Dire Wolf “De-Extinction” Facilitates Non-Invasive Cloning Of The World’s Most Endangered Wolf

Who’d have thought we’d get Game Of Thrones in real life before George R R Martin had time to finish the books? To be fair, probably everyone.

“Colossal has de-extincted the dire wolf,” said Colossal Biosciences CEO Ben Lamm to IFLScience. “We took a 13,000 year old tooth, a 74,000 year old skull, and made puppies, and then we also used some of that same tech to help save the red wolves, which is really exciting.”

“We’ve made three dire wolf puppies. There’s Romulus and Remus, which are five months old and around 80 pounds. They’re beautiful. Then there’s Khaleesi, a little over six weeks old. She’s a baby, our first girl.”

De-extincting the dire wolf

In the same way that Colossal didn’t just shove mammoth DNA into a mouse to create the woolly mouse, the approach to de-extinction for the dire wolf involved gene editing and a surrogate: the gray wolf. The goal isn’t to create a living clone of a dire wolf, but almost to create a “dire wolf 2.0” that’s functionally similar to the original animal. So, it’s true that Colossal’s de-extinction is different from bringing something back from the dead, and there’s a good reason we can’t yet – and may never be able to – do that.

“To truly de-extinct something, you would have to clone it,” said Associate Professor Nic Rawlence of the Otago Palaeogenetics Lab at University of Otago in a Science Media Centre reaction. “The problem is we can’t clone extinct animals because the DNA is not well enough preserved. Even if you sequence the genome, you can’t extract DNA from extinct animals in long enough chunks like you could with a living animal. So, the only way to ‘de-extinct’ an animal is to use the new synthetic biology technology like CRISPR-cas9 where it acts like molecular scissors, and you can go and chop out a little bit of DNA and insert a new piece of DNA that effectively results in a genetic change.”

So, how did Colossal create their dire wolf?

We’ve shown that we can learn interesting information from a genome of something that went extinct more than 10,000 years ago

Beth Shapiro

“We got ancient DNA from these two remains, a tooth that’s around 13,000 years old, a skull that’s around 72,000 years old, and we sequenced a pretty good quality genome from both of them,” Colossal’s Chief Science Officer Beth Shapiro told IFLScience. “We then used that to figure out what it is that makes a dire wolf different. We know that dire wolves are similar genetically and phenotypically to a gray wolf. They’re larger, more muscular, and we also learned from their genome that they are light colored.”

The two dire wolf remains were separated in age by around 59,000 years, but they both showed variants of DNA in their genomes that suggest their coats would’ve been light colored, with dense, long, and thick fur. They also would’ve had more musculature, with larger jaws and heads, and in total Colossal made 20 edits to 14 loci in the gray wolf genome to bring about these traits. They then cloned that animal, and three pups were born.

“What I think is really exciting about this is that we’ve shown that we can learn interesting information from a genome of something that went extinct more than 10,000 years ago, and then used the tools of multiplex genome editing and computational biology to pull everything together and actually bring the functional equivalent of this animal back to life,” added Shapiro. “This is our dire wolf.”

These guys are just your typical rambunctious canids

Matt James

The burning question on our tongues? 

How does a dire wolf 2.0 puppy play?

“I’ve had great experience with a lot of exotic canids and these guys are just your typical rambunctious canids,” said Colossal’s Chief Animal Office Matt James to IFLScience. “Falling over each other, tackling each other, playing at any given opportunity.”

“What was really neat is when they started growing up, Romulus, who was sort of the leader when they were really young, when they were just a few weeks old, he was the bold one. He would go explore new things, new people, new stimuli. And now, as they’ve sort of matured, they’ve flipped. Now Remus is the bold, adventurous one and Romulus stays 20 paces back. He’s a little more shy and reclusive by nature. So, it’s neat to watch them mature and sort of find their standing within their social dynamic.”

The de-extinction toolkit and the red wolf

These remarkable puppies signify a pivotal moment in Colossal’s goal to create a de-extinction toolkit, innovating technologies that can support critically endangered species, and hopefully bring back those we don’t manage to save in time. This most recent news directly benefited the world’s most endangered wolf, the red wolf, as Colossal used the same technologies innovated to give life to dire wolf 2.0 to non-invasively clone four red wolf pups, named Hope, Blaze, Cinder, and Ash.

We cloned four red wolves from three founder lines, which represents about 25 percent more genetic diversity to the existing ~15 red wolves in the wild right now

Ben Lamm

“All of the technologies that we develop on the path to de-extinction, we make available for free to the world for conservation,” continued Lamm. “The most endangered wolf in the world is the only endemic American wolf, the red wolf. We cloned four red wolves from three founder lines, which represents about 25 percent more genetic diversity to the existing ~15 red wolves in the wild right now. There are others in captive breeding programs, and they come from about 12 founder lines. So, we have increased it by three founder lines.”

