Category Archives: DNA

Beyond just promise, CRISPR is delivering in the lab today

Ian Haydon, University of Washington

There’s a revolution happening in biology, and its name is CRISPR. The Conversation

CRISPR (pronounced “crisper”) is a powerful technique for editing DNA. It has received an enormous amount of attention in the scientific and popular press, largely based on the promise of what this powerful gene editing technology will someday do.

CRISPR was Science magazine’s 2015 Breakthrough of the Year; it’s been featured prominently in the New Yorker more than once; and The Hollywood Reporter revealed that Jennifer Lopez will be the executive producer on an upcoming CRISPR-themed NBC bio-crime drama. Not bad for a molecular biology laboratory technique.

Two of the CRISPR co-inventors, Emmanuelle Charpentier (middle-left) and Jennifer Doudna (middle-right), rubbing elbows with celebs after receiving the 2015 Breakthrough Prize in Life Sciences.
Breakthrough Prize Foundation, CC BY-ND

CRISPR is not the first molecular tool designed to edit DNA, but it gained its fame because it solves some longstanding problems in the field. First, it is highly specific. When properly set up, the molecular scissors that make up the CRISPR system will snip target DNA only where you want them to. It is also incredibly cheap. Unlike previous gene editing systems which could cost thousands of dollars, a relative novice can purchase a CRISPR toolkit for less than US$50.

Research labs around the world are in the process of turning the hype surrounding the CRISPR technique into real results. Addgene, a nonprofit supplier of scientific reagents, has shipped tens of thousands of CRISPR toolkits to researchers in more than 80 countries, and the scientific literature is now packed with thousands of CRISPR-related publications.

When you give scientists access to powerful tools, they can produce some pretty amazing results.

The CRISPR revolution in medicine

The most promising (and obvious) applications of gene editing are in medicine. As we learn more about the molecular underpinnings of various diseases, stunning progress has been made in correcting genetic diseases in the laboratory just over the past few years.

Take, for example, muscular dystrophy – a complex and devastating family of diseases characterized by the breakdown of a molecular component of muscle called dystrophin. For some types of muscular dystrophy, the cause of the breakdown is understood at the DNA level.

In 2014, researchers at the University of Texas showed that CRISPR could correct mutations associated with muscular dystrophy in isolated fertilized mouse eggs which, after being reimplanted, then grew into healthy mice. By February of this year, a team here at the University of Washington published results of a CRISPR-based gene replacement therapy which largely repaired the effects of Duchenne muscular dystrophy in adult mice. These mice showed significantly improved muscle strength – approaching normal levels – four months after receiving treatment.

Using CRISPR to correct disease-causing genetic mutations is certainly not a panacea. For starters, many diseases have causes outside the letters of our DNA. And even for diseases that are genetically encoded, making sense of the six billion DNA letters that comprise the human genome is no small task. But here CRISPR is again advancing science; by adding or removing new mutations – or even turning whole genes on or off – scientists are beginning to probe the basic code of life like never before.

CRISPR is already showing health applications beyond editing the DNA in our cells. A large team out of Harvard and MIT just debuted a CRISPR-based technology that enables precise detection of pathogens like Zika and dengue virus at extremely low cost – an estimated $0.61 per sample.

Using their system, the molecular components of CRISPR are dried up and smeared onto a strip of paper. Samples of bodily fluid (blood serum, urine or saliva) can be applied to these strips in the field and, because they linked CRISPR components to fluorescent particles, the amount of a specific virus in the sample can be quantified based on a visual readout. A sample that glows bright green could indicate a life-threatening dengue virus infection, for instance. The technology can also distinguish between bacterial species (useful for diagnosing infection) and could even determine mutations specific to an individual patient’s cancer (useful for personalized medicine).

Feng Zhang, another co-inventor of CRISPR technology, discussing its safety and ethical ramifications.
AP Photo/Susan Walsh

Almost all of CRISPR’s advances in improving human health remain in an early, experimental phase. We may not have to wait long to see this technology make its way into actual, living people though; the CEO of the biotech company Editas has announced plans to file paperwork with the Food and Drug Administration for an investigational new drug (a necessary legal step before beginning clinical trials) later this year. The company intends to use CRISPR to correct mutations in a gene associated with the most common cause of inherited childhood blindness.

CRISPR will soon affect what we eat

Physicians and medical researchers are not the only ones interested in making precise changes to DNA. In 2013, agricultural biotechnologists demonstrated that genes in rice and other crops could be modified using CRISPR – for instance, to silence a gene associated with susceptibility to bacterial blight. Less than a year later, a different group showed that CRISPR also worked in pigs. In this case, researchers sought to modify a gene related to blood coagulation, as leftover blood can promote bacterial growth in meat.

You won’t find CRISPR-modified food in your local grocery store just yet. As with medical applications, agricultural gene editing breakthroughs achieved in the laboratory take time to mature into commercially viable products, which must then be determined to be safe. Here again, though, CRISPR is changing things.

