Radical Eradication: A (Population) Crash Course in Genetic Engineering

Malaria is a life-threatening blood disease that plagues nearly two-thirds of the world’s population. The disease in manifested by parasites of the Plasmodium genus and transmitted to humans through the bite of female Anopheles mosquitoes, which serve as the primary disease vectors. Roughly 200 million people per year are infected with malaria, and approximately 400,000 deaths are reported annually, with children under the age of five comprising the majority of victims.

Africa disproportionately bears the global brunt of this devastating illness, with approximately 92% of all reported cases, as well as 93% of all reported deaths, originating from the continent. This can be partially attributed to the fact that the conditions for transmission are essentially ideal there: the principal vector species Anopheles gambiae are abundant in this region, and not only do they prefer to source their blood from humans over animals, but the mosquitoes also tend to have a longer lifespan, which allows the most common and deadly malaria parasite, Plasmodium falciparum, to complete its life cycle, which contributes to higher disease transmission efficacy.

Though malaria is a preventable disease, often the areas affected most lack access or resources, or are politically unstable, all factors that can contribute to the absence of consistent, functional malaria control programs. Though malaria is also a curable disease, it has long been debated whether eradication was even within the realm of possibility. There are four species of Plasmodium parasites responsible for the pathogenesis of malaria and each exhibit different forms of drug resistance and each responds differently to different medications. This alone makes the prospect of developing a single overarching vaccine for all strains of malaria an improbable achievement and the idea of eradication practically impossible.

A CRISP[E]R APPROACH

In a study recently published in Nature Biotechnology, a team of scientists were able to effectively implement a new, though indubitably controversial, type of genetic modification. The team was able to weaponize mosquitoes to take out…other mosquitoes! They were able to engineer male mosquitoes to rapidly pass down a fatal mutation through generations of their own species, effectively sterilizing all female offspring, eliminating the possibility of successful reproduction and resulting in a population crash.

Utilizing the dynamic technology known as CRISPR, the London-based team was able to genetically modify male mosquitoes of the malaria-transmitting species A. gambiae  with gene drive. The concept of gene drives and the idea to engineer them as a means of controlling insect populations that spread disease is not novel, however, up to this point in recent history, the technology to bring these ideas into reality has simply not existed.

In typical sexual reproduction, there are two possible versions of any given gene that the offspring can inherit, one from each parent. According to the traditional rules of genetics, there is then a 50% chance of the offspring inheriting either version of the gene. Gene drives completely negate these rules, and result in the rapid transmission of the desired engineered modification to almost all of the offspring.

The gene drive in this particular study selected a specific region of what is known in insects as the “doublesex” gene, which controls sexual determination and development. The region that they targeted would specifically impact female phenotypic sexual characteristics.

Female mosquitoes who initially have only one copy of the mutation would still present as healthy and normal, in terms of looks and behavior, and as a result were still reproductively able to continue spreading the mutation. Females born with two copies of the mutation would be phenotypically impacted, resulting in male-mouthed females that had lacked typical reproductive organs. These altered characteristics would render them sterile, unable to bite and by extension, unable to spread the malaria parasite.

For this research, two cage trials were run concurrently, utilizing 300 wild-type females, 150 wild-type males and 150 genetically-modified males. The aim of this experiment was for the gene drive to push the inheritance of this modified doublesex gene through multiple generations until the mutation reaches 100% prevalence in female offspring, leading to a population crash due to the inability to reproduce.

In the span of about six months, the team was able to achieve population crashes in both cage trials, as the populations reached 100% mutation frequency at generation 7 and generation 11, respectively.

GOING AU NATURALE 

Building upon the success of the initial study, a new phase of study, launched in Terni, Italy at the beginning of February, will get to test the model on a larger scale and under conditions designed to replicate as close to a natural environment as possible. Through this environmental imitation, the idea is for the surroundings to encourage natural behavior, thus providing more accurate insight into what the results of a gene drive release might look like if it were unfolding in their natural hot, humid habitats.

To set the stage for this experiment in the lab, the team of scientists are utilizing six huge cages that will each contain individual populations of A. gambiae mosquitoes. Every wall of each cage is lined floor to ceiling with mosquito netting, and each cage is supplied with stacks of hollow cylinders comprised of moist clay for the mosquitoes to utilize as shelter and heated canisters of cow’s blood, to help the specimens simulate drawing blood from a live animal.

