Gene-edited cattle have a major screwup in their DNA
Bid for barnyard revolution is set back after regulators find celebrity “hornless” bovines contaminated by bacterial genes.
by Antonio Regalado
Aug 29, 2019

They were the poster animals for the gene-editing revolution, appearing in story after story. By adding just a few letters of DNA to the genomes of dairy cattle, a US startup company had devised a way to make sure the animals never grew troublesome horns.

To Recombinetics—the St. Paul, Minnesota gene-editing company that made the hornless cattle—the animals were messengers of a new era of better, faster, molecular farming. “This same outcome could be achieved by breeding in the farmyard,” declared the company's then-CEO Tammy Lee Stanoch in 2017. “This is precision breeding.”

Except it wasn’t.

Food and Drug Administration scientists who had a closer look at the genome sequence of one of the edited animals, a bull named Buri, have discovered its genome contains a stretch of bacterial DNA including a gene conferring antibiotic resistance.

The “unintended” addition of DNA from a different species occurred during the gene-editing process itself, the government says. It went undetected by the company even as it touted the animals as 100% bovine and assailed the FDA for saying the animals needed to be regulated at all.

“It was not something expected, and we didn’t look for it” says Tad Sontesgard, CEO of Acceligen, a subsidiary of Recombinetics that owns the animals. He says a more complete check “should have been done.”

The blunder is a setback for Recombinetics, whose pioneering prototypes of gene-edited animals include heat-resistant cattle as well as pigs that never hit puberty. It’s also a strike against efforts to make such gene editing a routine practice in animal reproduction. All along, Recombinetics had noisily objected to oversight by the FDA, which categorizes gene-edited animals as new drugs that need extensive testing and approval. It even lobbied the Trump Administration to wrest oversight away from the health agency, saying it was holding back a barnyard revolution.

But gene editing isn’t yet as predictable or reliable as promoters say. Instead, the procedure, meant to make pinpoint changes to DNA, can introduce significant unexpected changes without anyone noticing. “As genome-editing technology evolves, so does our understanding of the unintended alterations it produces,” wrote the FDA scientists, led by Alexis Norris and Heather Lombardi, in a paper they released in July. They think gene-editing errors “are under reported” and a “blind spot” for scientists.

The risk of haphazard engineering isn’t just to barnyard animals. Genome-editing treatments to cure rare diseases are being tested on people and it is possible that patients will end up with unplanned genetic mutations. Unintended consequences are a particular concern in connection with attempts to modify human children before birth with gene editing—as occurred for the first time in China last year.

Independent scientists still haven’t had a chance to confirm whether the Chinese children—twin girls—also have unintended mistakes in their DNA. This year the World Health Organization said any further attempts to make gene-edited people would be “irresponsible” in part because of technical uncertainty.

Celebrity bovines

To make its hornless cows, Recombinetics started with skin cells from Holstein dairy bulls. That’s where the bacterial genes came in: to convey DNA instructions to the cell, the company, in work carried out in 2013, employed plasmids—a type of circular mini-chromosome present in bacteria.

The plasmids (meant to persist only temporarily) carried DNA instructions both for the genetic editing tool (known as TALENs) and the “no horns” genetic change, found in other breeds, and which involves swapping out about 200 genetic letters. Later, edited skin cells were used in a cloning procedure to generate two new animals, named Buri and Spotigy. These were copies of the original bulls, but they didn’t grow horns.

The hornless cattle, born in 2015, soon became gene-editing celebrities. While Spotigy was killed to analyze his tissues, Buri lived long enough to sire about 17 offspring, which are split between facilities at the University of California, Davis, and a farm in Australia. One hornless daughter cow posed for the cover of Wired in April.

“We know exactly where the gene should go, and we put it in its exact location,” Recombinetics executives told Bloomberg in 2017. “We have all the scientific data that proves that there are no off-target effects.”

Eat the animals

In part because it disagreed with regulations, the company never pursued formal approval for the hornless cattle in the US. However, its collaborator at the University of California, Davis, veterinary scientist Alison Van Eenennaam, opened a file on the animals with the FDA last year, she says, to trade information with the agency.

According to Sontesgard, Van Eenennaam then chose to test the FDA’s thinking by asking whether some of the surplus gene-edited animals taking up space at the Davis facility could go to a slaughterhouse, where they’d be made into steaks and hamburger.

Sontesgard calls the bid to win a “food exemption” an attempt to assess the FDA’s views. “Alison was curious whether the animals would be approved as food. I said ‘Okay, you can take a shot,’” says Sontesgard. “If they’d said okay, that would have been a nice outcome, and if they said no, they would have to come up with a logical reasoning. I just didn’t want them to go to waste because there’s really nothing wrong with them.”

Sontesgard thinks the animals are “safe to eat with or without the plasmid.”

Van Eenennaam had financial factors in mind. It costs 60 cents a pound to incinerate experimental animals, which weigh about a ton, which she considers a large expense for her university program. Better to sell them as hamburger.

