Colossal achieves multiple scientific firsts in progress towards thylacine de-extinction

Colossal, the de-extinction and species preservation company, has announced numerous breakthrough successes in all stages of the thylacine de-extinction effort that put the company much closer to returning the thylacine to Australia.

Thylacines, which have been extinct since 1936 due to human depredation, are a keystone species that is vital to the healthy function of the Tasmanian ecosystem but are also an ideal candidate for the Colossal mission. Given their relatively recent extinction, many thylacine specimens are exceptionally well preserved, allowing Colossal and its collaborators to push the boundaries of ancient DNA science and create the genomic blueprints for the thylacine’s return.

The world’s most complete ancient genome

Capitalizing on one of the best preserved specimens known for any extinct species, the thylacine team produced a newly reconstructed thylacine genome that is the most complete and contiguous ancient genome of any species to date.

The new thylacine genome, which is around 3 billion bases in length, is exceptional in its contiguity, as it is assembled to the level of chromosomes. The genome is also estimated to be >99.9% accurate, and even includes hard-to-assemble repetitive features such as centromeres and telomeres, which are challenging to reconstruct even for living species.

The genome has only 45 gaps, which will be closed by additional sequencing efforts in the coming months. The team is continuing to compare this high-quality genome to genomes from close relatives of the thylacine to identify thylacine-specific evolutionary changes.

The team was also able to isolate long RNA molecules from preserved soft tissues from a 110-year-old thylacine. RNA is a much less stable biomolecule compared to DNA and therefore its preservation is extremely rare in historic specimens. The sample is a complete head of an adult thylacine that was skinned and preserved in ethanol. The team was able to recover RNA fragments that were up to 2,000 bases in length. The preservation of a complete head means that RNA could be sampled from several important tissues, including the tongue, nasal cavity, brain, and eye.

Breaking records in genetic engineering

Thylacines were known for their distinctive jaw and skull morphology, similar to that seen in some canids – the family that includes wolves and dogs. To determine what genes underlie the thylacine craniofacial shape, the team compared genomes from thylacines with genomes from wolves and dogs with similar craniofacial shapes, and identified regions of the genomes that are evolving at an accelerated pace in both groups. These regions of the genome, which the team are calling 'Thylacine Wolf Accelerated Regions' (TWARs), are predicted to drive morphological similarities between thylacine and wolf.

The team set out to prove that the TWARs drive the evolution of skull shape in mammals. First, they used high throughput reporter assays in bone cell lines to examine the functions of each TWAR. This identified TWARs that were active in bone tissues of a developing thylacine.

Next, they determined when and where the TWARs activated gene expression by linking them to reporter genes and making transgenic mice with individual TWAR elements. Finally, they tested explicitly whether TWARs could change developmental outcomes by separately replacing three regions of the mouse genome with TWARs from the extinct thylacine – this 'knock-in' experiment replaced a component of mouse regulatory DNA with thylacine DNA. Each of these three DNA swaps impacted the development of the mouse skull in the predicted way, confirming that the identified TWARS drive changes in head shape.

After confirming that TWARs are responsible for changing craniofacial morphology, the thylacine team has now made these same genetic edits into a cell line of a fat-tailed dunnart, which is the species that is the foundation for Colossal’s future thylacines and future surrogate of thylacine embryos. This dunnart cell line is currently the most edited animal cell to date – with over 300 unique genetic changes edited into its genome.

First artificial reproductive technologies that induce ovulation and grow embryos

Another key suite of technologies needed for de-extinction success is what are known as assisted reproductive technologies (ART), for the species that will be the surrogate host. For the thylacine project, that host is the fat tailed dunnart, a small dasyurid marsupial and the closest living relative of the thylacine. Prior to beginning the thylacine de-extinction project, not only was there no ART available for the dunnart, but very little ART was available for any marsupial.

The thylacine team has discovered and optimized an approach to induce ovulation in a dunnart, which is a vital first for both marsupial conservation and thylacine de-extinction. This crucial technology makes it possible to control precisely when an animal will come into estrus (heat). The approach leads to ovulation of many eggs simultaneously. These eggs can then be used to create new embryos, and, eventually, these eggs will be host for our edited thylacine genomes.

The team has also been able to take fertilized single-cell embryos and culture them over half way through pregnancy in an artificial uterus device. This is far beyond any previous attempts to grow embryos for any marsupial.

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