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Using CRISPR to Stop Escaped Salmon Interbreeding

How can we protect wild salmon from interbreeding with farmed salmon? CRISPR gene editing is a solution

Upon an otherwise unruly landscape of choppy sea and craggy peaks, the salmon farms that dot many of Norway’s remote fjords impose a neat geometry. The circular pens are placid on the surface, but hold thousands of churning fish, separated by only a net from their wild counterparts. And that is precisely the conundrum. Although the pens help ensure the salmon’s welfare by mimicking the fish’s natural habitat, they also sometimes allow fish to escape, a problem for both the farm and the environment.

In an attempt to prevent escaped fish from interbreeding with their wild counterparts and threatening the latter’s genetic diversity, molecular biologist Anna Wargelius and her team at the Institute of Marine Research in Norway have spent years working on ways to induce sterility in Atlantic salmon. Farmed salmon that cannot reproduce, after all, pose no threat to the gene pool of wild stocks, and Wargelius has successfully developed a technique that uses the gene-editing technology Crispr to prevent the development of the cells that would otherwise generate functioning sex organs.

In fact, Wargelius’ team was a little too successful. To be financially viable, commercial fish farms need at least some of their stock to reproduce. So the scientists went a step further, developing a method of temporarily reversing the modification they had already made. They’ve created what they call “sterile parents.”

The term may sound like an oxymoron, but the sterile parents have the potential to solve one of the most pressing problems facing salmon aquaculture, both in Norway and around the world. Wargelius says it could be up to a decade before the results of her work are commercially available, but once they are, they have the potential to make an already burgeoning food source markedly more friendly on the environment. And by prioritizing environmental concerns and employing a technique that simply turns off a gene rather than introducing one from a different species, Wargelius and her team may contribute to a shift in how genetic engineering is perceived in Norway, a country with some of the strictest regulations regarding genetically modified organisms on the books.

Aquaculture, the cultivation of saltwater and freshwater organisms under controlled conditions, has long been a controversial industry. On the one hand, it’s been hailed as an answer to overfishing and to a growing global population’s demands for protein; on the other, it can pollute the water and spread disease among both farmed and wild fish. Despite these drawbacks, around the world, aquaculture is booming. In the two decades of this century, production has increased an average of 5.3 percent per year, and as of 2018, more than 126 million tons of the seafood consumed annually came from a farm. Of those 126 million, 1.4 million were Norwegian salmon.

In fact, Norway is the world’s largest producer of farmed salmon, raising millions of the fish in sea pens scattered throughout its jagged fjords. Aquaculture is the country’s second-largest industry, and those pens are an important part of its reputation among major seafood producers for relatively sustainable production methods. Up to 260 feet wide and 160 feet deep, they are open-net, allowing the chilly waters of the North Atlantic to circulate freely and more closely replicate part of the salmon’s habitat.

Exact numbers are difficult to pin down, but one estimate puts the total number of escaped fish at 2.1 million in the last decade, and another study found evidence of genes from farmed fish in wild specimens that were caught in 109 out of 147 Norwegian salmon rivers. Already under threat from overfishing — the population has been cut in half in the last 30 years — the threat to wild salmon biodiversity from this aquaculture gene stock is so great that farms are fined for fish that escape. In one case this year, Mowi, a company based in Bergen, Norway, was fined $450,000 for a 2018 incident in which 54,000 fish escaped.

In her early work, Wargelius focused on vaccines as a means of inducing the sterility that would minimize that threat. But her strategy changed soon after she learned about Crispr, the gene-editing technique that functions kind of like a Swiss Army knife, with different tools that make it possible to both insert material into a gene and snip it out.

Dorothy Dankel, a researcher at the University of Bergen who collaborates with Wargelius, recalled when Crispr first popped up. “Anna was saying, ‘Wow, there’s this new paper that just came out with something called Crispr,” Dankel said. “She felt like the vaccine approach, it’s really kind of hit or miss.” With Crispr, Wargelius thought, the team could hedge their bets.

But some scientists were skeptical, said Dankel. Genetically modified crops have encountered varying degrees of consumer resistance globally, in part because some modification techniques involve inserting genes from one species into another — a process that has provoked fears of Frankenstein foods. “People were freaking out, saying, ‘No, it’s GMO, we can’t do that in Norway,’” said Dankel.

But an exception exists for lab experiments. By using Crispr to treat newly fertilized fish eggs, Wargelius’s team was able to knock out a specific gene — called dead-end or dnd — that is responsible for the migration of germ cells to the gonad. Germ cells eventually give rise to gametes, or sexual reproductive cells, and without them, the thinking went, the fish in this initial cohort would not reach sexual maturity.

At the same time, the scientists also turned off a gene that controls for pigmentation, because the resulting albinism would make it significantly easier to keep track of which fish had been modified. Sure enough, many of the Crispr-treated embryos grew into yellow-hued salmon that lacked germ cells.

