10 times faster, requiring about three hours per batch as opposed to 30 or so; it is cheaper; and it brings forth a far purer product, approximately 50-65% pure.

A large vat containing a “soup” of nucleosides, the building blocks of RNA and DNA, at GreenLight's Rochester, New York, manufacturing plant.
A large vat containing a “soup” of nucleosides, the building blocks of RNA and DNA, at GreenLight’s Rochester, New York, manufacturing plant.

By 2017, that was all shown to work in the lab. But although it might be easy to make an 8-inch baguette in a home oven, it doesn’t mean you could successfully make a 65-foot-long loaf in a gigantic mega-oven.

Building up to a 1,250-litre reactor

Karthik Ramachandriya is a biochemical engineer and process development director at GreenLight. He holds up a small object, the size of a pen lid. “The first experiments were done in a 50-microlitre reactor,” he says—that is 0.05 millilitres, a barely visible quantity. “Then we scaled that up to 250ml.” He holds up a coffee cup. “It wasn’t that challenging, going from a microreactor to a coffee cup.” Then the team tried it in a bench-scale reactor that contained 10 litres. Finally, in 2019, the company started to build the Rochester plant, trying a 150-litre reactor and eventually a 1,250-litre one.

“We figured out how to do it all at the bench scale in Medford,” says Eric Otto, a biotechnologist and GreenLight’s vice president of manufacturing. But at both stages—making the plasmids and enzymes, and making the RNA—there were challenges associated with scaling up.

With enzyme and plasmid production, “our target audience was the good E. coli that we’re trying to make happy,” Otto says. “It’s like brewing beer, but a bit more complicated. You need to maintain what the organism needs and provide sufficient oxygen and other nutrients while removing the carbon dioxide so the fermentation doesn’t get sick.”

But maintaining those conditions is a different job in a 150-litre tank than it is in a one-litre one. “In the lab, you shake it in a tube,” says Shanebrook. “But at a manufacturing plant you can’t just shake it up when it’s a whole tank.”

“The mixing is different,” agrees Erin Cobb, a process engineer. “It’s not instantaneous any more. It’s not perfectly uniform. How you’re delivering oxygen to the cells is different; it’s easy at a small scale using bottled oxygen, but that is not economical when you scale up, and it’s a combustion risk, so you have to use pressurized air.” You can simply shake a one-litre flask to get all the nutrients to where they need to be, and the whole thing will automatically be at a uniform temperature. 

Pumps, fork trucks, and conveyor belts

Cobb adds that there are also logistical issues. If you’re adding a kilo of salt to a reactor, she says, “I can lift that with my hand. Can I do that with something 10 times bigger? I can pick up the 10-litre tank and dump it out; I can’t with the 1,000-litre. I need pumps, fork trucks, conveyor belts.”

Even prosaic tasks like cleaning become more complicated. “The spatula that I scooped the raw material into the small tank with,” she says: “I can take it over to the sink, wash it in the autoclave,” a machine that sterilizes equipment with steam. “You can’t put a 1,000-litre tank in an autoclave. You need a whole sterile system, you have to heat it to 120°C, you need sterilized air going into the system.”

There’s also a human resources issue. As the process becomes larger and reliant on many more skillsets, you need different kinds of labor. Setting up the Rochester plant was a case in point. “You need to know about utilities, fire alarms, sprinklers, heating, ventilation, floor drains,” says Shanebrook. “You’re dealing with hundreds of people in skilled trades. People who fix the roof, coat the floor.” 

Inspecting clear containers for formulations at GreenLight's Rochester, New York, manufacturing facility.
Inspecting clear containers for formulations at GreenLight’s Rochester, New York, manufacturing facility.

Within GreenLight itself, there are many brands of expertise—engineers, biotechnologists, chemists, entomologists, agronomists—all of whom need to understand each other’s specialty to some degree. “The most important thing in scaling up and scaling down is understanding the people who have worked on the process, being really comfortable asking them questions,” says Cobb. “Can I call anytime and ask, does this look weird? Is it supposed to look like this?” 

