Eriona Hysolli is a former Postdoctoral Researcher at the George Church Lab in Harvard Medical School. Hysolli was involved in the Woolly Mammoth Revival Project, which aims to genetically engineer the genes of the Asian elephant with genes from the woolly mammoth to reintroduce this creature back into the tundra in order to combat climate change.
Please note the transcript has been edited for brevity and clarity.
FLG: Hello, everyone, and hello, Eriona. Thank you so much for joining me today as we go down the rabbit hole and take a look at some of the unusual applications of genomics. Today, we’re going to be talking about the Woolly Mammoth Project. So, Eriona, if you could just introduce yourself and tell us a little bit about what you do.
Eriona: Thank you, Shannon. I’m happy to be here. My name is Eriona Hysolli. I am currently a Postdoctoral Researcher at the lab of George Church at Harvard Medical School, and I did my PhD in stem cell biology.
FLG: So, you’ve probably discussed this a lot, but it would be great if you could just briefly summarise what the Woolly Mammoth Revival Project is and what you are trying to achieve.
Eriona: Yeah, I think I’m going to give a little bit of background first. So, the first sequence of the woolly mammoth came around 2006, where they sequenced the mitochondrial DNA, and then later on genomic DNA, and we have now sequences of several woolly mammoths. The fact that we are here and discussing this topic is probably because of the pioneering thoughts of my professor, George Church. He, ever since the sequences of ancient species came along, especially woolly mammoth, was interested in using genome editing tools to revive the species. And this in combination with some other work.
Sergey Zimov over at Northeast Science Centre in Siberia – he was very much interested in reviving a lost ecosystem of the woolly mammoths, which was grasslands, very biodiverse. And he actually has a piece of land where he’s trying to actually test his hypothesis in real life, where he tries to make a very biodiverse environment in order to see whether increased vegetation and biodiversity could be the key to slowing down global warming, or at least the effects of global warming in the future.
So, in the lab, what we do is we take elephant cells and we try to use a variety of genome editing tools in order to change the sequence from the sequence of the elephant to the sequence of the woolly mammoth. We have obviously done a lot of background work on comparing the sequences of a lot of African elephants and Asian elephants to the current genomes we have from several specimens of the woolly mammoth. So, we have compiled a list of changes. Mostly they are just single nucleotide changes dispersed throughout both the sequence the coding regions and non-coding regions of the genome. And we focus on several genes that we think are associated with some of the phenotypes that gave the woolly mammoths the ability to withstand cold temperatures. And so, we hope to engineer those features into an elephant cell.
FLG: What is the ultimate goal of this project and where might it be useful within society?
Eriona: Yeah, so I think we see this as a sort of like a pilot or a landmark genome project. That is, we would like to see how we can use the current technology for conservation efforts or for restoration of lost ecosystems by bringing back some of these lost species. Now, there are caveats. Of course, we cannot do that with every lost species. Obviously, the woolly mammoth, they roamed at least an isolated island in Siberia as recently as 4000-5000 years ago. So, the ecosystem is similar to what it used to be. And it’s possible to revive the species and basically return it to the ecosystem it used to live in because a lot of it has been preserved. But I think, in the future as we face, due to human-animal encroachment, poaching, due to climate change, we’re losing species at an alarming rate. So, we’d like to build a framework of how we can return or revive some of these species and preserve biodiversity in the future.
FLG: What other animals are being looked at within de-extinction projects?
Eriona: I strongly recommend people to look up this non-profit organisation called Revive and Restore. They have listed there the projects they are interested in. It’s a combination of trying to increase genetic variability in some of the species that are threatened or are undergoing extinction, and also reviving some of the lost species. For example, the complete revival would be the woolly mammoth or the passenger pigeon. But there are also projects like the Heath Hen, Prezewalski’s Horse or the Black-footed Ferret. These are projects with species that are still alive, but we can increase genetic diversity so that we give them a chance and they don’t go extinct.
FLG: What are some of the dangers of bringing these species back?
