Hacking the Code of Life

by Nessa Carey

  15. https://www.biorxiv.org/content/biorxiv/early/2018/07/07/362558.full.pdf

  16. https://www.doc.govt.nz/nature/pests-and-threats/predator-free-2050/

  17. Loss, S.R., Will, T., Marra, P.P. ‘The impact of free-ranging domestic cats on wildlife of the United States’. Nat. Commun. (2013); 4: 1396.

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  PICK A QUESTION, ANY QUESTION

  One of the reasons why the new gene editing technologies are having such an impact in basic science is because they can be applied to just about any species really easily and cheaply. This wasn’t true of any of the previous approaches, because they required very specialist molecular reagents, fine-tuned to a single species. If a researcher wanted to work on an unusual animal or plant, it could take them several years simply to develop the genetic tools they wanted. But not any more. The new technologies have democratised biological science. No matter how obscure your species of choice, you can create the molecular reagents to probe the questions that really interest you. It’s pure curiosity-driven research at its finest, and it’s yielding incredibly powerful results.

  Take the clonal raider ant, for example. It’s tiny, but it’s mighty. It’s a stocky little creature, about 2mm long, and it lives in colonies of a few hundred individuals. Its diminutive size is no barrier to the clonal raider ants’ ambitions. The insects spend their lives underground, raiding the nests of other ant species, and taking away the young grubs to eat for dinner.

  If your life depends on forming successful raiding parties that all move together and can also make it back to safety following an afternoon of marauding, you need to be able to communicate with the others in your troop. Researchers were fairly sure that the clonal raider ants did this by following chemical trails laid down by their colony members as they set out on their missions. But exactly how they did this wasn’t clear. Ants are relatively simple creatures, whose actions are essentially hard-wired. There are a limited number of options they can make in response to any given situation. These responses are instinctive, not cognitive – the ant doesn’t make conscious decisions. The range of actions it can make in response to a stimulus is governed by its genes. The problem for researchers was identifying exactly which ant gene, or combination of genes, was vital for specific responses.

  In 2017, scientists at Rockefeller University in New York were able to do exactly the experiments they wanted. They suspected that a particular gene was vital for communication in clonal raider ants. They tested their hypothesis by editing the gene and stopping it from working, after which they examined the ants’ behaviour. You actually feel sorry for the little creatures. They couldn’t follow the trails left by other ants so were always wandering off and getting lost. Even when they managed to stay with other colony members, they were hopeless at following social clues and so also became isolated.1 They were like that kid in all our childhood school memories, the one who was always picked last for sports and inevitably got lost on the field trips.

  The gene editing of the clonal raider ants converted the high school jock into the chess club geek. So perhaps it’s no surprise that the technology has also been used to investigate the glamorous nature of the prom queen of the insect world.

  Butterfly minds

  There are nearly 180,000 species in the insect order Lepidoptera. About 10% of these are butterflies, and the rest are moths. In the battle for the popular vote, butterflies are right up there with ladybirds in the public’s list of insects it actually likes. It’s hard not to love butterflies – they don’t bite us, they don’t destroy our crops (at least, not in their adult stages) and many of them look gorgeous. They come in vast numbers of patterns and colours, often extraordinarily flamboyant and beautiful. But the vast range of wing patterns and colours that allow us to distinguish butterfly species so readily creates a rather strange conundrum. How can such enormous diversity in appearance occur when all the species use basically the same genes? Attempts to answer this have been stymied because it was very difficult to perform genetic experiments on butterflies. But that’s all changed with the latest generation of easy-to-use gene editing technology.

  A group at Cornell University was interested in a specific gene which had been implicated in the development of wing colour pattern. This gene was identified through a variety of laborious studies over many, many years. But even though the researchers thought this gene was important, they had hit a wall in how they could test this conclusively. But along came the new gene editing technologies, and suddenly it was playtime for curious lepidopterists.

  The researchers used the new approaches to disrupt the expression of the gene in four different species of butterflies. The resulting butterflies lost the natural red colours in their wings, and replaced them with black. The scientists inferred that the gene they were investigating acts as a switch, controlling whether the butterfly’s cells produce a coloured pigment or the black pigment, melanin. They found consistent results in different species of butterflies that had diverged over 80 million years ago, suggesting that this system of controlling whether the wings are colourful or dark is a remarkably fundamental one.

  But in at least one species, the gene modification experiments showed that this gene has other roles as well. One of the reasons that butterflies were much more popular with collectors in the 19th century than other equally colourful insects such as dragonflies is because of the way some of their most dramatic colour patterns are created. The bright jewel-like colours of dragonflies are usually generated by pigments, specific protein molecules in the cells. After death, these proteins break down. When that happens, the organism’s colours fade, reducing the originally resplendent insect to a dull shadow of its living self. The same is true of some species of butterflies. After collection they may fade like a painting left in bright sunlight.

