De-extinction and Species preservation: New milestones for resurrection of Thylacine (Tasmanian tiger)

The thylacine de-extinction project announced in 2022 has achieved new milestones in generation of the highest quality ancient genome, marsupial genome editing and new assisted reproductive technologies (ARTs) for marsupials. These advancements will not only support resurrection of Tasmanian tigers (that are extinct since 1936 due to human depredation) but will also help in preservation of species at risk of extinction. Resurrection and returning of thylacines back into the native Tasmania will restore healthy functioning of the local ecosystem. The newly acquired capabilities will also help in preservation of critically endangred species.

The newly reconstructed thylacine genome, which is around 3 billion bases in length, is the most complete and contiguous ancient genome of any species to date. It is assembled to the level of chromosomes and is estimated to be >99.9% accurate. It includes hard-to-assemble repetitive features such as centromeres and telomeres, which are difficult to reconstruct even for living species. The genome has only 45 gaps, which will be closed by additional sequencing efforts in the coming months.

Most ancient specimens retain only short DNA sequences with little to no RNA, due to degradation after death of an organism. The new thylacine genome is exceptional in the unusual preservation of long DNA sequences and RNA. RNA degrades much quickly hence preservation of RNA is rare in historical samples. In this case, the research team successfully isolated long RNA molecules from preserved soft tissues from a 110-year-old sample. This is important because expression of RNA varies in tissues hence presence of RNAs in tissues give an idea of active genes required for proper functioning of tissues. The new RNA layer makes the thylacine genome built from DNA much more useful in de-extinction.

After reconstructing thylacine genome, the next logical step was to identify genes that underlie the core thylacine trait of the distinctive jaw and skull morphology. For determining this, the research team compared genomes from thylacines with genomes from wolves and dogs with similar craniofacial shapes and identified regions of the genome called “Thylacine Wolf Accelerated Regions” (TWARs) which were later found to drive the evolution of skull shape in mammals.

Following confirmation that TWARs are responsible for craniofacial morphology, the research team made same genetic edits numbering over 300 into a cell line of a fat-tailed dunnart, which is the closest living relative of thylacine and to be the future surrogate of thylacine embryos.

Next is development of assisted reproductive technologies (ARTs) for the dunnart species that will be the surrogate thylacine. Prior to the thylacine de-extinction project, there was practically no ART for any marsupial. The reserach has now developed a crucial technology to induce controlled ovulation of many eggs simultaneously in a dunnart. The eggs can be used to create new embryos to host edited thylacine genomes. The researchers were also able to take fertilized single-cell embryos and culture them over half way through pregnancy in an artificial uterus device. The new ART capabilities can be applied across marsupial family for de-extinction of thylacine as well as for improving breeding cappacity of endangered marsupial species.

Resurrection and returning of thylacines back into the native Tasmania will restore healthy functioning of the local ecosystem. The newly acquired capabilities will also help in preservation of critically endangred species.

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