By March, though, an FDA bioinformatician running tests on the bulls’ genomes had stumbled on the presence of the plasmid—it had somehow integrated into Buri’s genome. Agency officials that month arranged a conference call break the bad news, Van Eenennaam says. The animals contained bacterial genes and were GMOs, by any definition.

“We were surprised, but when you get new information, you proceed ahead,” says Van Eenennaam. “That is what science does.” She says that half Buri's offspring would be expected to have also inherited the bacteria genes.

It could seem hard to explain why the company and UC Davis failed to see the bacterial genes, which ended up right next to their ‘no-horns’ edit in the animals’ genomes. However, to study DNA, researchers first have to decide which bits to isolate or check for—in effect, they’re often looking in the lamplight, a process that introduces assumptions and biases, according to the FDA scientists.

It’s not clear if the bacterial DNA poses a larger risk. It’s unlikely to affect the cow or a person who eats it; instead, the concern is that the antibiotic resistance gene could be taken up by any of the billions of bacteria present in a cow’s gut or body. John Heritage, a retired microbiologist from Leeds University, says he doesn’t see a large chance of the gene jumping further but says its presence in a cow could create unpredictable opportunities for it to spread.

The discovery that some of the hornless animals have unwanted DNA from another species has sealed their fate. No one is going to win regulatory approval for them now. Already, regulators in Brazil rejected a revised petition by Recombinetics connected to the animals, Sontesgard confirmed.

“It’s novel DNA, so right there that throws it into the whole GMO debate,” says Van Eenennaam. “It doesn’t worry me from a food safety perspective, but regulatory—well, you wouldn’t even try. It’s not a product that would move forward.”

Van Eenennaam says since the FDA discovery, UC Davis has incinerated three of the five males who were living at its farms. The animal who posed for the cover of Wired is pregnant and will be spared for the time being.

https://www.technologyreview.com/s/61423...or-dna-screwup/

CRISPR gene editing is not quite as precise and as safe as thought
Health
16 July 2018
By Michael Le Page

A study of CRIPSR suggests we shouldn’t rush into trying out CRISPR genome editing inside people’s bodies just yet. The technique can cause big deletions or rearrangements of DNA, says Allan Bradley of the Wellcome Sanger Institute in the UK, meaning some therapies based on CRISPR may not be quite as safe as we thought.

The CRISPR genome editing technique is revolutionising biology, enabling us to create new varieties of plants and animals and develop treatments for a wide range of diseases.

The CRISPR Cas9 protein works by cutting the DNA of a cell in a specific place. When the cell repairs the damage, a few DNA letters get changed at this spot – an effect that can be exploited to disable genes.

At least, that’s how it is supposed to work. But in studies of mice and human cells, Bradley’s team has found that in around a fifth of cells, CRISPR causes deletions or rearrangements more than 100 DNA letters long. These surprising changes are sometimes thousands of letters long.

Cancer risk

This finding is not a problem for the purposes for which CRISPR is currently being used. But some groups are developing treatments that would involve using CRISPR to edit billions of cells inside the human body. If Bradley is right, there’s a chance that a few of these cells might turn cancerous.

“There’s a risk of causing cancer sometime in a patient’s lifetime,” says Bradley. “We need to understand more before rushing into human clinical trials.”

“I do believe the findings are robust,” says Gaetan Burgio of the Australian National University, an expert on CRISPR who has debunked previous studies questioning the method’s safety. “This is a well-performed study and fairly significant.”

The finding comes just a month after another team reported that there was a cancer risk from gene-editing embryonic stem cells, for a quite different reason. It was also claimed last year that CRISPR causes lots of mutations far from the target site, but Burgio and others pointed out that the study was seriously flawed and it has since been retracted.
Newer techniques

“Overall, this adds more safety challenges to CRISPR-based gene therapies but it doesn’t compromise its use,” says Burgio of the new findings.

Many variations of the CRISPR technique are currently being developed, including base editors that alter specific DNA letters and nickases that do not completely sever the DNA. These methods may prove to be far safer. Some groups are also using modified forms of CRISPR to control how active a gene is instead of directly altering a gene’s DNA.

So why have the thousands of teams using CRISPR failed to discover this before? Because they have been looking for small mutations in a narrow region around the target site. If that whole region is deleted, this approach makes it appear as if there have been no mutations at all. “You find what you look for,” says Bradley.

Journal reference: Nature Biotechnology, DOI: 10.1038/nbt.4192

https://www.newscientist.com/article/217...afe-as-thought/

Back in the 50's when Watson and Cricks discovered the double helix it was believed that there was one gene for each trait. It was speculated that by replacing that one gene for a bad trait, e.g. diabetes, one could cure diabetes. Since then out knowledge has expanded exponentially. Those four molecules (cytosine [C], guanine [G], adenine [A] or thymine [T]) that make up our genome function interact in a very complex way. Multiple genes interact to control one trait. What was referred to as junk DNA is now recognized as the Epigenome, a sort of template that regulates gene expression. responds to environment, and whose changes are sometimes passed on.

And yet there are those who are arrogant enough to play God with DNA by creating Genetically Modified Organisms, and to also assume they can anticipate all the outcomes of this manipulation.