The dire wolf (Aenocyon dirus) was a North American species of canine that went extinct approximately 13,000 years ago during the extinctions of the Late Pleistocene epoch.
Popularized by HBO’s “Game of Thrones” and George R.R. Martin’s “A Song of Ice and Fire” book series, the dire wolf has long been in the spotlight of popular culture. Recently, however, it has become a focal point of discussion among the scientific community due to the efforts of researchers at Colossal Biosciences Inc.
In a recent April 7, 2025, press release, American biotechnology and genetic engineering company, Colossal Biosciences, claimed that they had successfully brought the dire wolf back from extinction. With the successful birth and continued survival of three genetically-engineered puppies — named Khaleesi, Romulus and Remus — Colossal has stated on their website that the company “[accomplished] something that’s never been done before: the revival of a species from its longstanding population of zero.”
To breed the puppies, researchers at Colossal created two dire wolf genomes from DNA preserved within dire wolf specimens — an approximately 13,000-year-old tooth and a 72,000-year-old skull. After comparing the dire wolf genomes with those of the gray wolf (Canis lupus), the researchers identified 20 differences in 14 genes responsible for producing the phenotypical differences observed between the two species.
To express the dire wolf’s traits, the researchers harvested endothelial progenitor cells (EPCs) from living gray wolves before editing the genetic material within the nuclei of the EPCs with CRISPR technology. The nuclei of the cells were then transferred from the EPCs into a denucleated gray wolf ova to grow into embryos, which were eventually transplanted into surrogate wombs. The three puppies were born during the fall and winter seasons of late 2024 and early 2025.
Officials at Colossal have celebrated the recent births as a massive advancement for de-extinction efforts and evidence of the necessity of Colossal’s efforts, especially as biodiversity loss from anthropogenic climate change continues to increase. Ben Lamm, a co-founder of the company and the current CEO, stated that “this massive milestone is the first of many coming examples demonstrating that our end-to-end de-extinction technology stack works.”
Dr. Beth Shapiro, Colossal’s chief science officer, added on by remarking that “[humanity is] an evolutionary force at this point” and that we should be “giving ourselves the opportunity to see what our big brains can do to reverse some of the bad things that we’ve done to the world already.”
De-extinction is not a new concept. In 2003, a team of European scientists unsuccessfully attempted to produce a clone of the Pyrenean ibex, which had gone extinct in 2000, through a process similar to Colossal’s. The group took preserved DNA from a Pyrenean ibex and selected a surrogate from a genetically similar species in an attempt to facilitate its revival. The effort failed, though.
What sets Colossal apart from previous efforts, however, is its methodology. The company focuses on long-extinct species and attempts to facilitate their reintroduction by modifying close genetic relatives of extinct species in order to produce offspring that look and act similar to the previously extinct species.
Since its founding in 2021, Colossal has worked towards what they call the “functional de-extinction” of several species of now-extinct organisms, including the woolly mammoth and the dodo. The company defines functional de-extinction on their website as “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.”
Many experts have responded with criticism to Colossal’s assertion that the newborn puppies are dire wolves. Dr. Philip, a zoologist, from the University of Otago said that the puppies are “genetically engineered gray wolves.” Paleogeneticist Dr. Nic Rawlence, from the same university, added on by clarifying that dire wolves are “in a completely different genus to grey wolves,” and that Colossal had “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.”
“What Colossal is trying to do is genetically [engineer] animals to look like extinct creatures,” Rawlence remarked. “They look cute and cuddly, but … they’re not a dire wolf.”
Colossal has attempted to defend its methods, with Shapiro assuring that the company is “using a morphological species concept.” Shapiro conceded that it “is not really possible” to create something identical to the dire wolf, but explained that “it’s also not the goal” and that they don’t “have to have something that is 100 percent genetically identical.”
In a similar line, Colossal’s official Reddit account responded to criticism of the company on Reddit with “the reality is that ‘species’ is a human idea” and that the company prefers “a phenotypic definition of species. Our dire wolves look and act like dire wolves, so we believe it’s accurate to call them dire wolves.”
University of Maine paleoecologist Dr. Jacquelyn Gill disagreed with Colossal’s species definition, insisting, “I have more than 14 Neandertal genes in me, and we wouldn’t call me a Neandertal” and further cast doubt on the idea that the puppies could act like dire wolves by stating that they “don’t have any traits that would allow us to understand the dire wolf any better than we did yesterday.”
Cornell University geneticist Dr. Adam Boyko similarly stated that the puppies “are not being raised in dire-wolf packs, where they could learn dire-wolf behavior” and additionally “aren’t eating an ancient diet, so they are not acquiring their ancestors’ unique suite of intestinal microbes.”
The International Union for Conservation of Nature (IUCN) Species Survival Commission (SSC) noted in an April 18 statement that “the dire wolf and the grey wolf are genetically distinct by thousands of genes while the gene editing by Colossal was done on only a handful of genes of the grey wolf genome to re-create phenotypic traits of a dire wolf.”
The IUCN went further to declare, “The three animals produced by Colossal are not dire wolves. Nor are they proxies of the dire wolf based on the IUCN SSC guiding principles of extinct species for conservation benefit. First, there is no evidence that the genetically modified animals are phenotypically distinct from the grey wolf and phenotypically resemble the dire wolf. Second, our knowledge of the behaviour, phenotype, and ecology is inherently limited because the dire wolf is extinct.”
Dr. Jeremy Austin, an evolutionary biologist at the University of Adelaide and the Director of the Australian Centre for Ancient DNA, commented that while Colossal’s recent success does have real world applications in the fields of conservation and genetics, the puppies were not dire wolves “under any definition of a species ever.”