A common perception of what it means to genetically modify a crop involves swapping genes from one organism to another – putting a fish gene into a tomato, for example. While this type of genetic modification – known as transfection – has actually been used, there are other ways to change DNA. CRISPR has the advantage of being much more programmable than previous gene editing technologies, meaning very specific changes can be made in just a few DNA letters.

This precision led Yinong Yang – a plant biologist at Penn State – to write a letter to the USDA in 2015 seeking clarification on a current research project. He was in the process of modifying an edible white mushroom so it would brown less on the shelf. This could be accomplished, he discovered, by turning down the volume of just one gene.

White Agaricus bisporus mushrooms with no browning are more visually appealing.
Olha Afanasieva/

Yang was doing this work using CRISPR, and because his process did not introduce any foreign DNA into the mushrooms, he wanted to know if the product would be considered a “regulated article” by the Animal and Plant Health Inspection Service, a division of the U.S. Department of Agriculture tasked with regulating GMOs.

“APHIS does not consider CRISPR/Cas9-edited white button mushrooms as described in your October 30, 2015 letter to be regulated,” they replied.

Yang’s mushrooms were not the first genetically modified crop deemed exempt from current USDA regulation, but they were the first made using CRISPR. The heightened attention that CRISPR has brought to the gene editing field is forcing policymakers in the U.S. and abroad to update some of their thinking around what it means to genetically modify food.

New frontiers for CRISPR

One particularly controversial application of this powerful gene editing technology is the possibility of driving certain species to extinction – such as the most lethal animal on Earth, the malaria-causing Anopheles gambiae mosquito. This is, as far as scientists can tell, actually possible, and some serious players like the Bill and Melinda Gates Foundation are already investing in the project. (The BMGF funds The Conversation Africa.)

Most CRISPR applications are not nearly as ethically fraught. Here at the University of Washington, CRISPR is helping researchers understand how embryonic stem cells mature, how DNA can be spatially reorganized inside living cells and why some frogs can regrow their spinal cords (an ability we humans do not share).

It is safe to say CRISPR is more than just hype. Centuries ago we were writing on clay tablets – in this century we will write the stuff of life.

Ian Haydon, Doctoral Student in Biochemistry, University of Washington

Fishing for DNA: Free-floating eDNA identifies presence and abundance of ocean life

Mark Stoeckle, The Rockefeller University

Ocean life is largely hidden from view. Monitoring what lives where is costly – typically requiring big boats, big nets, skilled personnel and plenty of time. An emerging technology using what’s called environmental DNA gets around some of those limitations, providing a quick, affordable way to figure out what’s present beneath the water’s surface. The Conversation

Fish and other animals shed DNA into the water, in the form of cells, secretions or excreta. About 10 years ago, researchers in Europe first demonstrated that small volumes of pond water contained enough free-floating DNA to detect resident animals.

Researchers have subsequently looked for aquatic eDNA in multiple freshwater systems, and more recently in vastly larger and more complex marine environments. While the principle of aquatic eDNA is well-established, we’re just beginning to explore its potential for detecting fish and their abundance in particular marine settings. The technology promises many practical and scientific applications, from helping set sustainable fish quotas and evaluating protections for endangered species to assessing the impacts of offshore wind farms.

Who’s in the Hudson, when?

In our new study, my colleagues and I tested how well aquatic eDNA could detect fish in the Hudson River estuary surrounding New York City. Despite being the most heavily urbanized estuary in North America, water quality has improved dramatically over the past decades, and the estuary has partly recovered its role as essential habitat for many fish species. The improved health of local waters is highlighted by the now regular fall appearance of humpback whales feeding on large schools of Atlantic menhaden at the borders of New York harbor, within site of the Empire State Building.

Preparing to hurl the collecting bucket into the river.
Mark Stoeckle, CC BY-ND

Our study is the first recording of spring migration of ocean fish by conducting DNA tests on water samples. We collected one liter (about a quart) water samples weekly at two city sites from January to July 2016. Because the Manhattan shoreline is armored and elevated, we tossed a bucket on a rope into the water. Wintertime samples had little or no fish eDNA. Beginning in April there was a steady increase in fish detected, with about 10 to 15 species per sample by early summer. The eDNA findings largely matched our existing knowledge of fish movements, hard won from decades of traditional seining surveys.

Our results demonstrate the “Goldilocks” quality of aquatic eDNA – it seems to last just the right amount of time to be useful. If it disappeared too quickly, we wouldn’t be able to detect it. If it lasted for too long, we wouldn’t detect seasonal differences and would likely find DNAs of many freshwater and open ocean species as well as those of local estuary fish. Research suggests DNA decays over hours to days, depending on temperature, currents and so on.

