A gene-edited bull didn't only get a new gene for hornlessness. An unexpected piece of bacteria DNA had also entered his genome

Here's an interesting article on a story covered by GMWatch earlier this month. GMWatch reported that cows gene-edited to be hornless were found to contain two copies of the DNA construct (plasmid) that carried the desired hornless gene variant into the target site. The plasmid contained antibiotic resistance genes.

This was an unintended outcome of the gene editing, by the biotech company Recombinetics. The company did not find and publish this discovery – that was left to scientists at the US FDA. The episode shows why it's crucial to strictly regulate this technology, contrary to the lobbying push to de-regulate it.

The words of Tad Sonstegard, CEO of Recombinetics’ agriculture subsidiary, Acceligen, which was running the research, should be engraved in our minds: “We weren’t looking for plasmid integrations. We should have.”
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Brazil's plans for gene-edited cows got scrapped – here's why

MEGAN MOLTENI
Wired, 26 Aug 2019
https://www.wired.com/story/brazils-plans-for-gene-edited-cows-got-scrappedheres-why/

* A gene-edited bull didn't only get a new gene for hornlessness. An unexpected piece of bacteria DNA had also entered his genome.

UP UNTIL A few months ago, Brazil was all set to create the country’s first herd of genetically dehorned dairy cows. In October 2018, Brazilian regulators had determined that an American biotechnology company’s efforts to produce such an animal didn’t require any special oversight. It was, after all, merely trying to edit into dairy cattle a naturally occurring trait for hornlessness commonly found in beef breeds.

The company, Minnesota-based Recombinetics, started preparing shipments of sperm from one of their two gene-edited Holstein bulls, Buri. With it, breeders planned to create about 10 calves to prove the edit could be passed down, and to study their health for a few years while they lived in Brazil. If it all went well, they’d try the edits in a more elite dairy stud (sorry, Buri) and move into the market. But now, WIRED has learned, those plans have been abruptly dropped.

Buri, it turns out, had more than just the hornlessness gene slipped into his genome. Part of the editing machinery, the piece of bacterial DNA that delivered the desired gene into Buri’s cells, called a plasmid, had accidentally gotten pasted into his genome. He was, in fact, part bacteria — a teeny tiny part, around 4,000 base pairs out of about 3 billion.

That fact alone is not necessarily problematic; scientists believe that bacteria are constantly swapping genes with the organisms they live on, including cows and humans. But this transfer had happened in a lab, not a pasture. And the bacterial bits included a few genes for antibiotic resistance commonly found in plasmids. So in the eyes of Brazilian regulators, he was bacteria enough that Buri—and any of his progeny—could no longer be viewed as not GMO. At least for now, the project was doomed.

Recombinetics didn’t discover this on its own. Earlier this year, scientists at the US Food and Drug Administration stumbled across the glitch when they ran Buri’s publicly available genome sequence through new screening software they were working on. They posted these results in the bioRxiv pre-print server last month.

When Recombinetics had created Buri (and his hornless half-brother Spotigy) in a pair of petri dishes, back in 2014, the scientists had checked to make sure the gene had in fact inserted, and they had checked to see if the editing machinery—a precursor to Crispr, called TALENs—didn’t make any cuts where it wasn’t supposed to. When the company’s researchers reported their results in the journal Nature Biotechnology in 2016, they concluded that the animals were free of any unexpected alterations.

“We weren’t looking for plasmid integrations,” says Tad Sonstegard, CEO of Recombinetics’ agriculture subsidiary, Acceligen, which was running the research with a Brazilian consulting partner. “We should have.”

Sonstegard says that when Acceligen found out about the accidental edit in March, the company’s executives informed CTNBio, the Comissão Técnica Nacional de Biossegurança in Brazil, and ended their plans for the experimental hornless herd. The FDA also contacted CTNBio about its discovery. None of Buri’s semen was ever exported, according to Acceligen. CTNBio could not be reached for comment.

The company hopes to revisit the now-abandoned project in the future with an improved, plasmid-free cell line. Sonstegard says in the years since Buri was created, Recombinetics since moved on to an editing system that no longer uses plasmids to cart over the hornless gene. “We still believe the system used in Brazil is one example of a good model for gene-editing regulation,” the executive adds.

In 2016, Recombinetics petitioned the FDA to designate its gene-edited cows “generally recognized as safe,” which would have allowed the animals to enter the food supply without much oversight. The federal regulator declined. A few months later, in January 2017, the agency announced it would begin regulating any intentional edits made in animal genes as new drugs. That’s when Recombinetics started looking abroad. “We don’t really need the US,” the company’s chief commercial officer, Mitch Abrahamsen, told Nature earlier this year. In addition to its work in Brazil, Acceligen is pursuing partnerships to introduce its gene-edited cows in Canada and Australia, though it has not yet applied for approval in those countries.

Its hopes for a hornless dairy herd may be dashed in Brazil for now, but another project it has there to create gene-edited, heat-tolerant beef cattle is still moving forward. Acceligen says it has found no trace of bacterial DNA in those animals, and that no approvals have been revoked. In fact, the company intends to intensify its presence in Brazil, with plans to establish an office there sometime next year.