Colossal Biosciences progresses towards thylacine de-extinction

Species preservation company Colossal Biosciences has announced numerous breakthroughs in all stages of its thylacine de-extinction project, putting the company much closer to returning the iconic marsupial to Australia. Colossal’s success in developing de-extinction technologies is expected to not only support the thylacine effort but also to help combat the world’s extinction crisis.

Reconstructing an ancient genome

Capitalising on one of the best preserved specimens known for any extinct species, the Colossal team has produced a newly reconstructed thylacine genome that is claimed to be the most complete and contiguous ancient genome of any species to date. The genome is assembled to the level of chromosomes and is estimated to be >99.9% accurate; it even includes hard-to-assemble repetitive features such as centromeres and telomeres, which are challenging to reconstruct even for living species. It also has only 45 gaps, which will be closed by additional sequencing efforts in the coming months.

“The thylacine samples used for our new reference genome are among the best preserved ancient specimens my team has worked with,” said Beth Shapiro, Colossal’s Chief Science Officer and the Director of the UCSC Paleogenomics Lab, where the samples were processed. “It’s rare to have a sample that allows you to push the envelope in ancient DNA methods to such an extent. We’ve delivered a record-breaking ancient genome that will accelerate our thylacine de-extinction project.”

The high quality of the new thylacine genome, which is around three billion bases in length, is due to the unusual preservation of long DNA sequences. Most ancient specimens retain only short DNA sequences with little to no RNA, due to degradation that happens after the organism dies.

The team were 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 — a preservation method that probably led to the exceptional preservation of RNA. The team were able to recover RNA fragments that were up to 2000 bases in length.

Unlike DNA, which is the same in almost every nucleus in every cell in our body, the RNA that is expressed varies within each tissue, and contains a complete readout of all the active genes required for the functioning of a particular tissue. The preservation of a complete head means that RNA could be sampled from several important tissues, including the tongue, nasal cavity, brain and eye.

“This exceptional sample provides a fantastic opportunity for us to understand gene expression in thylacines,” said Dr Andrew Pask, a member of Colossal’s Scientific Advisory Board and Head of the Thylacine Integrated Genetic Restoration Research (TIGRR) Lab at The University of Melbourne. “With this new resource in hand we will be able to determine what a thylacine could taste, what it could smell, what kind of vision it had and even how its brain functioned.”

Together with the thylacine genome built from DNA, this new layer of RNA is expected to lead to the first complete, new annotated extinct animal genome ever produced.

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 Colossal 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’ or 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.

“We’re beyond pleased to learn that these regions of the genome are impacting the craniofacial phenotype as we predicted,” said Sara Ord, Director of Species Restoration at Colossal. “This is crucial evidence of the power of Colossal’s approach and an important step toward thylacine de-extinction.”

After confirming that TWARs are responsible for changing craniofacial morphology, the thylacine team have 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.

“We are really pushing forward the frontier of de-extinction technologies — from innovative ways of finding the regions of the genome driving evolution to novel methods to determine gene function,” Pask said. “We are in the best place ever to rebuild this species using the most thorough genome resources and the best informed experiments to determine function.”

Artificial reproductive technologies for marsupials

Another key suite of technologies needed for de-extinction success is what are known as assisted reproductive technologies, or ART, for the species that will be the surrogate host. For the thylacine project, that host is the fat-tailed dunnart, 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 now discovered and optimised an approach to induce ovulation in a dunnart, which is important 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 the edited thylacine genomes.

In another first, the team have been able to take fertilised 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.

“Not only are these major milestones for the thylacine de-extinction project, but Colossal’s advancements for improving assisted reproductive technologies in marsupials can be applied across the marsupial family tree,” Pask said. “These technologies will, for example, improve the breeding capacity of critically endangered species in captive populations — such as the closely related Tasmanian devils being bred to help fight against their extinction from the devil facial tumour disease (DFTD).”

Update on the northern quoll

In other news, Colossal has provided an update on its efforts to engineer resistance to cane toad toxin into marsupial cells. The company’s refined strategy for engineering resistance can now create over 6000-fold increased resistance in the northern quoll with just one edit to the genome.

“By changing a single base in a three billion base pair genome, we can make the endangered northern quoll go from completely susceptible to cane toad toxin to among the most resistant species to this toxin on earth,” Pask said.

The team has also now derived the first induced pluripotent stem cell (iPSC) lines for quolls, which they created using the approach developed to make iPSCs for the fat-tailed dunnart. These iPSCs are essential for making a living quoll that carries the cane toad toxin resistance in its genome. iPSCs are also essential in conservation efforts including biobanking, cloning, breeding and genetic engineering work.

“It's incredible to see how all these new technologies (ART, iPSC derivation, marsupial gene editing) are already being used to solve major conservation issues for marsupials, as well hitting all of our major milestones for bringing back the thylacine,” Pask said. “We’re getting closer every day to being able to place the thylacine back into the ecosystem — which of course is a major conservation benefit as well.”

Image credit: iStock.com/CoreyFord