Salmon grow slowly, so it would take just over a year before the biologists could confirm the impact of the missing cells, but by 2016, it was clear: 100 percent of the albino fish failed to reach sexual maturity. They were all sterile.

Using Crispr to change a gene that causes sex organs to develop, scientists have created salmon that are sterile. But, with the right treatment, those same fish may have their fertility returned, and thus breed sterile offspring that still contain the edited gene. Video: Institute of Marine Research

“It was a very elegant experiment,” says Yonathan Zohar, a professor of marine biotechnology at the University of Maryland, Baltimore County and an expert in aquaculture and fish reproduction technologies. Linking the dead-end gene to an albino gene provided “a very good visual indication” of which fish had been treated, he said. “Her approach made a lot of sense.”

There was only one problem: The element of Crispr they were using to produce the sterile salmon required a technician to manually inject each embryo with a protein that cut the dnd gene — a labor-intensive method that is hardly viable for commercial fisheries. Wargelius wanted to find a way to reverse the impact of the genetic modification without removing it from the salmon’s DNA. After all, the goal was pass sterility along to the next generation. A fish farm could then keep its brood stock separate from the rest of the salmon.

“We thought, okay, maybe the simplest way to produce enough sterile salmon is to enable some of the sterile fish to reproduce,” Wargelius explained.

She was skeptical the idea would work. But a year after her team injected a certain mRNA from wild salmon into newly fertilized eggs in an effort to effectively turn the fish’s fertility back on, Wargelius received a text from the research station with photos that proved the technique had worked. It read: “We have many fish with germ cells here!”

Eventually, the treated fish developed gonads and reached sexual maturity, producing offspring that inherited their parents’ genetic sterility. The scientists won’t have the full results until this fall, after the first generation is 8 to 10 months old — the age at which salmon normally develop gonads. But so far, they say, everything is on track. “Theoretically, yes we should get 100 percent sterility,” Wargelius says.

Of course, certain safeguards need to be in place. A Crispr experiment to breed hornless cattle in the U.S. was initially hailed as a major success, but was later discovered to have introduced an unintended stretch of bacterial DNA into the cows’ genome. “The producers thought that only their edit was being introduced,” said Jennifer Kuzma, a professor and co-director of the Genetic Engineering and Society Center at North Carolina State University. “You have to be cautious that you’re not getting any off-target” — or unintended — “effects,” she said. One way guard against this: Sequence the offspring’s entire genome and look carefully for unintended changes in the DNA.

The Norwegian team is taking care to do this, and Kuzma sees their work as, in many ways, exemplary. “The work has a societal benefit, it has biosafety mechanisms in place, and it’s being done in collaboration with ethicists,” she says. “It’s being done under a pretty solid, good governance model.”

The Norwegian scientists haven’t yet sequenced the salmon’s genomes to look for any secondary effects, and it’s still relatively early in the salmon’s lifespan, so they won’t know about behavioral changes until the sterile offspring are transferred to the sea pens; currently the juveniles are living in tanks in the lab. “Anna will have to demonstrate that when you take those fish to the net pens, they perform as well as the non-treated ones,” says Zohar. And, he adds, she’ll have to scale everything up.

Those are significant hurdles, but the biggest hurdle, Zohar points out, is regulatory. From the time that genetically engineered crops first became widely available in the 1990s, their production has been regulated to different degrees, with some countries, such as the United States, merely demanding that the crops meet the same health and environmental standards as their conventionally bred counterparts.

Other countries have imposed stricter regulations on selected crops. In Mexico, for example, genetically engineered corn is banned because it poses a threat to the biodiversity of native maize. And other countries — especially those in Europe — have banned all genetically engineered crops intended for human consumption, as a food safety precaution. (To date, the safety concerns associated with GMOs have not been borne out.)

Norway has some of the most stringent restrictions in the world when it comes to genetically modified organisms: Farmers are barred from cultivating GMO crops and no genetically modified food products can be imported. Those policies, codified in the 1993 Gene Technology Act, were a reflection of both a powerful and fiercely protectionist agricultural sector and a public that is deeply conservationist and prides itself on its close connection to nature.

“It was black or white,” says Aina Bartmann, CEO of GMO-Network, an umbrella organization of nongovernmental organizations and corporations that represents 1.7 million consumers. “It was so obvious, I think, for everyone in Norway, in Scandinavia, and also in the European Union,” she said, that GMOs offered “no contribution to anything we want.”

Under the current legislation in Europe, Crispr is considered a gene modification technique, and no products created through it can be sold in Norway. (It is, however, authorized for research, and is being tested on lettuce and strawberries, in addition to salmon.) But that may be changing. Bjørn Kåre Myskja, a professor of ethics at Norwegian University of Science and Technology, is working on a study of the conditions that would make gene editing technology socially and morally acceptable to Norwegians.

