Colossal announces 'de-extinction' of the dire wolf

US de-extinction company Colossal Biosciences has announced what it describes as the rebirth of the dire wolf, which would make it the world’s first successfully de-extincted animal.

Dire wolves were distributed across the American midcontinent during the Pleistocene ice ages. Dire wolves were as much as 25% larger than grey wolves and had a slightly wider head, light thick fur and stronger jaw. Dire wolves went extinct at the end of the most recent ice age, around 13,000 years ago, although they continue to live on in popular culture, including role-playing games such as Dungeons & Dragons and fantasy series such as Game of Thrones.

To de-extinct the dire wolf, Colossal extracted ancient DNA from two dire wolf fossils: a tooth from Sheridan Pit, Ohio, that is around 13,000 years old, and an inner ear bone from American Falls, Idaho, around 72,000 years old. The team deeply sequenced the extracted DNA and used Colossal’s novel approach to iteratively assemble high-quality ancient genomes, resulting in a 3.4-fold coverage genome from the tooth and 12.8-fold coverage genome from the inner ear bone. Together, this data provided more than 500x more coverage of the dire wolf genome than was available previously.

Colossal’s computational analysis of the reconstructed dire wolf genome revealed that the grey wolf is the closest living relative of dire wolves — with dire wolves and grey wolves sharing 99.5% of their DNA code. The analysis also revealed that the dire wolf lineage emerged between 3.5 and 2.5 million years ago as a consequence of hybridisation between two ancient canid lineages: an ancient and early member of the tribe Canini, which may be represented in the fossil record as Eucyon or Xenocyon, and a lineage that was part of the early diversification of wolf-like lineages including wolves, dholes, jackals and African wild dogs.

The team identified multiple genes undergoing positive selection that are linked to dire wolf skeletal, muscular, circulatory and sensory adaptation. They discovered dire-wolf-specific variants in essential pigmentation genes revealing that dire wolves had a white coat colour — a fact that is impossible to glean from fossil remains alone. The team also identified dire-wolf-specific variants in regulatory regions that alter the expression of genes. From this list, Colossal used its proprietary computational pipeline and software to select 20 gene edits across 14 distinct loci as targets for dire wolf de-extinction, focusing on the core traits that made dire wolves unique including size, musculature, hair colour, hair texture, hair length and coat patterning.

Based on Colossal’s genomic analysis, the team used grey wolves as the donor species for establishing cell lines. Using Colossal’s novel approach to establish cell lines from a standard blood draw, the team collected blood during a normal veterinary procedure and established cell lines from blood epithelial progenitor cells (EPCs). The team then performed multiplex genome editing of these cells followed by whole genome sequencing to confirm editing efficiency and identify any alterations to the genome arising during extended cell culture. The dire wolf team selected high-quality cells with normal karyotypes for cloning by somatic cell nuclear transfer into donor oocytes, followed by short-term culture to confirm cleavage. Healthy developing embryos were then transferred into surrogates for interspecies gestation. Three pregnancies led to births of two males (Romulus and Remus) and one female (Khaleesi).

Colossal edited 15 extinct dire wolf variants into the donor grey wolf genome, creating dire wolves that express genes that have not been expressed for more than 10,000 years. These target genes were selected because each is linked to one or more key traits that made dire wolves unique among canids. For example, Colossal targeted CORIN, a serine protease that is expressed in hair follicles and suppresses the agouti pathway, impacting coat colour and patterning. The dire wolf CORIN variants impact pigmentation in a way that leads to a light coat colour.

Colossal also edited dire-wolf-specific variants in a multi-gene regulatory module that has been linked to variation in body size as well as ear, skull and facial morphology. The region encodes eight genes that establish species-specific constraints in skeletal size and structure, and has been linked to features including differences in human height and the diverse beak shapes among finch species. One gene encoded by this module — HMGA2 — is directly associated with body size in dogs and wolves. Another gene in this module — MSRB3 — has been linked to variation in ear and skull shape among canines and other mammals. Given the role of these genes in establishing species-specific size and morphology, the dire wolf team edited dire-wolf-specific variants into gene enhancers (DNA sequences that make it more likely that the gene will be transcribed into RNA) in this genomic region.

For each high-impact variant identified as linked to a target phenotype, the dire wolf team created a detailed profile of all potential impacts on a donor grey wolf genome. To ensure healthy outcomes, the team discarded variants that would incur some risk outside of the predicted phenotype or prioritised variants already evolved in grey wolves with the predicted phenotype. For example, Colossal edited the protein coding region of LCORL, a transcription factor that regulates gene expression by influencing whether a gene is transcribed. Variations in LCORL have been linked to variation in body size in many species, including humans, horses and canids. The dire wolf has three changes to the LCORL protein sequence that are predicted via 3D modelling to alter the way the protein folds precisely at the location where LCORL should bind to a major gene silencing complex known as the PRC2 domain. Interestingly, large dog breeds (which are domesticated grey wolves) have a variant of LCORL that is missing the PRC2 domain entirely. As the dire wolf version is predicted to have a similar phenotypic impact as the variant found in larger dog breeds, and because of the potential for LCORL to interact with other genes in the grey wolf genetic background that are not edited, Colossal’s dire wolves express the protein that is found in the largest grey wolves. This choice allows for the predicted phenotypic impact and without any additional risk.

