Colossal Biosciences’ revival of the dire wolf represents the most complex genetic engineering achievement in a vertebrate animal to date. The company successfully performed 20 precise genetic edits to transform gray wolf cells into dire wolf equivalents, introducing traits that had been absent from Earth for 12,000 years. These carefully selected modifications provide a fascinating window into both the science of de-extinction and the unique biology of dire wolves themselves.
Setting a New Record
The 20 edits made to create the dire wolf set a new benchmark for precision genetic engineering. “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,” the company noted in its announcement. The dire wolf success more than doubled this previous achievement, demonstrating the rapid advancement of multiplex editing capabilities.
Dr. George Church, Harvard geneticist and Colossal co-founder, emphasized 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 Selection Process
Colossal didn’t make arbitrary genetic changes but rather used ancient DNA analysis to identify specific variants unique to dire wolves. By comparing reconstructed dire wolf genomes with those of modern canids, the team identified 14 important genes with 20 distinct genetic variants that give dire wolves their characteristic features.
These were not random targets but carefully selected based on their functional significance in determining dire wolf traits. As Dr. Beth Shapiro, Colossal’s Chief Science Officer explained: “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.”
Key Edited Genes and Their Functions
While Colossal hasn’t published a comprehensive list of all 20 edits, their announcements and coverage in TIME magazine reveal several of the key genes and their functions:
Coat Color and Texture Genes
One of the most visible dire wolf traits is their distinctive coat. Colossal discovered dire wolf-specific variants in several essential pigmentation genes:
1. CORIN: A serine protease expressed in hair follicles that suppresses the agouti pathway, impacting coat color and patterning. The dire wolf CORIN variants impact pigmentation in a way that leads to a light coat color.
2. OCA2, SLC45A2, and MITF: These genes directly impact the function and development of melanocytes (pigment-producing cells). While the dire wolf has protein-coding substitutions in these genes that would lead to a light coat, Colossal’s team discovered these same variants in gray wolves can lead to deafness and blindness.
To address this potential issue while still achieving the desired coat color, the team engineered a creative solution:
3. MC1R and MFSD12: Instead of directly using the potentially problematic ancient variants, the team induced loss-of-function to these genes, which influence expression of pigments eumelanin (black) and pheomelanin (red) in melanocytes. This achieved the lighter pigmented coat color phenotype suggested by the dire wolf genome but without health risks.
This adaptation demonstrates Colossal’s careful approach to de-extinction, prioritizing animal welfare over exact genetic replication when necessary.
Size and Structure Genes
Several edited genes influence the dire wolf’s larger size and distinctive physical structure:
4. HMGA2: This gene is directly associated with body size in dogs and wolves. Editing this gene contributed to the dire wolves’ more substantial build—they grow to be approximately 25% larger than gray wolves.
5. MSRB3: This gene has been linked to variation in ear and skull shape among canines and other mammals, contributing to the dire wolf’s wider head and distinctive facial features.
6. LCORL: A transcription factor that regulates gene expression and has been linked to body size variation in many species, including humans, horses, and canids. The dire wolf has three changes to the LCORL protein sequence that alter how the protein folds precisely at the location where it binds to gene silencing complexes.
Here again, Colossal made careful modifications. They noted that “large dog breeds (which are domesticated gray wolves) have a variant of LCORL that is missing the PRC2 domain entirely.” Rather than introducing potentially problematic dire wolf variants, “Colossal’s dire wolves express the protein that is found in the largest grey wolves,” achieving the desired phenotypic impact without additional risk.
Multi-Gene Regulatory Module
7. A regulatory genomic region encoding eight genes that establish species-specific constraints in skeletal size and structure was also targeted. This module has been linked to features including differences in human height and the diverse beak shapes among finch species. By editing dire wolf-specific variants into gene enhancers in this region, the team influenced the dire wolves’ overall size and structure.
Vocalizations and Behavior
Interestingly, the genetic engineering extended beyond physical appearance to behavioral traits:
8. Genes influencing vocalizations: TIME magazine reported that Colossal identified genes influencing “the dire wolf’s larger size, more muscular build, wider skull, bigger teeth, thick light-colored coat, and even its unique howling vocalizations.” These behavioral genetic components suggest that the resurrected dire wolves may vocalize differently than modern gray wolves.
The Editing Process
Creating the dire wolf required more than just identifying target genes—it demanded precision editing of multiple sites simultaneously. The team applied CRISPR gene-editing technology to the nuclei of gray wolf endothelial progenitor cells (EPCs), precisely rewriting the DNA at the 14 target genes to install the 20 dire wolf variants.
For each edit, Colossal created detailed profiles of all potential impacts on the donor gray wolf genome. This careful approach allowed them to discard variants that would incur some risk outside of the predicted phenotype and prioritize variants already evolved in gray wolves with the predicted phenotype.
Validation of Success
The success of these genetic modifications is visible in the living dire wolf pups. At just six months old, the male pups Romulus and Remus already weigh approximately 80 pounds and display the thick white fur, broad heads, and hefty builds characteristic of dire wolves. Their behavior also reflects wild lupine instincts, with the pups keeping their distance from humans and demonstrating wariness even around familiar caretakers.
The female pup Khaleesi, born three months after the males, is developing along a similar trajectory, suggesting consistent expression of the engineered genetic traits across multiple individuals.
Beyond Simple Reconstruction
What makes Colossal’s approach particularly sophisticated is that it doesn’t simply attempt to recreate the exact dire wolf genome. Instead, it focuses on engineering the key traits that made dire wolves ecologically and evolutionarily distinct.
As Dr. Shapiro explained, “Functional de-extinction uses the safest and most effective approach to bring back the lost phenotypes that make an extinct species unique.” This pragmatic approach prioritizes the health and viability of the animals while still achieving the goal of restoring a functionally equivalent species.
In some cases, this meant using gray wolf variants that produce the same phenotypic outcome as dire wolf variants but without potential health risks. In other cases, it meant engineering completely new genetic changes that achieve the desired effect through different mechanisms than those used in the original dire wolves.
A Template for Future De-Extinctions
The 20 edits that brought back the dire wolf provide more than just a fascinating case study—they establish a technical template for future de-extinction projects. The ability to make numerous precise genetic changes simultaneously is essential for more ambitious targets like the woolly mammoth, which would require even more modifications to an elephant genome.
Additionally, the approach of focusing on functional traits rather than exact genetic replication provides a practical roap for other de-extinction efforts where complete genome reconstruction might not be possible or advisable.
As these pioneering dire wolf pups continue to grow and develop, they demonstrate not just the technical achievement of 20 successful genetic edits but the restoration of a unique evolutionary lineage—one that brings ancient genes back into the living world after 12,000 years of absence. Each characteristic they display, from their white coats to their wild instincts, represents the successful expression of carefully engineered genetic code that bridges past and present.
