How Colossal Biosciences Achieved 20 Genetic Edits
The resurrection of the dire wolf (Aenocyon dirus) by Colossal Biosciences represents a landmark achievement in genetic engineering, setting a new record with 20 precise genomic edits in a vertebrate animal.
This scientific breakthrough not only brought back an iconic Ice Age predator but also demonstrated the feasibility of sophisticated genetic interventions that could revolutionize conservation efforts for endangered species worldwide.
Unprecedented Genetic Engineering
The dire wolf revival marks the first time scientists have successfully de-extinct an animal through genetic engineering, establishing a new benchmark in the field.
As Colossal’s announcement explained, the dire wolf achievement surpassed their previous record of 8 edits in the “woolly mouse” project, more than doubling the complexity of the genetic intervention.
“This news comes on the heels of the recent announcement of the Colossal woolly mouse, which previously held the record for unique germline edits in an animal with 8 precision edits. With the dire wolves, Colossal has made 20 unique precision germline edits including 15 edits from the ancient gene variants that have not existed in over 12,000 years, setting a new bar for precision germline editing in any animal,” the company reported.
The Five-Step Methodology
Colossal’s achievement arose from a meticulous five-step process that combined ancient DNA analysis, modern genomic tools, and reproductive technology:
1. Ancient DNA Extraction and Genome Reconstruction
The process began with extracting genetic material from two dire wolf fossils: a 13,000-year-old tooth from Sheridan Pit, Ohio, and a 72,000-year-old inner ear bone from American Falls, Idaho. Using novel sequencing approaches, Colossal’s scientists assembled high-quality ancient genomes from these fragments.
“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,” according to Colossal’s technical documentation.
2. Identification of Key Genetic Traits
Through comprehensive genomic analysis, the team discovered that gray wolves are the closest living relatives to dire wolves, sharing 99.5% of their DNA.
The scientists then identified the crucial genetic differences responsible for the dire wolf’s distinctive traits – including body size, skull morphology, and coat characteristics.
Dr. Beth Shapiro, Colossal’s Chief Science Officer, explained: “Our novel approach to iteratively improve our ancient genome in the absence of a perfect reference sets a new standard for paleogenome reconstruction.
Together with improved approaches to recover ancient DNA, these computational advances allowed us to resolve the evolutionary history of dire wolves and establish the genomic foundation for de-extinction.”
3. CRISPR Gene Editing
With a precise genetic blueprint in hand, Colossal applied CRISPR technology to edit living cells from modern gray wolves. Rather than using invasive tissue sampling, scientists collected blood from gray wolves during routine veterinary procedures and isolated endothelial progenitor cells (EPCs).
Using multiplex genome editing, they installed 20 precise edits across 14 different genes. These included modifications to genes controlling coat color (CORIN, MC1R, MFSD12), body size (LCORL, HMGA2), and skeletal structure (MSRB3).
Notably, the team employed sophisticated genetic engineering strategies to achieve the desired phenotypic outcomes while avoiding potential health issues. For example, when addressing coat color:
“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. The team therefore engineered a light colored coat in Colossal’s dire wolves via a path known to be safe in gray wolves.”
4. Somatic Cell Nuclear Transfer (Cloning)
Once the cells were genetically modified, the team employed cloning techniques via somatic cell nuclear transfer. The edited cell nuclei were inserted into egg cells that had their original nuclei removed, creating viable embryos that carried the dire wolf genetic profile.
“The Colossal 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,” the technical documentation states.
5. Surrogate Pregnancies and Birth
In the final step, the engineered embryos were implanted into domestic dog surrogates selected for their genetic compatibility with canids. Two initial surrogates carried embryos to term, resulting in the birth of males Romulus and Remus in October 2024, followed by female Khaleesi in January 2025.
“Three pregnancies led to births of the first de-extinct species,” Colossal reported, noting the remarkable success rate with “no miscarriages or stillbirths” during the trials – an exceptional outcome for such pioneering reproductive technology.
Evolutionary Insights
Beyond the technical achievement, Colossal’s research yielded significant scientific insights about dire wolf evolution. Their analysis revealed that the dire wolf lineage emerged between 3.5 and 2.5 million years ago through hybridization between two ancient canid lineages – explaining previous uncertainty about their evolutionary history.
Dr. Elinor Karlsson of UMass Chan Medical School and the Broad Institute of MIT and Harvard praised the careful approach to gene editing: “When I learned of Colossal’s approach to engineering the light coat color into their dire wolves, I was simultaneously impressed and relieved. 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.”
Beyond De-Extinction: Conservation Applications
The technologies developed for the dire wolf revival have immediate applications for endangered species conservation. Using the same methods, Colossal has already produced four healthy red wolf pups – a species with fewer than 20 individuals remaining in the wild.
Dr. Christopher Mason, a scientific advisor to Colossal, emphasized the broader impact: “The same technologies that created the dire wolf can directly help save a variety of other endangered animals as well. This is an extraordinary technological leap in genetic engineering efforts for both science and for conservation.”
One particularly promising innovation is the non-invasive blood cloning technique, which allows scientists to establish cell lines from routine blood draws without invasive tissue sampling. This approach creates new possibilities for biobanking endangered species’ genetic material.
“Biobanking and cloning EPCs from threatened or endangered populations of wild wolves provides a safety net to preserve the genomic diversity present today from further loss and extinction,” noted Colossal’s documentation.
The dire wolf revival demonstrates that de-extinction technology is not merely about bringing back lost species but developing powerful new tools to prevent further extinctions. As these techniques continue to advance, they may provide crucial support for conservation efforts worldwide, offering new ways to preserve genetic diversity and protect vulnerable species.