  But some of the most dramatic butterfly species create colour in a different way. Instead of using pigments, the scales on their wings are extraordinarily complex in their physical structure. These structures influence how the scales interact with light, bending the rays to create astonishing colours, such as an insanely vivid blue. This is known as iridescence and is an example of structural colouration. Because it’s dependent on the physical structure of the scales, and not the presence of pigments, it doesn’t break down and fade after death. There are iridescent butterfly specimens in museum collections that were collected well over a century ago and are as startlingly bright and vivid as the day they were first netted, killed and pinned. It was this permanent vividness that made them highly prized among collectors.

  The team from Cornell were astonished when they discovered that in one of the four butterfly species they tested, the genetic modification of the test gene didn’t simply result in a transition from coloured to black. In the buckeye butterfly, the brown and yellow colours in the wings were replaced by brilliant iridescent blue. No one had anticipated this outcome. It suggests that the target gene normally has two effects. It represses the production of melanin and it prevents the development of the structural features that create iridescence.

  How can disrupting the expression of one gene have such dramatically different effects in closely related species? It’s likely that the target gene controls multiple other genes that work together to influence colours in the different species. The researchers examined which genes are active in the normal and modified butterflies and identified various candidates that may be responsible for the final effects. They weren’t able to test their hypotheses in the original paper,2 but it’s a fairly safe bet to speculate that they are now using the gene editing techniques to explore further.

  You can wait half your life for a butterfly molecular genetics paper to appear, and then two are published at once. Released back-to-back in the same journal, the second paper was written by biologists from seven different universities, spread across the US and the UK. They also used gene editing technology to investigate wing patterns and colours in butterflies, but they were interested in a different gene
from the Cornell group.3 They used the latest technology to inactivate the relevant gene in seven different species of butterfly. This inactivation led to easily identified changes in the marking patterns on the wings. The authors were able to conclude that in a single species, this gene is responsible for development of patterns in different regions of the wing. This creates the specific design of stripes, spots and blotches that are so diagnostic of the different species. In a beautiful comment, one of the senior authors described this gene as acting like a sketching tool that draws the patterns on the wings, whereas the gene analysed by the Cornell group is the paintbrush that fills in the colour.4

  They also showed that the sketching gene is responsible for the different complex patterns that occur naturally when comparing species. This implies that the target gene operates subtly differently in different species, possibly because of slight variations in other genes that it influences. This model of interacting subtle differences is consistent with an evolutionary theory which posits that huge inter-species variation can be driven by apparently minor alterations in sets of interacting genes.

  The data published in the two papers generated quite a bit of interest in the popular press because just about everyone likes butterflies. The work has also given evolutionary biologists a real fillip, as it helps to explain some of the astonishing diversity in the insect world. But the most startling aspect is really the fact that these kinds of experiments have become feasible, and incredibly quickly. As one of the senior authors pointed out, in a statement that seems to combine awe and a slight sense of personal redundancy: ‘These are experiments we could only have dreamed of years ago. The most challenging task in my career has become an undergraduate project overnight.’5

  Salamander secrets

  The axolotl is a lovely creature, one that makes you happy when you look at it. It’s an amphibian, a member of the salamander family, and it has one of those faces that seems to be smiling. Even though we know that our response is completely anthropomorphic, it’s very hard to resist smiling back.

  The axolotl is in a very odd situation, in that it is critically endangered and yet there are millions of them on the planet. This is because it has almost been wiped out in the wild, but is thriving in captivity. One of the reasons there are so many axolotls is because they make rather cute, and easy to keep, pets. The other reason is that they have powers of regeneration that seem almost miraculous to us humans. And that has made them a very popular model organism among scientists.

  If a human loses their little toe, or part of an earlobe, or the tip of their nose, it’s gone for ever. An axolotl can lose an entire limb and it cares not a jot. It can regrow it in about a month and a half. No mammal or bird can do anything like this. For both curiosity and for potential medical improvements, we’d love to know how these adorable little salamanders pull this trick. And we’d like to know if we can adapt their abilities in order to improve human regenerative medicine. This is becoming an area of intense focus, because of the ageing human population. Many of our tissues didn’t evolve to keep going for the long periods for which many of us now live. Medical science isn’t trying to help us grow new limbs, just to improve the functions of worn-out body parts. Creaking knees, agonising hips, arthritic fingers – we’d love to be able to improve their function without surgical intervention, perhaps by encouraging rejuvenation of tired tissues like old cartilage and bone. Perhaps we can learn from the regenerative talents of the axolotl.