Photo Credit: Firkin

Each chamber is additionally equipped with large black boxes with white backgrounds, and computer-regulated lighting systems. The boxes and their backgrounds play an important role in this experiment, as they are meant to mimic swarming, a key part of the mosquito mating ritual, and the controlled lighting is critical in simulating day and night cycles, particularly sunset as that is the typical time mating occurs.

To initiate the study, lab technicians methodically introduced small glass dishes to each mosquito-infested chamber. These glass dishes contained dozens of pupal-stage modified mosquitoes, which will quickly develop and assimilate into the already-thriving populations of the hundreds of normal mosquitoes that inhabit each cage. Two of the cages received an amount of modified insects equivalent to 25% of the normal population, another two received an amount of modified mosquitoes equivalent to approximately 50% of the normal population and the final two received no modified mosquito pupals and will serve as control populations.

Scientists will be collecting thousands of eggs from the chambers every week to monitor the spread of the lethal sterilizing mutation throughout each model population, and hoping that after about six months to a year, they will be able to see the effects of the engineered mutation play out.

EXAMINING EXPERIMENTATION EFFORTS

At least one thing has been made clear from these studies. The gravity of this situation – with just how controversial this technology and the implication of its successes in the laboratory is – is emphatically not lost on any of the scientists involved with these projects, which is a good indicator that this technology is in the best possible hands

As with most new and groundbreaking technologies, CRISPR carries the weight of great power but those who use it must also bear the baggage of great responsibility. Part of that responsibility is the thorough examination of the pros and cons and meticulous analysis of every “what if”.

When it comes to the decision to move this experimentation out of the lab and into real world applications, there is obviously some rightful pushback. This is not a topic to be taken lightly, and there is a very real possibility that we could see severe consequences, should anyone decide to continue full-speed ahead without evaluating all the possibilities.

It’s almost too easy to build a case against the advancement of these genetic engineering mechanisms simply by looking at the magnitude of all the unknown factors. Because there really is no precedent for this applied technology, there seem to be innumerable potential risks no matter which way you slice it.

From a con point of view, let’s say we were to press on with this particular experimentation in real-world environments. The potential eradication of the A. gambiae mosquitoes using this technology could present a variety of plausible undesirable effects. Delicate ecosystems could be thrown out of whack. The technology could stretch beyond the desired species and inadvertently lead to the destruction of non-disease-transmitting mosquitoes, or even the destruction of important pollinators.

Furthermore, what if the population crash of A. gambiae leaves an empty niche in an ecosystem that could be filled by another more problematic disease-peddling species?  And on an even broader scale, what if this gene-editing technology falls into the wrong hands? What if those wrong hands belong to terrorists who use the technology to develop biological warfare agents?

Photo Credit: GDJ

Though all these unknowns are valid questions that undeniably should be posed and thoroughly discussed, its equally important to not get so absorbed in what could go wrong, that we can no longer see what could go right. What if the large scale study is wildly successful? What if we can then apply the study to real life populations, and we eliminate huge populations of malaria-causing A. gambiae, effectively eliminating, if not entirely eradicating, the disease? We could then turn our focus to ending other insect-borne diseases plaguing humanity, such as Zika, Lyme Disease, West Nile, sleeping sickness, and yellow fever. What if we are able to spare millions of people from agonizing, unnecessary suffering?

What if we start to apply the gene drive model to other categories of global-scale issues? We could help endangered ecosystems thrive again, by targeting invasive rodents, fungi and plants. We could clean up harmful oil spills in our oceans utilizing genetically modified bacteria. We could engineer more efficient crops to aide the global hunger crisis, and minimize the need for harmful pesticides and herbicides by modifying bugs to stop consuming crops in the first place.

Depending on your perspective, everywhere you look you could either see potential problems or see potential solutions. Don’t disregard the problems as they arise–welcome them! Sit with them, make them a cup of tea, get to know them better. Just try to keep your eyes on the solutions–the advances, the unveiling of previous mysteries, the cures–as those are what make the pursuit of science worthwhile.

The Secret Fluorescent Life of Flying Squirrels

flying squirrel specimen
A flying squirrel museum specimen under normal light versus ultraviolet light. Photo courtesy of AM Kohler, et al.