Eriona: Yeah, we definitely need to have ethical discussions on bringing back a lot of species. I think in our lab, currently, we are working at the in vitro model with cells. When we get more advanced, and we move on to potentially bringing back an actual fully grown animal, a lot of conversations have to happen. I think there are several things you have to consider. First, is there an ecosystem for the lost species? And we believe, at least for the woolly mammoth, that there is because they’ve only recently been extinct. Obviously, we cannot talk about bringing back something or an animal that we lost millions of years ago, for many reasons. Primarily, because we don’t have the sequence. Obviously, you need to have the genetic information of the lost species. And for the woolly mammoth, we have a lot of specimens that are well preserved, so we’re able to construct the whole genome.
A lot of people, for example, ask about the dinosaurs. But sadly, we don’t have genetic information for dinosaurs so we cannot. Rest assured that at least now, it’s not quite possible to bring back the dinosaurs. We’re talking about hundreds of millions of years ago and DNA withstands a lot of degradation over time, but it’s not that robust. So, you have to have the genetic information of the species. And also, you need to have the ecosystem as I talked about, and for the woolly mammoth we do. For some of the lost species, it’s no longer there. So, in that case, you really have to discuss the feasibility, and whether it’s a good idea to actually bring back a species that has no habitat. So, these are some of the factors along with, of course, the ethical discussions of bringing back species that we believe are more complex, for example, the woolly mammoth or elephant, in which case, we have to consider their wellbeing in nature.
FLG: I feel like a lot of dinosaur lovers are going to be very heartbroken about this news. A few months ago, a record was broken for sequencing the oldest ancient genome from permafrost-preserved teeth from a mammoth. What are the challenges with working with this material?
Eriona: Yeah, so I think some of the challenges for sequencing ancient DNA is the specimen itself. So obviously, the better preserved the specimen, the better sequences we will get. We have obviously sequenced specimen from our own species, like Homo Neanderthalensis and Denisovans and so on. And it’s been a bit more challenging there because all you have is bone. So, you have to grind a lot of bone to get some DNA from the specimen. We have been a bit more fortunate with the woolly mammoth that there are so many of them preserved in permafrost. But obviously, if you want to get as good quality sample as you can, you want to keep it preserved and frozen during transportation. And one other major challenge in sequencing ancient genomes is contamination of the sample itself with the flora and fauna of the environment where it’s found. So, most of the DNA sequences from those samples are actually contaminant DNA. And this also includes human contaminants because during handling. PCR is actually very sensitive; it is a very sensitive technique. So, you can actually sequence very well even one molecule of DNA. So, you have to be aware of contaminants. And while sequencing ancient genomes, you can actually go through an enrichment step where you try to use strands that are similar or that are going to be complimentary to the sequence you will find so as to enrich it from the contaminants found in the specimen itself. That way you increase the quality and you enrich for the reads you want.
After that, usually the DNA is highly degraded and deaminated so some of it may not sequence well. So, you have to go through a repair step in order to get the DNA to sequence well, and then attach adapters or these universal sequences that can be used to amplify one specific molecule many, many times – hundreds or thousands of times. And you end up with these short fragments of DNA. You don’t get much information from those, so obviously, you need computational tools and programmes to stitch them together in silico. So that’s what happens next. You have millions of these short reads, and then you actually try to stitch them together. And you can only go so far, of course, especially with novel species, you actually have to build the genome de novo. But in the case of the woolly mammoth, it is great because we have a very similar genome in the elephant. So, you can actually use the elephant genome as a scaffold. So, you can stitch the smaller fragments into contigs that are longer and then try to use the scaffold genome of the elephant. So, you can actually map where the sequences are and try to find out more information, whether it’s coding or non-coding, whether it’s a gene, whether it’s a regulatory region, or whether it’s outside of those altogether. So, it’s a field that is kind of separate and a lot of advancement has been made there to make it possible now to mainstream sequencing of ancient samples, because it’s definitely more challenging and more difficult with old, degraded DNA than with fresh DNA.
FLG: Your lab is working on an elephant-mammoth hybrid – how similar are their genomes? Are there any notable differences?