  Once again, the new techniques in gene editing are enabling scientists to optimise how they use the axolotl as an experimental system. It’s easy to use the technology to change the DNA of axolotls and to investigate which genes and processes are crucial in the regenerative process. It also helps that axolotls produce enormous eggs, making it very easy to introduce the gene editing reagents into the organism in the first place. Using this approach, researchers have already shown that a specific gene, in a select population of cells, is absolutely critical for creating new muscle when the axolotl grows a new limb.6

  No one is expecting that these experiments will lead to complete limb regeneration in humans any time soon. The barriers are too high and the complexity too great to make this likely in the lifetime of anyone reading this book. Dr Curt Connors in Spiderman – aka The Lizard – is not on anyone’s therapeutic horizon.* But axolotls can also regenerate their spinal cord after severe injury and this is a much more appealing regenerative medicine opportunity.

  Genetic modification has been used to probe the importance of specific genes in spinal cord regeneration in axolotls.7 The hope is that eventually this will lead to a detailed understanding of how the axolotl repairs this vital tissue, and which parts of the process are missing/acting differently in humans. It is perfectly feasible that we may be able to use this knowledge, and similar genetic modification techniques, to modify the behaviour and actions of the nerve cells and associated tissues in human spinal trauma patients. A gap of just a few millimetres in the human spinal cord can create lifelong paralysis and disability. It’s not ridiculous to hope that we will be able to bridge this gap within the next couple of decades.

  When Sally Met Sally, and Harry Met Harry

  To create a new baby, you need a man to supply a sperm cell and a woman to supply an egg. It’s the basic requirement. This may happen in the traditional way, or in an IVF clinic where it is followed by culturing the developing embryo in the laboratory before implanting it into a woman’s uterus. But however it’s done, there’s got to be a sperm and an egg. Of course.

  Of course. One of the most belligerent phrases in science. Because if asked ‘why’ at certain times in a scientific field, there are usually two possible responses. The first is ‘because’. This is generally not considered helpful. The second is ‘I don’t know but I am going to find out’. This is generally more useful, but often only a select few have the imagination to say it, and to find a way of making good on their claim.

  In the 1980s, Azim Surani did exactly this at the University of Cambridge. He is a quiet, soft-spoken man who revolutionised our understanding of mammalian reproductive biology. Professor Surani wanted to know why mammals can only reproduce if both a sperm and an egg are involved in the process. After all, lots of other animals, from stick insects to Komodo dragons, have no such absolute barrier. Their females don’t have much trouble producing young without a daddy. So what’s so special about mammals?

  Azim Surani’s experiments were so beautifully elegant that one can almost forget how mind-blowing they were. He used the techniques of IVF, but working in mice rather than humans. Here’s basically what he did. He obtained mouse eggs and removed the nucleus. Then he injected other nuclei into the ‘empty’ eggs. In some eggs he injected two egg nuclei. In others he injected two sperm nuclei. In the third set he injected one nucleus from an egg and one from a sperm. Then he cultured the manipulated eggs.

  The nuclei fused in all three experimental conditions. Once they are inside an egg, two sperm nuclei or two egg nuclei can get together just as effectively as an egg and sperm. Professor Surani implanted the various developing embryos into female mice, only one type of embryo into each female. Then he waited. The females that had received embryos created from an egg and a sperm gave birth to healthy live young. The ones that had received sperm-only or egg-only embryos didn’t have any pups. When he retrieved these embryos from the females into which they had been implanted, he found that there had been some development but it had gone haywire.

  We might think this just told us what we already knew – you need an egg and a sperm to create a mammal. But there was a fabulous detail in the way these experiments were designed that told us so much more. Researchers have had access to genetically identical mice for decades now, as a result of extensive tightly controlled inbreeding programmes. Azim Surani took advantage of this when he carried out his experiments. In all three of his experimental conditions he used exactly the same strains of mice. The DNA in the egg nuclei was exactl
y the same as the DNA in the sperm nuclei. At a genetic level, there was no difference at all between the three experimental situations, and yet the outcomes were entirely different. So much for DNA being the sole arbiter of destiny.

  Professor Surani had demonstrated that mammalian reproduction relies on the inheritance of something else beyond DNA. He provided preliminary evidence that this ‘something else’ is a set of chemical additions to DNA, which are referred to as epigenetic modifications. At certain key positions in the genome, DNA is differentially tagged with these modifications, depending on whether the copy was inherited from the egg or the sperm. Having the right balance of these is critical. In the experimental situation where there were two eggs or two sperm, this balance was wrong and it adversely affected the development of the embryo.8

  These epigenetic modifications don’t have the same role in non-mammalian species, which is one of the reasons why Komodo dragons and other parthenogenetic organisms are able to reproduce without any input from sperm. But these chemical modifications are vitally important in all placental mammals, including humans, in which there are around 100 of these critical regions.

  There was extensive media interest in this field in 2018 when a group from Beijing used the latest gene editing technology to break through this reproductive barrier in mice. They were able to remove specific regions from the mouse genome, which would normally carry the additional epigenetic information. Depending on the regions they deleted, they were able to produce live mice which had two mothers or two fathers.9 The pups with two genetic mothers were even able to mature and have offspring of their own, but the pups with two fathers didn’t survive to adulthood.