The de-extinction company Colossal Biosciences made headlines recently when it introduced the world’s first “woolly mouse” – a mouse genetically engineered to express woolly mammoth genes.
Colossal has now announced what it describes as the first successful example of animal de-extinction. In exciting news for Game of Thrones fans, the animal is the dire wolf – a canine that was native to the Americas until its extinction roughly ~12,500 years ago.
In a press release distributed to Technology Networks, Colossal said the birth of three pups – two males and one female – illustrates “another leap forward in Colossal’s de-extinction technologies and is a critical step on the pathway to the de-extinction of other target species.”
The pups’ names are Romulus, Remus and Khaleesi.
A brief history of Colossal
Colossal was co-founded in 2021 by renowned geneticist Professor George Church and serial entrepreneur Ben Lamm. In an interview with Technology Networks that same year, Church explained how the idea for the company arose in 2006–2007 when two journalists asked whether it would be possible to use synthetic biology approaches to read – and then re-build – the mammoth genome.
It seems that Church pondered the question quite seriously. Colossal is the world’s first and only de-extinction company, valued at an estimated $10 billion.
Through its ever-expanding de-extinction portfolio, Colossal is harnessing advancements in ancient DNA extraction, next-generation sequencing, stem cell research and multiplex genome editing – among other sophisticated technologies – to “revive” a version of an extinct species. This “version” is not a clone – it does not possess the exact same genetic material as the extinct organism. Rather, it is a genetic hybrid.
What is multiplexed genome editing?
Multiplexed genome editing is a technique that enables scientists to make multiple changes to DNA at different locations in the genome simultaneously, using tools such as CRISPR-Cas9 technology.
Here’s how Colossal approaches creating such hybrids. By assembling the genome of an ancient extinct organism and comparing it to the genome of that organism’s closest living relative, researchers work to identify the gene variants specific to the extinct organism and, therefore, the genes that underpin the extinct organism’s phenotype.
Using multiplex gene-editing tools, a donor genome from the closest living relative is edited to include the extinct variants. The edited cell lines are then screened, and high-quality cell lines are cloned by somatic cell nuclear transfer into donor egg cells. Healthy embryos are then transplanted into the closest living relative to act as a surrogate carrier for the hybrid organism.
What is somatic cell nuclear transfer?
A technique used in cloning where the nucleus of a somatic cell (a non-reproductive cell) is transferred into an egg cell that has had its nucleus removed. This reprogrammed egg cell is then stimulated to divide and develop into an embryo, which possesses the same DNA as the donor of the somatic cell.
The making of Romulus, Remus and Khaleesi
Romulus, Remus and Khaleesi were created using blood epithelial progenitor cells from a gray wolf, obtained through a simple blood draw. This novel approach developed by Colossal has also been used to create red wolf embryos, the company said in the press release.
Colossal scientists made 20 unique precision germline edits to the gray wolf-derived cell lines, 15 of which were ancient dire wolf variants. This sets a new record for the number of precise edits made in any animal and breaks the record previously held by the woolly mouse, which possesses eight edits.
The dire wolf variants were identified by extracting and sequencing DNA from two fossils: a 13,000-year-old tooth from Sheridan Pit, Ohio, and a 72,000-year-old dire wolf ear bone from American Falls, Idaho. Using a new method to assemble high-quality genomes, Colossal created a 3.4-fold coverage genome from the tooth and a 12.8-fold coverage genome from the ear bone; collectively these offer 500x greater coverage of the dire wolf genome than has been previously possible. This deep level of sequencing also helped establish that the dire wolf lineage emerged roughly 2.5–3.5 million years ago as a result of hybridization between two ancient canid lineages.
“Our novel approach to iteratively improve our ancient genome in the absence of a perfect reference sets a new standard for paleogenome reconstruction,” Dr. Beth Shapiro, Colossal’s chief science officer, said in a press release.
