This is how progress is going to revive the extinct Tasmanian tiger according to the company that tries to resurrect mammoths

Colossalthe company founded to try return the mammoth to the arctic tundrahas also decided to address other species that have become extinct relatively recently: he dodo and he thylacine. Because of important biological differences, one of which is the generation time of proboscideans, these other efforts can reach many critical milestones well before the mammoth work.

At the end of last week, Colossal published a progress report about the work involved in the resurrection of the thylacinealso known as the Tasmanian wolf or tiger, which became extinct when the last known survivor died in a zoo in 1936. The biology of marsupials has some characteristics that can make de-extinction somewhat easier, but we have much less sophisticated forms of manipulating it compared to the technology we have developed to work with stem cells and the reproduction of placental mammals. But based on these new announcements, the technology available for working with marsupials is expanding rapidly.

The resistance of the cane toad

In the case of marsupial predators, the de-extinction effort is incorporating work that will benefit existing marsupial predators: building resistance to toxins found in the cane toad (Rhinella marina), an invasive species that has spread widely throughout Australia.

The main threat to cane toads comes from ‘bufotoxins’, a group of complex and related chemicals that bind to a protein found on the surface of cells called ATP1A1. This protein helps control the trafficking of ions across the cell membrane. Andrew Pask, who leads Colossal’s marsupial efforts, told Ars Technica that animals in the cane toad’s native range in Africa share a mutation in ATP1A1 that greatly reduces bufotoxin binding. Now, the team has engineered that change into the genome of a marsupial stem cell line and shown that it increases resistance by a factor of more than 6,000. (A manuscript describing some of this work is available.)

For the de-extinction process, the goal would be to ensure that the thylacine could survive in the presence of the cane toad. But Colossal has also started a conservation effort, called the Colossal Foundation, that aims to prevent threatened species from needing its services in the future. As part of that effort, the research team has generated stem cell lines from a surviving Australian carnivore, the Dasiyuros maculatus or tiger cat, which has officially become an endangered species, largely due to ingestion of the extremely poisonous cane toad. The goal is to ultimately engineer cane toad resistance into dasyurus stem cells and have the gene circulate among the wild population, allowing them to survive contact with their invasive neighbors.

Almost complete genome

Meanwhile, that editing and more have been taking place in stem cells derived from the fat-tailed dunnart, the thylacine’s closest living relative. Pask’s team is announcing that they have made more than 300 different edits to their genome. Ben Lamm, CEO of Colossal, told Ars They have discovered ways to do more edits at once without off-target effects (where editing occurs in the wrong place) and have been doing multiple rounds of editing on a single cell line.

The result of these editions has been the product of several efforts. For a start, Colossal has obtained an almost complete genome sequence of a thylacine sample which was preserved in ethanol a little over a century ago. According to Pask, this sample contains both the short fragments typical of older DNA samples (typically just a few hundred base pairs in length), but also some DNA molecules that were more than 10,000 bases in length. This allowed them to do short- and long-read sequencing, leaving them with just 45 holes in the total genome sequence, which the team hopes to close shortly.

This is exceptionally comprehensive for an extinct species and may help provide a greater degree of confidence that the team has identified all the DNA differences between the thylacine and its closest living relatives.