Hacking the Code of Life

by Nessa Carey

  Back in 2002, Oxitec bred their first Friendly Mosquitoes. These mosquitoes have been genetically modified to contain a suicide gene. When it’s activated, this suicide gene disrupts the activity of the insect’s cells, and causes death. Sensibly, the company did not call their genetically modified beasties anything stupid like ‘Suicide Mosquitoes’. The last thing that a company selling a gene-modified product needs to do is to give that product a frightening name.

  The company now produces these modified mosquitoes by the millions, by breeding them in the laboratory. These insects have been released into the wild in a number of locations where there have been outbreaks of relevant diseases. For example, 8 million have been released in a specific area of the Cayman Islands.

  The mosquitoes that are released in such huge numbers are all males. Once they are flying free they do what male mosquitoes always do. They try to find females with whom to mate. Assuming they are successful, all their offspring contain the suicide gene. The expression of this gene leads to the accumulation of a lethal toxin, and the offspring die at the immature larval or pupal stages. The results in the Cayman Islands trial have been very encouraging. After repeated releases in the wild, the numbers of eggs detected over a season dropped by 88% and the numbers of virus-carrying mosquitoes fell by 62%.

  These genetically engineered little insects are a remarkably elegant technological solution, for a number of reasons. In addition to the suicide gene, the mosquitoes also pass on a gene that codes for a particular fluorescent protein. Researchers in the field can use the fluorescence to identify specimens that have inherited the modified genetic material. The suicide gene itself has been engineered into the genome as part of a positive feedback loop. Once the suicide gene is switched on, it drives up increased expression of itself. This means the toxin reaches lethal levels very quickly.

  The most beautiful part of the technology is the bit that solves a quite basic problem. If the suicide gene is lethal, why aren’t the males killed by it before they reach adulthood and are released into the wild to ruin the dreams of motherhood of their lady friends? The reason is because the company that breeds the millions of males is able to control what they eat. They supplement the food with an antibiotic called tetracycline. This binds to the suicide gene and switches it off. Tetracycline isn’t found in the natural world, and so the gene is only switched on in the males after release. But the pre-existing repression lasts long enough for the males to find a female and mate with her. The offspring inherit the suicide gene but can’t switch it off because there is no tetracycline in their food. And so they die, as a result of their own lethal genetic inheritance.

  There is much to admire about this technology. It has the potential to cut down on use of chemical insecticides that are often unfortunately promiscuous in the insects they target. In anti-mosquito campaigns that use chemicals it can be difficult for humans to find all the tiny little reservoirs of stagnant water that the female mosquitoes love so much. But this isn’t an issue for the genetically modified males – 100 million years of evolution have made them masters of this activity. Oxitec’s technology is targeted at just one species of mosquito, so it won’t affect others that don’t carry diseases. In the Cayman Islands the species that has been targeted is one that didn’t occur there originally. It was accidentally imported by human actions. The technology is self-limiting – once the released males and their poisoned offspring have died, the suicide gene disappears from the population. All of these factors minimise the disruption to the ecosystem.

  Driving to extinction

  With the development of the latest gene editing techniques, it’s possible to devise even more sophisticated approaches to control of mosquitoes and other insect pest species. These can also be developed and implemented much faster than the kinds of technology that Oxitec was dependent upon when it created the Friendly Mosquito.

  Research conducted at Imperial College in London has created a fascinating model for this.7 The group worked on a mosquito species that is very common in sub-Saharan Africa and is a major carrier of malaria. They used gene editing to create something very odd, which is rarely seen in nature. Essentially, they subverted one of the fundamental principles of genetics.

  Like us, mosquitoes have two copies of most genes, one inherited from the mother and one from the father. When a male mosquito produces sperm, only one copy of each gene enters each sperm. A similar situation occurs in the female when she creates eggs. When the egg and sperm fuse to start a new individual, the double dose of each gene is restored.

  Let’s generate a hypothetical male mosquito and pick a random gene, which we’ll call RANDOM. Let’s assume the RANDOM gene comes in two colours, maybe red and yellow, and our imaginary little buzzing pest contains one of each colour. When our hypothetical male mosquito produces sperm, half of them will contain the RANDOM-red version and half will contain RANDOM-yellow. We will also expect that half of his offspring will inherit the RANDOM-red and half will inherit RANDOM-yellow. It’s just the law of averages in action.

  Now let’s imagine that RANDOM-red is quite a rare version of the RANDOM gene. Maybe only one out of ten mosquitoes possesses a red copy. If these ten mosquitoes have 100 offspring each, only 50 in every 1,000 of the next generation possess RANDOM-red. The chances are that RANDOM-red will never reach a high level in the subsequent generations, because it will keep being swamped out by the RANDOM-yellow versions. It’s the law of averages again.

  But what if we could influence the roll of the genetic dice, so that RANDOM-red is over-represented in each generation, and spreads to a really high level? Normally this could only happen if RANDOM-red gave the mosquito that contained it a strong selective advantage over its RANDOM-yellow competitors. That’s essentially what the team at Imperial College achieved. They found a way of favouring the transmission of one version of a key gene over another. This meant that they were able to accelerate the speed with which this version spread through a mosquito population, pushing its level up far beyond that predicted by the law of averages. This phenomenon is known as gene drive.