“Functional de-extinction uses the safest and most effective approach to bring back the lost phenotypes that make an extinct species unique,” said Dr Beth Shapiro, Colossal’s Chief Science Officer. “We turn to ancient DNA to learn as much as we can about each species and, whenever possible, to link specific extinct DNA sequence variants to each key trait. In some cases, we learn that variants already present in the surrogate species can be used to engineer that key trait. In those cases, engineering existing variants into the donor genome is an optimal path, as that path provides strong confidence in the outcome with minimal risk to the animal.”

The dire wolf genome has protein-coding substitutions in three essential pigmentation genes: OCA2, SLC45A2 and MITF, which directly impact the function and development of melanocytes. While these variants would have led to a light coat in dire wolves, variation in these genes in grey wolves can lead to deafness and blindness. The team therefore engineered a light coloured coat in Colossal’s dire wolves via a path known to be safe in grey wolves: by inducing loss-of-function to MC1R and MFSD12. These genes influence expression of pigments eumelanin (black) and pheomelanin (red) in melanocytes that deposit to the coat, achieving the lighter pigmented coat colour phenotype suggested by the dire wolf genome but without any potential health impacts.

According to Colossal, the birth of dire wolf pups proves the efficacy of the company’s de-extinction protocols and the feasibility of creating a standardised toolkit for de-extinction. The dire wolf pups set the record for number of precise genetic edits in any living species, with the company performing 20 precise edits to the genome — all modifications derived from analysis of the dire wolf genome — with 15 of those edits being the exact extinct variants. The Colossal Woolly Mouse previously held the record for unique germline edits in an animal, with eight precision edits.

The research undertaken to birth the dire wolf has already been paralleled to the birth of two litters of the red wolf, using a new approach to non-invasive blood cloning. Fewer than 20 red wolves remain in North America, which makes them the most endangered wolves on the planet.

Colossal successfully birthed two litters of red wolves — including one female and three males — from a total of three different cell lines. The company generated the cell lines, collected from the south-west Louisiana population, using its novel method of insolating EPCs following a standard blood draw. The pups were born after somatic cell nuclear transfer into a donor oocyte followed by embryogenesis and embryo transfer into a surrogate mother. Both embryo transfers resulted in the birth of healthy red wolf pups, providing evidence of the link between de-extinction efforts and Colossal’s capacity to support conservation.

“The same technologies that created the dire wolf can directly help save a variety of other endangered animals as well,” said Dr Christopher Mason, a scientific advisor and member of the board of observers for Colossal. “This is an extraordinary technological leap in genetic engineering efforts for both science and for conservation as well as preservation of life, and a wonderful example of the power of biotechnology to protect species, both extant and extinct.”

It should be noted that, according to expert commentators, Colossal Biosciences has not really de-extincted the dire wolf — rather, the company has created a grey wolf with dire wolf-like characteristics. As noted by Associate Professor Nic Rawlence, Director of the Otago Palaeogenetics Laboratory at the University of Otago, “This is not a de-extincted dire wolf, rather it’s a ‘hybrid’. And importantly, it’s what they think are the important dire-wolf-like characteristics.

“Dire wolves diverged from grey wolves anywhere between 2.5 to 6 million years ago,” Rawlence continued. “It’s in a completely different genus to grey wolves. Colossal compared the genomes of the dire wolf and the grey wolf, and from about 19,000 genes, they determined that 20 changes in 14 genes gave them a dire wolf.”

Associate Professor Michael Knapp, also from the University of Otago, was a bit more positive, agreeing that Colossal’s technology is suitable to contribute to conservation of threatened species. “Possibilities include editing harmful mutations out of the populations of threatened species and introducing traits that may help rare species adapt to environmental change,” he said.

“On the other hand, the technology still has its limitations. Genes that may be introduced to give a species more fur might have other and unwanted functions as well. Also, often not only the species, but the ecosystems they used to live in are extinct. This is for example the case for mammoths, which used to live in a ‘mammoth steppe’ environment — vegetation type that has fallen victim to prehistoric climate change and no longer exists anywhere on the planet. So where do we put these ‘homeless’ de-extinct animals?

“Ignoring all justified ethical concerns raised, it is undeniable that the birth of these wolves is a major breakthrough in genetics. Whether or not this is an avenue that should be further pursued is a highly complex question.”

Image caption: Colossal’s dire wolves, Romulus and Remus, at one month of age. The wolves were born on 10 January 2024.