A plant that can scale up

There are other complications. Although the Rochester plant is a large targeted RNA manufacturing plant, it is itself a pilot for a much bigger facility—hundreds of times larger, 20,000 litres or more. So there’s no point solving a problem at a 150-litre scale if it wouldn’t then work at a much greater one. For instance, it might be easy to find ways to remove waste products from a one-litre tank, so that the bacteria can thrive. But unless the solution would also work at a much larger scale, then it’s not a solution at all. The pilot plant is designed as far as possible to mimic the realities of a larger, commercial plant. “You need to understand the limitations of a 20,000-litre reactor when we’re developing a process at a one-litre reactor,” says Ramachandriya.

“This is our playground,” says Shanebrook. “In the commercial plant, it’ll be designed for purpose: your pump goes from A to B, it’s dedicated to this function.

“But in the pilot plant, you don’t know what you need, and you need to accept that. You need to be flexible, use hoses that go from A to B, or A to C, or D to A. You take the changes in stride.”

When you do meet a problem in the pilot plant, says Ramachandriya, what’s important is going back to the smaller scales and understanding it properly. “If it’s a biological function, we go back to the tiny scale,” he says. “If it’s a process function, we do it at the coffee-cup or the one-litre size.” If your E. coli are unhappy and producing too much acid, you can probably explain that in the microreactor. If your pipes are clogging because the RNA solution gets too viscous, you’ll need the larger reactors, with their own pipes and pumps, to see where it’s going wrong.

GreenLight’s reactor energizes nucleotiodes

What’s also important as you scale up, says Ramachandriya, is keeping costs down. For instance, when you’re ordering thousands of litres of nucleotide solution or nutrients for your E. coli, you don’t want to be spending too much per litre. The nucleotides are made from yeast byproducts and energized by enzymes in GreenLight’s own reactor; they can be bought in bulk. “The yeast industry is very large and produces a variety of products from its specialized process, and we gain from the economies of scale,” says Otto.

Rows of clear bottles filled with formulations at GreenLight's Rochester manufacturing facility.
At GreenLight’s Rochester manufacturing facility, clear bottles are filled with formulations of RNA solutions.

Shanebrook adds that the scaling project isn’t done yet. You have to foresee future demand: if, as GreenLight hopes, its products become widely used, then even the planned 20,000-litre reactor will not be enough. “It’s reading the tea leaves and planning for expansion,” he says. “As the manufacturing person, you don’t want to be the limiting step. If the business side says, ‘We want a bigger plant,’ you want to be able to say, ‘We have a plan for this;’ it’s a common problem that business says we want this next month, and the technologists say it’ll take two years.”

At some point, scaling up further in a single plant becomes impossible. “In baking,” says Shanebrook, returning to his earlier metaphor, “sometimes you reach a scale, X number of tons, where you can’t scale up any more. Instead you number up.” That is, you build more plants, doing the same thing. It has advantages of redundancy in case of disaster.

GreenLight is not at that stage yet. But Shanebrook, in particular, is already thinking about ways to continue the scale-up process. At the moment, at each scale—whether 50-microlitre or 1,250-litre—the RNA is produced in batches: a reactor is filled with raw materials, left to do its thing, and the final product is harvested. But perhaps the future is continuous production: “You can put all that stuff in the pot of soup for three hours,” he says, “or you can have a long pipe that takes three hours to flow from the input to the output.” 

The ability to run continuously is a scenario for the future, he hopes. But the scale-up has already been spectacular, dealing in hundreds of thousands of times the volume the GreenLight team did just a few years ago. 

“It’s sort of like I sat with the home chefs,” says Cobb, “and then learned to cater the wedding.”

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a potato beetle shows its understide while chewing on a leaf

Sustaining ladybug life

How RNA can target pests like the Colorado potato beetle and save beneficial insects

We have a problem. Insect populations worldwide seem to be dying and although solid research remains scant and populations vary year to year, scientists agree that insects are at risk and the pesticides we use to keep crops healthy are probably partly responsible.