Eriona: Yeah, I mean, obviously just by looking at an image of a woolly mammoth versus an elephant, you can clearly see differences in morphology. And I think we’ve estimated that the closest similarity in DNA a woolly mammoth has to any other species is actually the Asian elephant, roughly 99.6% similar. And from our computational work, we’ve estimated that there are around 5 million changes throughout the genome. So, we have to make basically 5 million changes to derive some sort of like a base elephant, without, of course, the genetic variation that happens between individuals. But roughly we estimate, we have to do 5 million changes first.
FLG: Would you be able to discuss the techniques you are using to achieve this?
Eriona: Yeah, the field of gene editing has advanced pretty fast. So, we use CRISPR-Cas9 and its derived variations, like base editors, where you make a single base pair change. It is pretty precise. Obviously, there are off-target effects, but I think it’s one of the best tools we have, as well as prime editing where you can actually insert or delete small sequences. So, these are some of the major tools we use now where we’re only focusing on a few genes. Later on, as you increase the number of edits, we will need better tools. We don’t have great tools; we have good tools. I think we’d have to also use a combination of synthesis assembly and then swapping into the elephant genome. So, for example, if you need to make many changes in a particular locus of DNA in the elephant cells, and you find that there are so many changes that maybe a base editor or CRISPR is not exactly the most efficient tool there. We predict that we can actually synthesise that fragment of DNA, synthesise maybe many of them, put them together, assemble them together, and then swap them through different kinds of enzymes into the elephant genome. So, these are some of the tools we use now. But we’re developing and optimising better tools for the future, so we’ll wait and see.
FLG: You are specifically exploring mammoth genes that would enable it to adapt to cold climates – why is that you selected those genes? Are you hoping to explore any other genes?
Eriona: We selected those genes because they popped up when we did the comparison. And when we looked at the annotation of those genes and some other previous publications, there is a strong indication that they might give us the phenotype we see in the woolly mammoth, such as cold resistance, hair and brown fat deposits and so on. But on top of that, we see a lot of changes in some of the regulatory regions. We’re specifically looking at coding regions, but we can extend that work into regulatory regions, regions that actually regulate the expression of any given gene. So, we’ve seen some differences there. And actually, we’ve seen a lot of things that are unannotated – that means that we don’t know the function of those genes. Therefore, it would be very cool in the future to explore the role and the function of what those genes do in the background of an elephant or woolly mammoth as we go along with our research. But what most excites me now is actually some of these regulatory regions and unannotated genes.
FLG: What other traits are you hoping to incorporate into this hybrid?
Eriona: The phenotype of cold resistance, of course, is an absolute must. But we don’t even know whether the genes we’re going to edit are going to fully give us that phenotype. So, the more changes you make, obviously, the closer you will get to the woolly mammoth background. We are looking at, as I said, some of the easy picks – the low hanging fruit, so to speak – and then extend that search to the unannotated genes and other sequences that we don’t know the function of. But obviously, we’re going to definitely look into any gene that is in involved in the developmental process. We see the elephants and the woolly mammoth do have changes or differences in their morphology – smaller ears, smaller stature, and so on – so we predict a lot of developmental genes to be involved there. So, we will definitely look there.
FLG: Are you looking at any sort of genes that could help it potentially be more resistant or give it an advantage?
Eriona: Yeah, yeah. But I think in order to achieve that, we also need a lot more information, for example, for the genes themselves. So, for example, for climate change, I suppose if you’re going to make a hybrid elephant-woolly mammoth (‘elemoth’) maybe now that the climate is getting warmer, potentially, you can look into maybe preserving some of the adaptation that elephants have to warmer climates, rather than you know, conferring full cold resistance. But we’ll have to see how the climate evolves by the time we generate, first, the cells and then hopefully, the woolly mammoth. I think what would be cool is actually to engineer disease resistance into the new woolly mammoths or hybrids. And for that, we’ll probably need to know a lot about what potentially could have affected the woolly mammoths, but also what diseases afflict the extant elephant species. And we know of one, such as the elephant endotheliotropic herpesvirus, which is fatal to young elephants. And because the sequences of the woolly mammoth and the Asian elephants are so close, we might predict the woolly mammoth might also be afflicted by infection with this virus. And so, when you build or you engineer the genome of the hybrid or the woolly mammoth, as we understand more of the diseases, we can engineer resistance into the genome as well.