In May 2017, a surprising discovery was made in the woods of Bayfield County, Wisconsin, just about a 5-hour drive north of Promega headquarters. Jonathan Martin, Associate Professor of Forestry at Northland College, was exploring the forest with an ultraviolet (UV) light in search of fluorescent lichen or plant life. What he found instead was a bright pink glow coming from a most unexpected source—a flying squirrel.

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It’s Almost iGEM Season—Help Is On The Way!

The 2019 iGEM Competition is on the horizon and team registration opens this month. We’re excited to partner with the iGEM Foundation again this year and offer our support to the young scientists who participate. If you’re starting an iGEM project, there are going to be things you need along the way. We are pleased to share a number of different ways we can help your iGEM team from now until the Giant Jamboree.

Grant Sponsorship

Tell us about your iGEM project and your team could win a 2019 Promega iGEM Grant Sponsorship. Ten winning teams will each receive $2000 in free Promega products to use for their iGEM projects. Tell us about your project—What problem are you addressing? What is your proposed solution? What challenges does your team face? Last year’s winning teams selected from a wide range of reagents and supplies, including master mix, restriction enzymes, ligase, DNA purification kits, expression systems, DNA ladders and markers, buffers and agarose. Click here to apply! Continue reading “It’s Almost iGEM Season—Help Is On The Way!”

Extra extra: Read All About Tautonyms

If you’re active on #sciencetwitter, you may have seen a thread recently about tautonyms. “Tautonym” is a cool word for scientific names where the genus and species are the same word, For example, Vulpes vulpes is the scientific name for the red fox.

I have taken great delight in sharing these tautonyms with friends, colleagues, and random strangers on the bus. However, the problem that I keep having is that people want more details about something than the name. If you’ve had that problem, too, then this blog is for you. Continue reading “Extra extra: Read All About Tautonyms”

Goodbye to the Most Famous Bird in Maine

When Wisconsin plunged into a deep freeze during last week’s polar vortex, I built a roaring fire in my fireplace and settled into my armchair with a thick blanket and a video game controller. Except for the twenty minutes I spent driving to and from the office, I stayed warm and toasty.

Birds, however, don’t have it quite as easy. To survive freezing temperatures, non-migratory birds have developed many interesting adaptations. Many species grow extra down layers and huddle together for wind protection. Others, like the black-capped chickadee, use a process called regulated hypothermia to drop their resting body temperature by as much as 22°F to conserve energy. I’m particularly fascinated by the process of regional hypothermia—many species of ducks and gulls use a countercurrent heat exchange system to keep vital organs warm while letting temperatures fall in extremities.

Birds that aren’t accustomed to cold weather don’t have these adaptations, though. When a bird—or any animal—ends up far outside of its natural habitat, the consequences can be deadly.

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Meet Měnglà Virus: the newest cousin in the Ebola and Marburg virus family tree

Ebola virus (EBOV) and Marburg virus (MARV) are two closely-related viruses in the family Filoviridae. Filoviruses are often pathogenic, causing hemorrhagic fever disease in human hosts. The Ebola outbreak of 2014 caught the world by surprise by spreading so quickly and severely that public health organizations were unprepared. The devastating outcome was a total of over 11,000 deaths by the time the outbreak ended in 2016. Research that provides further understanding of filoviruses and their potential for transmission is important in preventing future outbreaks from occurring. But what if the outbreak comes from a virus we’ve never seen before?

fruit_bat
Měnglà virus was discovered among filoviruses isolated from Old World fruit bats (Rousettus)

All in the viral family

A recent study published in the journal Nature Microbiology provides evidence of a newly identified filovirus species. Using serum samples taken from bats, a well-known host for filoviruses, Yang et al. isolated and identified viral RNA for an unclassified viral genome sequence using next generation sequencing analysis. This new virus genome sequence was organized with the same open reading frames as other filoviruses, encoding for nucleoprotein (NP), viral protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L). This new genome sequence shared up to 54% of the nucleotide sequences for the filovirus species Lloviu virus (LLOV), EBOV and MARV, with MARV being the most similar. Their analysis suggested that this novel virus should be classified within the Filoviridae family tree as a separate genus, Dianlovirus, and was named Měnglà virus (MLAV).