The solution, though, isn’t to simply stop the use of pesticides, a vital part of modern agriculture. So how do we eliminate intended targets without killing beneficial insects that keep our ecosystems healthy? In order to keep feeding the world, we need more sustainable ways of getting rid of pests. Products with RNA offer one pathway to pesticide replacement. By allowing the targeting of a specific organism, they may provide farmers with a revolutionary new tool. 

“There’s a ton of evidence that, broadly, insect populations in Europe and North America are declining,” says David Goulson, a professor of biology at the University of Sussex in the UK. “Some studies suggest frighteningly fast.” Professor Helen Roy, an entomologist at the UK’s Centre for Ecology and Hydrology, agrees that some species are definitely struggling. Vital to ecosystems and agriculture, insects “do amazing things in the soil, are pollinators and pest controllers,” she says. 

Defoliated potato plant field
A defoliated potato plant field as a result of the Colorado potato beetle. Traditional pesticides used to control these pests harm beneficial pollinators like ladybugs, honeybees and others.

The Intergovernmental Panel on Biodiversity and Ecosystem Services lists five main drivers of biodiversity loss: land-use change, climate change, pollution, natural resource use and exploitation, and invasive species. Pesticide use would come under pollution. Kelly Jowett of the Rothamsted Research Institute studies carabids, a species of predatory beetle that preys on several pest insects. She says that researchers often don’t ask the right questions, so lab studies might say “our pesticide doesn’t kill the carabids. [but don’t] measure them six months later to see if they’re less fecund, whether they still breed or behave properly.”   

Goulson agrees “there are examples [of studies] that stand out, especially with regard to neonicotinoids. There’s fairly clear evidence that patterns of bee decline seem to be closely linked to patterns of neonicotinoid use, and that freshwater pollution with the insecticide in the Netherlands predicts loss of water insects. In Lake Shinji in Japan, there was a massive collapse in invertebrate life, which precisely coincided with the introduction of neonicotinoids into the surrounding fields.” Since then, these pesticides have largely been banned in Europe. In this breach lies the opportunity for solutions that target pests while supporting surrounding biodiversity.  

Brian Manley, director of product biology at GreenLight, points out that pesticides can kill beneficial creatures that help crops and even cause the pest species to flare up. “An example is spider mites,” he says. “They’re a significant pest in drier conditions. Several beneficial insects feed on spider mites, and they do a great job of managing the population without the need for insecticide programs.” The use of broad-spectrum insecticides “flares the mite population, because they kill the beneficials that manage them.”

Colorado Potato Beetle on a leaf
Colorado potato beetles are major pests across North America

Another example is aphids. “Ladybird beetles eat aphids and do a good job managing them, so farmers don’t have to worry about spraying for aphids,” he says. “If you knock out the ladybug, you flare the aphid populations.”

There’s a wider problem, which is that the repeated use of any pesticide will create strains of insect resistant to that pesticide, via the same mechanism that creates microbes resistant to antibiotics.

Ron Flannagan, GreenLight’s vice president for plant health research and development, cites the weed-killer Roundup as the classic example. “It initially controlled the vast majority of weed species, but with repeated use year after year across the farm without alternating modes of action, RoundUp is now far less effective due to resistance development.”

Insecticides such as DDT and organophosphate were widely used after the Second World War and led to large jumps in crop yields, says Goulson. But they then became less effective, so farmers had to use increasing amounts to achieve the same effect. “Farmers are in a tricky position,” he says. “They’re squeezed on profitability and struggle to make a living, so they can’t afford to lose crops to pests. One factor is risk aversion: the safest thing if you’re not sure is to spray, so if there’s a chance he’ll lose the crop if he doesn’t spray, he’ll spray.”

Pests can be controlled through other means—natural enemies, crop rotation, and the gold standard of “integrated pest management,” which encourages those methods and uses pesticides only as a last resort. “According to EU law, all farms have to use it,” Goulson says, “but it’s so loosely defined that it’s barely enforced. An added challenge is that no-pesticide systems can take time before beneficials are established.  