  The scientists achieved this by using gene editing. They created mosquitoes where one copy of a key gene had been altered in a very clever way. They introduced a whole gene editing cassette into a specific part of the mosquito, on just one copy of a selected gene. When the mosquito bred, it passed on the gene editing cassette to 50% of its offspring.

  The gene editing cassette was designed so that it would be activated at a certain point in the development of the offspring. Once it was activated, it would reach out and cut the version inherited from the other parent, and then convert it to the same version as itself. Essentially, it’s like RANDOM-red converting the RANDOM-yellow gene. The resulting mosquito may start life with one each of the red and yellow versions of the genes, but as it develops they all shift to red.

  Once this happens, the mosquito population has higher levels of the edited version of the key gene than we would expect. Gene drive has started.

  There was another trick up the researchers’ lab coat sleeves. The gene they altered was a really odd one, called doublesex. Mosquitoes with one normal and one edited version of doublesex develop just fine. But once a mosquito has two edited versions, things get weird. 50% of these individuals develop as perfectly healthy and fertile males. But the other 50% develop as very messed up females, with a strange mixture of male and female reproductive organs. They are infertile and can’t produce eggs. Because they don’t produce eggs, they don’t need to feed on blood, so this immediately makes them less dangerous to humans as disease vectors.

  This version of gene editing therefore has multiple benefits in control of mosquito populations. The females don’t feed on blood and they are infertile, and the edited gene which leads to this female infertility spreads through a population much faster than normal.

  A secure mosquito breeding colony was created, containing 300 normal males, 150 normal females and 150 males who possessed one normal doubl
esex gene and one edited one. It was predicted by mathematical modelling that the spread of the edited doublesex gene, and its consequent effects on fertility, would result in collapse of the colony in nine to thirteen generations. In a number of independent repeats of this experiment, the actual results were always within the limits of the mathematical projections.

  These results don’t necessarily guarantee that such dramatic effects will be seen in the natural world. There might be unsuspected weaknesses in the mosquitoes that carry just one edited version of doublesex, which only become apparent in the rough and tumble of complex competitive environments. Extensive field trials lie ahead, but it is very likely that this approach will be adapted for other pest insect species as well.

  Does ‘could’ mean ‘should’?

  Using gene drive to obliterate mosquito species is an example of scientific intervention at an ecosystem level. Worryingly, attempts to control undesired species in this way have frequently highlighted the phenomenon of unintended consequences.

  The widespread over-use of the pesticide DDT, from the 1940s to the 1970s, led to environmental near-catastrophe. DDT was very promiscuous in action, killing vast numbers of insects from multiple species, distorting food webs disastrously, and leading to collapses in bird populations, especially of the raptors at the top of the avian food chain.

  More recently, the neonicotinoid class of pesticides has been implicated in the huge declines in numbers of pollinating insects such as bees. The European Food Safety Authority now controls the use of these compounds very strictly.8

  It’s not just chemicals that have caused problems when we have introduced them into the environment. In 1935, 3,000 cane toads were released into Australia to control cane beetles which were damaging sugar cane crops. The toads were native to South America, but turned out to be supremely well suited to their new home. They are poisonous to everything that might eat them, and there’s a huge number of Australian invertebrates that they love to eat themselves. Ironically, cane beetles are not one of them. There are now millions of cane toads in Australia, and they are undermining many fragile and unique ecosystems.9

  There have of course been real successes, especially in the control of invasive species. The introduced and rampant prickly pear cactus in Australia was brought under control by the introduction of a species of moth that found it irresistible.10 In the mid-20th century, nearly half a million acres of US farmland were overrun with St John’s wort, a plant that had never occurred naturally on that continent. This has all but disappeared now, thanks to the introduction of beetles from Australia.11

  The problem is that we often only get the full picture about ecosystem-scale consequences after we have made our intervention. If gene editing is used to cause population crashes in mosquitoes, what might the consequences be? Will we see major drops in the numbers of their predators such as dragonflies and bats? Will this allow other species of mosquitoes or other insects to expand their range into newly-vacated territories, bringing other and different disease vectors with them? Certain species of bats are significant pollinators of plants (if you like tequila, you need to thank a bat that pollinates the agave plant), so disruptions in bat populations may have unanticipated knock-on effects for important food crops.12

  Of course, your perspective on this is likely to be influenced by where you live and the diseases to which you are exposed. If you’re a resident of a temperate region, a potential collapse in bat numbers is likely to matter more to you than if you live in the tropics and have lost family members to malaria.

  The attraction of the gene drive technologies that have been enabled by gene editing is the way they spread rapidly through a population after just a single introduction. This is why certain funders are investing large sums of money in this field. The Bill and Melinda Gates Foundation has invested $75 million in these technologies, and DARPA, the US Defense Advanced Research Projects Agency, has poured in $100 million. But it’s the very rapid spread and persistence of the gene drive approach that perhaps should worry us most. Once they’re out there in the wild, it will be very difficult to put the gene-edited mosquitoes back into the test tube.

