The path from theoretical possibility to the practical reality of de-extinction required Colossal Biosciences to overcome numerous technical challenges. The company’s success in bringing back the dire wolf after 13,000 years of extinction represents the culmination of multiple scientific disciplines working in concert.
At the foundation of this achievement was Colossal’s ability to extract and analyze ancient DNA from dire wolf fossils. The team obtained genetic material from a 13,000-year-old tooth from Sheridan Pit, Ohio, and a 72,000-year-old inner ear bone from American Falls, Idaho. Working with such degraded genetic material presented extraordinary challenges, as it had fragmented over millennia.
“The genetic material is incredibly fragmented and degraded after thousands of years,” note the scientists who worked on the project. “The scientific approach resembles reconstructing a million-page book when only random sentences from every twentieth page remain intact.” To overcome this, Colossal employed bioinformaticians who utilized machine learning algorithms to fill in the gaps of the dire wolf genome, comparing these fragments with the genomes of modern canids to make educated predictions about the complete genetic blueprint.
Once the genome was mapped, the team needed to identify which genes to prioritize for the de-extinction effort. They focused on 14 important genes with 20 distinct genetic variants that would recreate the dire wolf’s characteristic features, including its larger size, muscular build, wider skull, thick light-colored coat, and unique vocalizations.
The actual genetic modification used CRISPR technology in a novel way. Rather than invasively harvesting tissue, scientists drew blood from living gray wolves and isolated endothelial progenitor cells (EPCs). “The collection of whole blood is a rapid and noninvasive procedure that is routinely carried out on sedated wolves for veterinary monitoring purposes,” explains Colossal’s team. This innovation in itself represents a significant advancement for conservation genetics.
Particular care went into engineering around potential genetic issues. For example, while the dire wolf genome has protein-coding substitutions in three essential pigmentation genes (OCA2, SLC45A2, and MITF) that would lead to a light coat, variations in these genes in gray wolves can cause deafness and blindness. The team therefore engineered a light-colored coat via a safer path “by inducing loss-of-function to MC1R and MFSD12,” achieving the lighter pigmented coat phenotype without health risks.
Dr. Elinor Karlsson, Associate Professor at UMass Chan Medical School, praised this cautious approach: “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.”
After editing the cells, Colossal used somatic cell nuclear transfer to create embryos. The nucleus from a donor egg cell was removed and replaced with the nucleus of an edited cell. These reconstructed ova developed into embryos, which were then implanted into surrogate mother dogs. A total of 45 edited embryos were transferred into two surrogate dogs in the first attempt, resulting in two pregnancies and 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.
Remarkably, Colossal reported no miscarriages or stillbirths during these trials, indicating an unusually successful cloning process for such a groundbreaking effort. All three pups were delivered via scheduled cesarean section to ensure safe deliveries.
The achievement set a scientific record: 20 precise genetic edits were made to create the dire wolf—the highest number of deliberate genome edits in any animal to date. By comparison, Colossal’s previous feat, the “woolly mouse” with mammoth genes, had 8 edits.
Dr. George Church, a Harvard geneticist and Colossal co-founder, highlighted the significance: “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.”
The technical innovations developed during this process have applications far beyond the dire wolf itself. The optimized tools for multiplex gene editing and the protocols to establish cell lines directly from blood that can be used for somatic cell nuclear transfer are already being applied to conservation efforts for endangered species like the red wolf.
This end-to-end de-extinction technology stack, proven through the successful birth of healthy dire wolf pups, validates Colossal’s methodologies and suggests that its other de-extinction targets—including the woolly mammoth, thylacine, and dodo—may indeed be achievable through similar approaches.