The advantage of RNA pesticides  

GreenLight’s pesticides–based on double-stranded RNA that targets the destructive pest–can respond to some of these field and mindset challenges. A key feature of the approach is that the RNA sequences target particular processes related to a specific insect, meaning that it should have no effect on other animals. “We can design with great specificity from the outset, looking at a particular process in the target insect,” says Ken Narva, head of entomology at GreenLight. 

Mixed with water and sprayed using conventional methods over crops, the RNA enters the cells of a target pest—–specifically the Colorado potato beetle—at a rate of a few grams per hectare (equivalent to 1 teaspoon per football field). This amounts to less than one-tenth the amount of conventional industrial chemicals normally used on fields. The Colorado potato beetles eat the treated foliage and ingest the material, which selectively targets the pest and does not affect any other organisms. The liquid formulation convinces the defoliator beetle’s digestive system that it has made the protein necessary for excretion, which causes it to stop eating potato leaves and eventually expire from its own toxins. Beneficial insects—including bees, butterflies, and the closely related ladybug—are unaffected. 

How the dsRNA mixture is delivered to the Colorado Potato Beetle
How the dsRNA mixture is delivered to the Colorado Potato Beetle

Growers often face more than one pest. “In the case of a potato farm, thousands of acres might need treating on a weekly basis,” says Manley, “so cheaper and easier is what they want. The Colorado potato is the number 1 insect pest to potatoes and can affect other plants in the nightshade family–like eggplant.”

Placed in direct comparison with their traditional counterparts, RNA pesticides appear to be as effective as a market-leading rival at killing the Colorado potato beetle. “From the trials we’ve performed so far,” says Flannagan, “it’s been statistically indistinguishable from the control, in terms of foliage protection and yield protection.” It may take longer—most traditional pesticides are neurotoxins and kill their targets in a few hours, whereas the RNA solution takes a couple of days—but the crop protection appears to be comparable, according to Manley.

Untreated vs treated plots of potato plant
The success of RNA alternatives: untreated (where the pest decimated the plants to the ground) versus treated plots, where the potato plants remain.

Russ Groves is a professor and department of entomology chair at the University of Wisconsin-Madison, where field trials are being conducted. “When we started working with GreenLight and looking at the target, I was pleasantly surprised at the effectiveness of the compound compared with other biologics,” he says. “It maintains a very adequate level of protection of the commodity that to me is appropriate at large commercial scales.”

RNA has other advantages

Another advantage of RNA is its fragility. Long molecules like RNA break down more easily than the short ones used in traditional pesticides, and RNA is digested quickly, lasting only a few days when exposed to the environment. 

Because RNA is naturally occurring and is present in every living thing, “we eat RNA every day, and in most organisms, it degrades in the digestive system,” says Narva. 

Although RNA pesticides might not provide the magic bullet for plant-destroying pests, they can be a powerful tool in integrated crop management. “What we recommend is that if you have two generations of Colorado potato beetle,” says Flannagan, “you use this on the first generation and a pesticide with a different mode of action on the second in order to minimize the risk of selecting for resistant insects.”

It’s an approach that balances common sense, a judicious use of pest controls, and a respect for the environment. “Consumers are interested in thinking about supporting agriculture that is as sustainable as possible,” Groves says. “What GreenLight is working on looks like it will have a fit with the commercial industry. These new tools are the kinds of approaches that can potentially revolutionize what sustainability means.” 

Harnessing a fundamental process of a natural system without using synthetic chemistry, Groves says, holds a lot of promise. “It is part of the forefront of transforming agriculture.”

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Five peer-reviewed papers released in 2020

FASEB Journal

Bio-lethal effect of BAPCs-dsRNA formulations for genetics-based pest management

Environmental Microbiology

Multiple environmental parameters impact lipid cyclization in Sulfolobus acidocaldarius

Analytical Methods (Royal Chemistry Society)

Complete enzymatic digestion of double-stranded RNA to nucleosides enables accurate quantification of dsRNA

Frontiers in Plant Science

Safety Considerations for Humans and Other Vertebrates Regarding Agricultural Uses of Externally Applied RNA Molecules

Frontiers in Bioengineering & Biotechnology

Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops

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