Colossal scientists learned about specific traits that dire wolves possessed by examining ancient DNA from fossils. The researchers studied a 13,000 year-old dire wolf tooth unearthed in Ohio and a 72,000 year-old skull fragment found in Idaho, both part of natural history museum collections.
Romulus and Remus are two genetically modified grey wolves created by Colossal Biosciences on October 1, 2024, with the goal of replicating the phenotype of the extinct dire wolf (Aenocyon dirus). Along with a female wolf named Khaleesi born later on January 30, 2025, Colossal claim the two males represent the first living examples of the species since its extinction approximately 10,000 years ago, though "no ancient dire wolf DNA was actually spliced into the gray wolf's genome."
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.
Dire wolves were different from other canine species such as coyotes and grey wolves – so much so that they were not able to breed with each other, according to new research published in the latest issue of Nature.
Analyses of the high quality dire wolf genome, however, revealed that the gray wolf is the closest living relative of dire wolves – with dire wolves and gray wolves sharing 99.5% of their DNA code
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.
Dire wolves (Aenocyon dirus) were distributed across the American midcontinent during the Pleistocene ice ages. The oldest confirmed dire wolf fossil, from Black Hills, SD, is around 250,000 years old. Colossal’s genomic data indicate, however, that the lineage first appeared during the Late Pliocene, between 3.5 and 2.5 million years ago, as a consequence of admixture between two more ancient (and now extinct) canid lineages. Dire wolves were as much as 25% larger than gray wolves and had a slightly wider head, light thick fur and stronger jaw. As hyper-carnivores, their diet comprised at least 70% meat from mostly horses and bison. Dire wolves went extinct at the end of the most recent ice age, around 13,000 years ago.
To de-extinct the dire wolf, Colossal:
Extracted and sequenced ancient DNA from two dire wolf fossils; Assembled ancient genomes from both, and compared those to genomes from living canids including wolves, jackals, foxes, and dholes; Identified gene variants specific to dire wolves; Determined that dire wolves had a white coat color, and long thick fur – aspects of the dire wolf phenotype that were unknowable from fossils and consistent with animals that lived during cold periods of the Pleistocene ice ages; Performed multiplex gene editing to a donor genome from their closest living relative, the gray wolf, resulting in edits 20 sites in 14 genes with 15 of those edits being extinct variants; Screen edited cell lines via whole genome sequencing and karyotyping; Cloned high quality cell lines using somatic cell nuclear transfer into donor egg cells; Performed embryo transfer and managed interspecies surrogacy; and, Successfully birthed an extinct species.