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Twisted CRISPR: A Novel Activation Strategy to Treat Genetically Driven Obesity

Two Is Better Than One

Obese and normal mouse

Redundancy equips us to survive. We have more than one lung or one kidney for a reason—if one organ in a pair gets damaged, we can still manage if the other is functional. At the cellular level, we have two copies of each chromosome in every non-germline cell. Each copy was inherited originally from a single sperm and ovum, which are “haploid” cells. Consequently, there are two copies of any given gene in non-germline “diploid” cells. In many cases, should one copy of a gene be damaged, the cell can still survive with the other, functional copy of a gene. In plants, this redundancy is common, and many plants exhibit polyploidy. In an extreme example of polyploidy, the large (by bacterial standards) but otherwise unassuming species Epulopiscium contains tens of thousands of copies of its genome.

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Microsatellite Instability Symposium at Duke University

On January 23, doctors, scientists and researchers will gather for a symposium about Microsatellite Instability (MSI) at Duke University. During the one-day event, scientists from Duke University and The Ohio State University will share insight into their research on biomarkers, MSI status and GI cancer, Lynch Syndrome, and MSI and DNA mismatch repair deficiency (dMMR).

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Combatting Gun Violence with Synthetic Biology

Imagine you are a high school student living in a community devastated by gun violence and death. In the U.S., this could be one of many communities, but it happens to be Baltimore which had 301 deaths due to gun violence in 2017 (with a per capita rate well above other large cities). Then imagine you were part of an organization within that community that helped you, along with other students, gain knowledge and skills to come up with a viable solution to the problem using synthetic biology.

Baltimore Bio-Crew at the 2018 iGEM Giant Jamboree

This is exactly how the Baltimore Bio-Crew came up with their iGEM project, Coagulance Rx. The Baltimore Bio-Crew decided to tackle this community issue head-on. One team member, Mercedes Ferandes, reflected, “Living in Baltimore City, I have not only witnessed gun violence in front of me, but have had family members and friends die from it. I wanted to try to decrease the amount of deaths by gun violence using iGEM.”

After some research, they discovered that many of the gun deaths were due to blood loss and could have been prevented. The impoverished neighborhoods where this violence occurs lack the resources to provide timely emergency medical treatment. Many of these deaths can be attributed to delayed arrival of emergency response teams—wait times for an ambulance can be over an hour.

Although there were several contributing factors beyond their control, the team wanted to address this problem by focusing on blood clotting and how it could be helpful as a quick temporary treatment for open wounds. This solution could offer a reliable, cost efficient way to save lives by slowing or stopping blood loss until a victim could get medical attention. The team decided to pursue the use of snake venom after coming across some previous iGEM projects that had used it for clotting. Team member Henry Ryles pointed out that the need for snake venom powerful enough to clot blood quickly led them to choose the venom of the Russell’s Viper
(Daboia russelii).

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A Roadmap for PROTAC Development

PROTACs or Proteolysis-Targeting Chimeras are an emerging tool in protein degradation studies, potentially suited to any need involving the removal of a specific protein. These small-molecule chimeras are exciting due to: 1) their target specificity; and 2) their ability to enable target destruction versus target inhibition.

Destruction/Inhibition: Is There a Difference?
An analogy that microbiologists (and wrestlers or anyone that has ever spent time in a locker room shower) would understand, is fungicidal versus fungistatic compounds. A fungicidal compound kills fungus. A fungistatic compound just slows the fungus down a bit.

A small-molecule inhibitor attaches to its target protein, but for how long? What inhibitor testing must be done to determine how long the inhibition lasts?

On the other hand, a small-molecule agent that causes protein degradation first targets the protein of interest, then attaches ubiquitin to that target. Once a protein is marked with ubiquitin, it’s a dead man. E3 ligase must be involved, but if the ubiquitin is added by E3, the end is near. Next stop, Hades.

This ubiquitinated protein is headed to the proteasome and proteins that go there don’t come back. Ubiquitination was called the ‘molecular kiss of death’ when this discovery was awarded the Nobel prize in Chemistry in 2004.

PROTAC components: target protein ligand, E3 ligase and linker.

About PROTACs
PROTACs are degrader molecules composed of three parts: 1) a ligand that is specific for the target protein; 2) a ligand for E3 ligase; and 3) a linker molecule that connects the two ligands. The E3 ligase is one of three enzymes that can add ubiquitin to a cellular component, but only ubiquitins added by the E3 ligase cause targeting to the proteasome (Zoppi et al.).

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