Ethical de-extinction
Colossal’s dire wolf team carefully explored all the potential impacts that inserting dire wolf variants could have on the donor gray wolf genome. For instance, the dire wolf genome includes protein-coding substitutions in three genes, OCA2, SLC45A2 and MITF, which are pigmentation genes. These substitutions impact how melanocytes – cells that produce melanin – function and develop in the dire wolf. In the gray wolf, however, variations in such genes are associated with deafness and blindness. To safely engineer a lighter-colored coat in the dire wolves, Colossal scientists made edits via an alternate pathway, targeting genes MC1R and MFSD12.
“When I learned of Colossal’s approach to engineering the light coat color into their dire wolves, I was simultaneously impressed and relieved,” said Dr. Elinor Karlsson, associate professor in bioinformatics and integrative biology at the UMass Chan Medical School and director of vertebrate genomics at the Broad Institute of MIT and Harvard, in Colossal’s press release. “By choosing to engineer in variants that have already passed evolution’s clinical trial, Colossal is demonstrating their dedication to an ethical approach to de-extinction.”
Romulus, Remus and Khaleesi are said to be “thriving” on a 2,000+ acre ecological preserve at an undisclosed location in the US, where they are cared for by 10 full-time members of staff. The preserve is certified by the American Humane Society and registered with the United States Department of Agriculture. Colossal’s website offers admittedly adorable video updates on the pack’s first months of life. Eventually, the company said it hopes to restore the species in “secure and expansive ecological preservers” that are “potentially on indigenous land”.
As for the methods and technology finessed through the dire wolf project, Colossal hopes they can be directed to help the critically endangered red wolf species, native to North America. Focused breeding programs for this species can struggle due to a lack of genetic diversity; Colossal’s approach to revive a hybrid of the dire wolf represents a new way to genetically engineer diversity into a species. The company said it has already “successfully paralleled” the dire wolf research to birth two litters of red wolves, including one female and three males from three different cell lines.
Is extinction really forever?
While many members of the scientific community agree that the birth of Romulus, Remus and Khaleesi represents a milestone in genetics research, several are irked by the company’s claims that this project is an example of de-extinction.
“To truly de-extinct something, you would have to clone it. The problem is [that] we can’t clone extinct animals because the DNA is not well enough preserved,” said Associate Professor Nic Rawlence, director of the Otago Palaeogenetics Laboratory in the Department of Zoology, University of Otago in a statement to the New Zealand Science Media Centre.
“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,” he continued. “So, what Colossal Biosciences have produced is a gray wolf with dire wolf-like characteristics – this is not a de-extincted dire wolf, rather it’s a ‘hybrid’.”
Professor Philip Seddon from the Department of Zoology, University of Otago, echoed this sentiment: “They [Colossal] are claiming this as the world’s first de-extinction, but while no doubt it has required some amazing technological breakthroughs, the cute pups Romulus, Remus, and Khaleesi are not dire wolfs – they are genetically modified grey wolves.”
“Certainly, this involves advances in genetic technology, and these might have applications for the conservation of existing species – but the return of dire wolves? No. In the same way that Colossal’s plans for woolly mammoths and dodos will involve the genetic modification of related species. We have GMO [genetically modified organism] wolves and might one day have GMO Asian elephants, but for now extinction really is forever.”
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Colossal acknowledges that de-extinction is “complex, expansive, evolving and entirely new”. Consequently, the company suggests that older definitions of de-extinction are no longer applicable, and proposes its own: “The process of generating an organism that both resembles and is genetically similar to an extinct species by resurrecting its lost lineage of core genes; engineering natural resistances and enhancing adaptability that will allow it to thrive in today’s environment of climate change, dwindling resources, disease and human interference.”
The birth of Romulus, Remus and Khaleesi may not mark the return of the dire wolf in the traditional sense, but it certainly signals the beginning of a new chapter in genetic research and innovation.
Over the coming years, conversation and community engagement will be incredibly important to this realm of scientific development, Rawlence emphasized: “We need to have discussions around indigenous intellectual property, bio-prospecting, biopiracy, and what happens if animals are brought back from the dead and they are trademarked by these de-extinction companies?”
What feels like science fiction today may well become reality in the near future. At the current pace of change, it’s impossible to predict which species – or engineered hybrids – could be walking the planet by 2030.
Colossal told Technology Networks it will release the research from the dire wolf project via pre-print over the coming days.