FLG: Your lab is attempting to synthesise a virulent herpes strain in vitro – would you be able to expand on this work?
Eriona: First of all, it is only virulent we would say, in young elephants. I think humans are fairly safe. There are a lot of elephant handlers who deal with elephants in the zoos, and we don’t have any cases of transmission yet. And adult elephants do very well as well, in general. And they share this virus, continuously called EHHV, again, endotheliotropic herpesvirus. Something happens though, that when it is activated in young elephants (between the ages of zero, or at least neonates, and 10 or 12) it’s fairly fatal. So, for the afflicted individuals, about two thirds of them die. And so, it’s important to try to diagnose the disease as fast as possible, because they might have a chance. A lot of zoos, for example, do allocate resources to sequence the viral load of the virus in the young elephants continuously. And if they see spikes, they start treating with antivirals and IV. And so that can give young elephants a chance. But more often than not, they die because from the diagnosis, or from the observation, to that, is a very short time frame.
It seems to afflict more elephants in zoos than in the wild, though, of course, cases in the wild have been reported. It also seems to affect more Asian elephants than African elephants, again, variants of EHHV have also caused African elephant deaths. And so, we don’t know much about the mechanism of disease. So, we’re hoping to use synthetic biology, building the genome of the virus, and actually studying it in a cellular model to find out the viral life cycle and the proteins that it affects or how gene expression is affected within the cell. So basically, it’s a disaster; it’s a terrible disease. It perforates the arteries and veins, so these animals basically bleed out and haemorrhage. And we know about sequences of variants of this virus. But we don’t really know how to counter the disease. There are some efforts from the Laboratory of Dr. Paul Ling at Baylor. They’re trying to study the sequences, potentially make a vaccine. It’s still a work in progress. But we would also like to use our expertise and synthetic biology to build the genome and potentially find the right genome editing tools in order to treat these elephants in vivo, potentially with a vector like AAV carrying the gene editing tool and the guide RNA so it can chop up the virus genome in vivo. Those are obviously efforts for the future. We’re currently working on it, but it will take a little bit of time to make progress there. And obviously, working with zoos and veterinarians to actually make it happen. It’s not easy to establish protocols for exotic species.
FLG: How do you think this research will then impact conservation efforts for Asian elephants?
Eriona: The more we know about the disease, as I said, we can actually engineer virus resistance, even in elephants themselves. We don’t actually only have to engineer virus resistance in woolly mammoths or hybrids. So synthetic biology and gene editing can be quite helpful in preservation of the species. Obviously, we need a tonne of resources and willingness to save the elephants in the wild. But we always need a backup plan because they are highly endangered. And so, removing at least one endangering factor like disease could be very important for preservation of the species. That’s why we also work on the EHV not just the revival of the woolly mammoth.
FLG: That’s really interesting. Is there the possibility that there was something in the tundra, for example, a plant or microorganism that was beneficial for the mammoth, and now it is no longer present? Is their ecosystem really different now?
Eriona: Yeah. I mean, that’s a great question. Although extinction happened fairly recently compared to the age of the Earth, still, thousands of years have passed, and the flora and fauna have also changed. I’m a little bit afraid to just use the term geoengineering, because here we’re not creating an artificial system, we’re actually trying to restore a natural system. So, it’s not artificial, it’s just restoring what used to be there. And with the efforts of the Zimovs in Siberia, you can see that introducing more larger organisms into a patch of land, restores that grassland ecosystem that occurred during the Pleistocene time. So, at least some of that biodiversity can be captured by introducing some of the species that used to be there. I mean, it used to be a very, very, very rich ecosystem – the Zimovs estimate up to 10 tonnes of organic mass per square kilometre. So, they estimate about one mammoth per square kilometre that used to roam the plains of Siberia. So, we know from bone collections the large animals that existed there. But you are correct, we don’t really know the full gamut of the microorganisms that live there.