  Driving out our furry friends (or foes)

  We humans tend to create ecological havoc even when we don’t mean to. Our tendency to hack everything around us is probably equalled only by our obsession with looking around the next corner; beyond the next bend in the river; over the horizon. The history of humans has been one of travel and exploration, and we very rarely made these trips unaccompanied. Rodents in particular have been frequent stowaways on our vessels and have spread throughout the globe with terrifying speed.

  Remote isolated regions are particularly vulnerable to invasive species. Animals in these areas, and especially on islands, have evolved with few defences – behavioural or otherwise – to these invaders. Time and again, we have seen island populations devastated by introduced mammals. The seabirds on the remote Scottish Shiant Isles were suffering major predation from rats. This has now been brought under control by luring the rats into traps via the endearingly irresistible low-tech approach of chocolate powder and peanut butter.13 Four years of major airdrops of poisoned bait have finally rid the island of South Georgia of the rats and mice that have played havoc with its local bird population, including two species found nowhere else in the world.14

  While these successes are very welcome, there are situations where other methods than poisoning and trapping are required. Such traditional approaches are unfortunately only appropriate for geographically isolated regions, and where there are no native species that might also be affected by the toxic bait. We need alternative techniques that can be employed safely to control invasive vertebrates in other situations.

  It was obvious that researchers would quickly recognise that gene editing would allow them to design and test gene drive mechanisms with unprecedented speed. A team at the University of California, San Diego has used gene editing to create laboratory mice which contain a gene drive mechanism. They didn’t try to generate a lethal gene drive – they were just exploring if the principle would work, so they came up with a gene drive that changes the colour of the mouse’s fur. If the gene drive worked as expected, the numbers of white mice in their colonies should increase at a higher rate than in the non-edited population.

  Disappointingly for the scientists involved, they found that the white coat colour didn’t spread rapidly through the population when the mice bred. The numbers of white mice were much lower than hoped. The edited version of the gene didn’t spread at the rate that had occurred in the mosquito gene-drive experiments. It spread especially poorly from males, suggesting a particularly troubling hurdle during sperm production. The authors concluded: ‘It therefore appears that both the optimism and concern that gene drives may soon be used to reduce invasive rodent populations in the wild is likely premature.’15

  It’s inevitable that we will see many more attempts to create gene drives to control invasive species. The new gene editing technologies make it so much easier to create these weird genetic payloads and this will stimulate research into the field. This may also be taking place at a time when there is a renewed political will in certain parts of the world to tackle the problems caused by invasive species. New Zealand has launched an initiative called Predator Free 2050. The stated aims of this are ‘eradicating New Zealand’s most damaging introduced predators: rats, stoats and possums’.16 The focus at the moment is on trapping and other traditional methods. However, it wouldn’t be surprising if gene editing to create lethal gene drives is also employed as a weapon in the armoury.

  You may have noticed an animal missing from the hit list of New Zealand predators. There are about 1.5 million cats in New Zealand and the environmental toll of these is probably immense. A study in the US suggested that free-ranging cats there kill billions of prey items every year.17 But any governmental agency in any country that has tried to limit cat numbers has usually been met with extraordinary level
s of hostility and opposition. In pest control, as in most other areas of human activity, it appears we humans find it very hard to give up our belief in our dominion over our fellow denizens of the planet.

  Notes

  1. Reference to the King James Bible, Genesis 1:26: ‘And God said, Let us make man in our image, after our likeness: and let them have dominion over the fish of the sea, and over the fowl of the air, and over the cattle, and over all the earth, and over every creeping thing that creepeth upon the earth.’

  2. https://www.theguardian.com/environment/2015/sep/26/snakebites-kill-hundreds-of-thousands-worldwide

  3. https://www.gatesnotes.com/Health/Most-Lethal-Animal-Mosquito-Week

  4. http://www.who.int/en/news-room/fact-sheets/detail/malaria

  5. http://www.mosquitoworld.net/when-mosquitoes-bite/diseases/

  6. https://www.oxitec.com/friendly-mosquitoes/

  7. Kyrou, K., Hammond, A.M., Galizi, R., Kranjc, N., Burt, A., Beaghton, A.K., Nolan, T., Crisanti, A. ‘A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes’. Nat. Biotechnol. (24 September 2018); doi: 10.1038/nbt.4245.

  8. https://www.efsa.europa.eu/en/press/news/180228

  9. http://www.invasivespeciesinitiative.com/cane-toad/

  10. http://biology.anu.edu.au/successful-example-biological-control-and-its-explanation

  11. https://biocontrol.entomology.cornell.edu/success.php

  12. http://www.bats.org.uk/pages/why_bats_matter.html

  13. https://www.telegraph.co.uk/news/2018/03/02/remote-scottish-islands-declared-rat-free-rodents-lured-captivity/

  14. https://www.smithsonianmag.com/smart-news/after-worlds-largest-rodent-eradication-effort-island-officially-rodent-free-180969039/