His research is currently in progress, but he’s already seeing evidence, both in his work and anecdotally, that attitudes are changing — particularly when it comes to technologies like Crispr, which don’t always involve inserting the genes of one species into another. “When you do something that might happen in an ordinary naturally-occurring kind of mutation,” he said, “then there seems to be a larger percentage that will find that acceptable.”

Myskja has also observed in his research that opposition varies depending upon the perceived purpose of the modification. A modification that is intended to increase yields or to make an organism grow faster — and therefore increase the profits of the producer — is generally frowned upon in Norway. But a modification that achieves a broader good by increasing sustainability, for example, or improving animal welfare, might be tolerated. Therefore, a modification that benefits salmon, such as sterility or resistance to sea lice, “may fall on the acceptable part of the scale,” says Myskja.

His early findings are echoed in a survey conducted by GENEinnovate, a collaboration of private companies, research institutions, and the Norwegian Biotechnology Advisory Board, an independent committee made up of 15 members appointed by the Norwegian government. It found that a majority of Norwegian consumers had a positive attitude toward gene editing if it carried clear social benefits and was carefully labelled. Bartmann, of GMO-Network, has noticed the same even among her organization’s members. There are “a lot of uncertainties associated with many aspects of gene editing,” she said, and her members remain concerned about possible risks of releasing genetically modified crops — or animals — into the wild. “We support the research going on now in Norway,” she said, and “we think that the more knowledge we get about the new methods, the better.”

In the U.S., Kuzma has noted similar trends. “In surveys, people say they see edits or genes inserted from the same species as slightly more acceptable than transgenic,” she said, referring to genes inserted from different species. “In the marketplace, in part because there are so few products in the market, a significant proportion don’t really care. But there are still years of distrust to get over, and there’s a segment of around 20 percent that will reject GMOs in any form.”

For the moment, the aquaculture industry in Norway is hedging its bets. Historically, the industry has taken a hardline position against GMOs, conscious that the appeal of their products rests on a public perception of genetic purity. AquaGen, a breeding company that supplies fertilized Atlantic salmon eggs, sent a statement to Undark, writing that “producing sterile salmon by Crispr may be a future solution, but many technical, ethical, legislative, and commercial issues need to be solved before commercial implementation.” Cermaq, an international salmon farming company, similarly wrote to Undark that “farming sterile salmon may have advantages, and research in this area is very interesting,” but noted that the company is currently not planning to farm the gene-edited fish.

Yet Dankel has seen change among industry representatives. In 2014, she interviewed a senior manager at AquaGen, and asked if she saw a future for Crispr in her company. Dankel received a hard no: “’This is playing with fire,’” Dankel recalls being told. “’Our customers expect pure genetics; they don’t want anything modified.’” Just a few years later, she says, the company told her the technique is part of their research strategy.

The speed with which Crispr technology is developing and being adopted in laboratories around the world helps explain some of that transformation. But locally, Dankel’s own work plays a role, too. Within the Wargelius lab, Dankel is the representative for Responsible Research and Innovation, a position devoted to ensuring that ethical and social considerations are embedded into the research.

This involves doing outreach — explaining the research to the public — and what Dankel calls inreach — getting people who aren’t used to collaborating on a subject to work together. When it comes to something as complex as Crispr, she finds that with these interdisciplinary teams that combine biochemistry and molecular biology with social and economic assessments, it is essential to “create a common language” for what the goals are, and what success — and failure — might look like.

Dankel too is noticing a change in the discourse in the wake of the Crispr salmon. The pendulum has swung “and now people — even the Biotechnology Council of Norway — are only saying the good things about Crispr and not anything about off-target effects,” Dankel said, “or that once you start this technology you can never put it back.”

Yet perhaps the clearest indication that Norway may soon adjust its legislation to make room for Crispr is the government’s creation, in November 2020, of a new committee to review the field of genetic technologies. Headed by Wargelius, it will report on its findings and recommendations in June 2022. “Norway really wants to promote a public debate about the law that is based in science,” says Dankel. “They could have chosen a law professor to lead that. They could have chosen someone who’s not a biologist.”

And Wargelius still has a lot of biology to do. She and her team are just now beginning to work on their second generation of fish and also are planning to sequence the genome of the sterile fish to ensure there aren’t any unintended edits. Wargelius estimates that any commercial licensing for this application, provided it is approved, is five to 10 years away.

But she’s in no hurry. With Crispr, she suspects, Norway is moving toward an application-based process, where the technology will be approved in cases where the need for or benefit from a specific use is sufficient to outweigh the risks. Which is why, she says, she chooses an open and thorough approach for her own research. “We also now are trying to start a collaboration with both economic and ethical researchers to see what is the potential in the market, what will people think,” said Wargelius. “I would like to have a quite slow process, where we really have all the documentation that we need to be certain that it’s a solid product.”