One thing that can help is metagenomics of the soil, so trying to sequence as much as possible, and also sequence the guts of these well-preserved specimen. There have been a couple of studies there. I’m not well versed in the field, but you can actually look at using ribosomal RNA to sequence some of the microorganisms that lived in the guts of these ancient species. And of course, trying to compare what used to be a microorganism that lived thousands of years ago versus what’s populating or colonising the gut post death. So, there are definitely more studies that need to be done there. But we can actually use this information from sequencing to try to figure out some of the metagenomics biodiversity that existed at that time. But the truth of the matter is, we don’t really know. What we’re hoping is that introducing this species, these larger organisms, back into this huge habitat that basically has next to no large animals, at least compared to what it used to be. I think you just have to let nature run its course at that time. So, you are worried about trying to make it as close as possible, but at the same time, you just have to let life happen. Obviously, under observation, we learn more as we observe what happens. But the fact this Pleistocene Park in Siberia looks more like a grassland when you populate it with animals like muskox, reindeer, yaks, moose, gives us an indication that it’s possible to restore it to a more biodiverse ecosystem than it used to be a long time ago. Whether it will be fully captured like it used to be, that remains to be seen, potentially highly unlikely, but at least we can get very close.
FLG: Could there be cases of where mammoths get integrated, and they then make other organisms go extinct and they impact other biodiversity, such as that of microorganisms?
Eriona: Yeah, you will have to do that and observe. But as I mentioned, Pleistocene grasslands, the mammoth steppe, used to be very biodiverse. A lot of large organisms were living in the same square kilometre – it was very, very dense. So, it’s highly unlikely that the mammoths themselves would cause the extinction of any other species. There is competition, right? If there isn’t enough grass, of course, maybe you’ll see some of the larger species go extinct. Again, it’s hard to model it perfectly right now. It’s certainly a danger, but it will have to be gradual as well. And also, of course, usually a species expands depending on the nature and the environment surrounding it. So obviously, if there aren’t that many resources for it to survive, the ecosystem will not be as dense. I think one hypothesis is that it was humans that hunted a lot of mammoths and with the loss of the woolly mammoth, which trampled snow, allowed for this grassland ecosystem to happen. The land froze very quickly and therefore gradually all the other animals were lost. So, we can say the opposite, that the loss of the woolly mammoth actually contributed to the loss of biodiversity.
But, again, a lot of it is also speculation so we don’t know until we actually try. But it’s hard to see how the reintroduction of the woolly mammoth would have many negative effects. It used to be their ecosystem, their habitat. They are large species – it’s very easy to track them, even if there are negative effects from reintroduction in the wild. And they have a very, very long gestation time. They won’t populate the area as fast as we think. So, there’s plenty of time for observation. Their gestation time is almost two years. I think the problem would be to actually have more rather than fewer.
FLG: What do you think the future of this research will look like and what are you excited about as well?
Eriona: I think it’s very exciting to me that we have the possibility of restoring lost species. Right now, it might take us decades. But in the future, once we reach that inflection point, we can design, synthesise and assemble the genome of any species because synthetic biology has advanced and will continue to advance a lot. Again, with this power, comes great responsibility, so we’ll have to have those ethical discussions on what we do and when we are so powerful as to make these genomes very quickly. But as an animal lover, I think it really excites me that I can use science and technology to restore some of the balance of the ecosystems. Everyday we’re faced with loss of biodiversity and trying to preserve that for ourselves and for Earth I think it’s, as cliche as it sounds, very important. Studying species and studying biodiversity has helped us as a species quite a lot. And so, I feel like it’s only beneficial to try to keep our Earth as biodiverse as possible. And if we have to use some of the artificial tools to do so, so be it! Provided it is, of course, in an ethical and well discussed manner.
FLG: Thanks for joining me today, Eriona. It has been really exciting, and I look forward to seeing the progress your group makes. Thank you so much.
Eriona: Thank you so much, Shannon. It’s been a pleasure. I hope people look more into it, they get more excited about it, and they don’t necessarily fear science, but rather actually use it to our benefit. And hopefully we’ll see woolly mammoths roam again in Siberia!
FLG: Hopefully – thank you!