“In addition to that, we actually invented a new way to clone from isolating EPCs, or endothelial progenitor cells, that we find in blood. We’re certified by American Union Humane Society, all of our work, all of our facilities, everything. And we were actually able to isolate these EPCs just simply by taking a blood sample and then cloning them from that.”

Can clones support conservation?

If you’re raising your eyebrow at the concept of clones having a meaningful impact on conservation, recent research has suggested that this is an untapped resource for supporting endangered species. The study concluded that of the 56 species and subspecies cloned to date, 90 percent met or exceeded their natural lifespan, and 95 percent were fertile. Despite this, common misconceptions about limited longevity have held back the use of clones in conservation, a tool that could return crucial genetic diversity to dwindling wild populations.

Biodiversity loss is among the most pressing threats facing our planet today. As Shapiro explains in the below video, some scientists estimate that by 2050 half of all species that are alive today will be extinct. While extinction is a fact of life, the current rate is between 1,000 and 10,000 times faster than the base rate in the fossil record, so how can we – Homo sapiens, the species having the biggest impact on the planet – counteract the problem we have made for ourselves? Colossal’s goal is to safeguard against future losses with a de-extinction toolkit, while also teaming up with conservation bodies to try and prevent extinctions before they happen.

“Preserving, expanding and testing genetic diversity should be done well before important endangered animal species like the red wolf are lost,” said Harvard geneticist and co-founder of Colossal, Dr George Church, in a statement. “Another source of ecosystem variety stems from our new technologies to de-extinct lost genes, including deep ancient DNA sequencing, polyphyletic trait analyses, multiplex germline editing, and cloning. The dire wolf is an early example of this, including the largest number of precise genomic edits in a healthy vertebrate so far. A capability that is growing exponentially.”

Do we need de-extinction?

A recent analysis indicated that privately funding de-extinction is beneficial for biodiversity compared to if it were publicly funded, but also that if all the money that went to de-extinction went to conservation instead, the benefit would be far greater, and could potentially lead to eight times more species being saved. However, Colossal have maintained that their approach focuses on areas not currently being covered by conservation initiatives.

“Most of the money that goes into conservation goes to protect land,” Lamm told IFLScience. “It doesn’t go into [solving problems like] how do we biobank tissue samples? How do we build a genetic backup? How do we sequence everything and build reference genomes? How do we actually create induced pluripotent stem cells, so that if we do lose a species, we could bring it back?”

That de-extinction toolkit has so far included deriving induced pluripotent stem cells that could improve reproductive technologies for endangered animals. Then there’s vaccines, like the one they helped to create for the biggest killer of young elephants. They’re also working towards creating an artificial uterus that could facilitate rewilding without the need for surrogates.

As Lamm mentioned, all technologies are made available to the world of conservation, including their partners Re:Wild, in the hopes it can be rolled out to the species that need it most, before it’s too late. And if it does become too late? Well, perhaps one day we’ll have the toolkit needed to truly de-extinct them.

The puppies are going to continue to grow and develop in this great, expansive – I keep joking that it’s the Ritz Carlton of ecological preserves

Matt James

In the face of exciting, movie-worthy science, it’s always important to pause and reflect on the potential costs of such breakthroughs. As animal welfare expert Dr Heather Browning from the University of Southampton told IFLScience, problems can always arise when you have animals in captivity, and this could be especially true for those that can’t be raised by a mother that’s the same variant of gray/dire wolf as they are. 

The teams will continue to monitor the welfare of both their cloned red wolves and the dire wolf 2.0 pups. For now, it seems like they’re getting the VIP treatment, and there are hopes to bring Khaleesi, Romulus, and Remus all together the future.

“The puppies are going to continue to grow and develop in this great, expansive – I keep joking that it’s the Ritz Carlton of ecological preserves,” James told IFLScience. 

“Colossal has achieved American Humane Society Certification, the prestigious designation ensuring excellence in animal welfare and care,” said Robin Ganzert, PhD, CEO of American Humane Society, in a release emailed to IFLScience. “Optimal welfare is evidenced by spacious habitats with ample space and opportunity for animals to socialize, exercise, and exhibit natural behaviors. Staff are passionate, highly engaged and devoted to the animals in their care. We congratulate Colossal as a shining example of excellence in humane care and welfare. The technology they are pursuing may be the key to reversing the sixth mass extinction and making extinction events a thing of the past.”

Now if you’ll excuse us, we have a sudden urge to binge GOT.