Inside the Science: How Colossal Biosciences Resurrected the

The resurrection of the dire wolf by Colossal Biosciences represents one of the most remarkable achievements in genetic engineering to date. This historic breakthrough combines ancient DNA analysis, CRISPR gene editing, and advanced reproductive techniques to bring back traits not seen in living animals for over 12,000 years. Let's explore the scientific methodology that made this achievement possible.

Starting with Ancient DNA

The first challenge facing Colossal's scientists was obtaining usable genetic material from an extinct species. The team extracted ancient DNA from two dire wolf fossils: a tooth from Sheridan Pit, Ohio, approximately 13,000 years old, and an inner ear bone from American Falls, Idaho, around 72,000 years old.

Working with ancient DNA presents extraordinary challenges. After thousands of years, genetic material is incredibly fragmented and degraded. As Dr. Beth Shapiro, Colossal's Chief Science Officer and a leading expert in ancient DNA, explained, the process resembled "reconstructing a million-page book when only random sentences from every twentieth page remain intact."

The team deeply sequenced the extracted DNA and used Colossal's novel approach to iteratively assemble high-quality ancient genomes. This resulted 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 500 times more coverage of the dire wolf genome than was previously available.

"Our novel approach to iteratively improve our ancient genome in the absence of a perfect reference sets a new standard for paleogenome reconstruction," said Dr. Shapiro. These computational advances allowed the team to resolve the evolutionary history of dire wolves and establish the genomic foundation for de-extinction.

Surprising Evolutionary Insights

Colossal's computational analysis of the reconstructed dire wolf genome revealed several previously unknown aspects of dire wolf evolution. While previous work could not definitively determine the origin of dire wolves (leading to speculation that jackals might be their closest living relatives), analyses of the high-quality genome revealed that gray wolves are actually the closest living relatives of dire wolves—with dire wolves and gray wolves sharing 99.5% of their DNA code.

Interestingly, the analysis also revealed that dire wolves have a hybrid ancestry. The dire wolf lineage emerged between 3.5 and 2.5 million years ago as a consequence of hybridization between two ancient canid lineages:

This hybrid ancestry helps explain the previous uncertainty about dire wolf origins and provides new insights into the complex evolutionary history of canids in North America. It also demonstrates how hybridization can drive the emergence of new species with unique adaptations—in this case, the larger, more robust dire wolf adapted for hunting large Pleistocene megafauna.

Identifying Key Genetic Traits

With the dire wolf genome in hand, the next step was identifying the specific genes that gave dire wolves their distinctive characteristics. The team identified multiple genes undergoing positive selection linked to dire wolf skeletal, muscular, circulatory, and sensory adaptations.

Remarkably, they discovered dire wolf-specific variants in essential pigmentation genes revealing that dire wolves had a white coat color—a fact impossible to determine from fossil remains alone. They also identified dire wolf-specific variants in regulatory regions that alter gene expression.

From this genetic catalog, Colossal used its proprietary computational pipeline to select 20 gene edits across 14 distinct genetic loci as targets for dire wolf de-extinction. These edits focused on core traits that made dire wolves unique:

• Body size

• Musculature

• Hair color

• Hair texture

• Hair length

• Coat patterning

The Gene Editing Process

Based on their genomic analysis, the team used gray wolves—the closest living relative of dire wolves—as the donor species for establishing cell lines. Rather than invasively harvesting tissue, scientists drew blood from living gray wolves during normal veterinary procedures and isolated endothelial progenitor cells (EPCs) from the blood.

This represents a significant improvement over traditional methods. "The collection of whole blood is a rapid and noninvasive procedure that is routinely carried out on sedated wolves for veterinary monitoring purposes," explained the Colossal team. These isolated EPCs can be frozen for later genomic analyses and, as now demonstrated, can be used to successfully clone wild canids.

The scientists then applied CRISPR gene-editing to install 20 dire wolf variants across 14 genes in the gray wolf cells. This set a scientific record: 20 precise edits is the highest number of deliberate genome edits in any vertebrate animal to date, far exceeding the previous record of 8 edits achieved in Colossal's "woolly mouse" with mammoth genes.

Careful Safety Considerations

For each genetic variant targeted, Colossal's team created a detailed profile of all potential impacts on the donor gray wolf genome. To ensure healthy outcomes, they discarded variants that would incur risks beyond the predicted phenotype or prioritized variants already evolved in gray wolves with the predicted phenotype.

For example, the team engineered around potential problems with coat color genes. 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 gray wolves can lead to deafness and blindness.

To avoid these potential health issues, the team engineered a light-colored coat in Colossal's dire wolves via a path known to be safe in gray wolves: by inducing loss-of-function to MC1R and MFSD12. These genes influence the expression of pigments eumelanin (black) and pheomelanin (red) in melanocytes, achieving the lighter pigmented coat color phenotype suggested by the dire wolf genome but without potential health impacts.

"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," noted Elinor Karlsson, Ph.D., Associate Professor at UMass Chan Medical School and Director of Vertebrate Genomics at the Broad Institute of MIT and Harvard.

From Cells to Living Animals

Once the cells were genetically modified into "dire wolf" cells, Colossal used cloning techniques to turn them into embryos. The 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 surrogate mothers—domestic dogs (hound mixes)—for gestation. Colossal transferred a total of 45 edited embryos into two surrogate dogs in the first attempt. Two pregnancies took hold (one in each dog), leading to the birth of Romulus and Remus after approximately 65 days of gestation. A few months later, a third surrogate carried another batch of edited embryos, resulting in the birth of Khaleesi.

All three pups were delivered via scheduled cesarean section to ensure safe delivery. Notably, Colossal reported no miscarriages or stillbirths during these trials, indicating a remarkably successful cloning process for an unprecedented de-extinction effort.

The Living Results

The three dire wolf pups—two males (Romulus and Remus) born in October 2024 and a female (Khaleesi) born in January 2025—are now thriving. At about 6 months and 3 months old respectively, they already exhibit classic dire wolf traits.

The pups have thick white fur, broad heads, and hefty builds, weighing around 80 pounds at just 6 months old. Their behavior is notably wild—unlike domestic puppies, Romulus and Remus keep their distance from humans, flinching or retreating even from familiar caretakers, demonstrating true wild lupine instincts.

Dr. George Church, a Harvard geneticist and Colossal co-founder, emphasized the significance of this achievement: "Preserving, expanding, and testing genetic diversity should be done well before important endangered animal species like the red wolf are lost. 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."

Technological Implications

The successful resurrection of the dire wolf has implications reaching far beyond this single species. The techniques developed include:

Dr. Christopher Mason, a scientific advisor to Colossal, called the achievement "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."

From Science Fiction to Reality

The resurrection of the dire wolf stands as a testament to how rapidly genetic technology is advancing. What once seemed like science fiction—bringing back extinct species—has become scientific fact through the convergence of paleogenomics, computational biology, and reproductive technology.

As Alta Charo, J.D., Professor of Law and Bioethics and Colossal Bioethics Lead, observed: "Whether due to natural or human-induced changes in climate, habitat and food source, the extinction of an untold number of species is a loss to our planet's history and biodiversity. Modern genetics lets us peer into the past, and modern genetic engineering lets us recover what was lost and might yet thrive."

With the successful birth of three dire wolves, Colossal Biosciences has demonstrated that their de-extinction methodology works. The company is now applying similar approaches to its other headline projects, including the woolly mammoth and the thylacine (Tasmanian tiger). More importantly, these same technologies are already being applied to help save critically endangered species that still exist, potentially transforming conservation efforts worldwide.