Fish identified via eDNA in one day’s sample from New York City’s East River.
New York State Department of Environmental Conservation: alewife (herring species), striped bass, American eel, mummichog; Massachusetts Department of Fish and Game: black sea bass, bluefish, Atlantic silverside; New Jersey Scuba Diving Association: oyster toadfish; Diane Rome Peeples: Atlantic menhaden, Tautog, Bay anchovy; H. Gervais: conger eel., CC BY-ND

Altogether, we obtained eDNAs matching 42 local marine fish species, including most (80 percent) of the locally abundant or common species. In addition, of species that we detected, abundant or common species were more frequently observed than were locally uncommon ones. That the species eDNA detected matched traditional observations of locally common fish in terms of abundance is good news for the method – it supports eDNA as an index of fish numbers. We expect we’ll eventually be able to detect all local species – by collecting larger volumes, at additional sites in the estuary and at different depths.

In addition to local marine species, we also found locally rare or absent species in a few samples. Most were fish we eat – Nile tilapia, Atlantic salmon, European sea bass (“branzino”). We speculate these came from wastewater – even though the Hudson is cleaner, sewage contamination persists. If that is how the DNA got into the estuary in this case, then it might be possible to determine if a community is consuming protected species by testing its wastewater. The remaining exotics we found were freshwater species, surprisingly few given the large, daily freshwater inflows into the saltwater estuary from the Hudson watershed.

Filtering the estuary water back in the lab.
Mark Stoeckle, CC BY-ND

Analyzing the naked DNA

Our protocol uses methods and equipment standard in a molecular biology laboratory, and follows the same procedures used to analyze human microbiomes, for example.

eDNA and other debris left on the filter after the estuary water passed through.
Mark Stoeckle, CC BY-ND

After collection, we run water samples through a small pore size (0.45 micron) filter that traps suspended material, including cells and cell fragments. We extract DNA from the filter, and amplify it using polymerase chain reaction (PCR). PCR is like “xeroxing” a particular DNA sequence, producing enough copies so that it can easily be analyzed.

We targeted mitochondrial DNA – the genetic material within the mitochondria, the organelle that generates the cell’s energy. Mitochondrial DNA is present in much higher concentrations than nuclear DNA, and so easier to detect. It also has regions that are the same in all vertebrates, which makes it easier for us to amplify multiple species.

We tagged each amplified sample, pooled the samples and sent them for next-generation sequencing. Rockefeller University scientist and co-author Zachary Charlop-Powers created the bioinformatic pipeline that assesses sequence quality and generates a list of the unique sequences and “read numbers” in each sample. That’s how many times we detected each unique sequence.

To identify species, each unique sequence is compared to those in the public database GenBank. Our results are consistent with read number being proportional to fish numbers, but more work is needed on the precise relationship of eDNA and fish abundance. For example, some fish may shed more DNA than others. The effects of fish mortality, water temperature, eggs and larval fish versus adult forms could also be at play.

Just like in television crime shows, eDNA identification relies on a comprehensive and accurate database. In a pilot study, we identified local species that were missing from the GenBank database, or had incomplete or mismatched sequences. To improve identifications, we sequenced 31 specimens representing 18 species from scientific collections at Monmouth University, and from bait stores and fish markets. This work was largely done by student researcher and co-author Lyubov Soboleva, a senior at John Bowne High School in New York City. We deposited these new sequences in GenBank, boosting the database’s coverage to about 80 percent of our local species.

Study’s collection sites in Manhattan.
Mark Stoeckle, CC BY-ND

We focused on fish and other vertebrates. Other research groups have applied an aquatic eDNA approach to invertebrates. In principle, the technique could assess the diversity of all animal, plant and microbial life in a particular habitat. In addition to detecting aquatic animals, eDNA reflects terrestrial animals in nearby watersheds. In our study, the commonest wild animal detected in New York City waters was the brown rat, a common urban denizen.

Future studies might employ autonomous vehicles to routinely sample remote and deep sites, helping us to better understand and manage the diversity of ocean life.

Mark Stoeckle, Senior Research Associate in the Program for the Human Environment, The Rockefeller University

This article was originally published on The Conversation. Read the original article.

To fight Zika, let’s genetically modify mosquitoes – the old-fashioned way

The near panic caused by the rapid spread of the Zika virus has brought new urgency to the question of how best to control mosquitoes that transmit human diseases. Aedes aegypti mosquitoes bite people across the globe, spreading three viral diseases: dengue, chikungunya and Zika. There are no proven effective vaccines or specific medications to treat patients after contracting these viruses.

Mosquito control is the only way, at present, to limit them. But that’s no easy task. Classical methods of control such as insecticides are falling out of favor – they can have adverse environmental effects as well as increase insecticide resistance in remaining mosquito populations. New mosquito control methods are needed – now.

The time is ripe, therefore, to explore a long-held dream of vector biologists, including me: to use genetics to stop or limit the spread of mosquito-borne diseases. While gene editing technologies have advanced dramatically in the last few decades, it is my belief that we’ve overlooked older, tried and true methods that could work just as well on these insects. We can accomplish the goal of producing mosquitoes incapable of transmitting human pathogens using the same kinds of selective breeding techniques people have been using for centuries on other animals and plants.

Technicians from Oxitec inspect genetically modified Aedes aegypti mosquitoes in Campinas, Brazil.
Paulo Whitaker/Reuters

Techniques on the table

One classic strategy for reducing insect populations has been to flood populations with sterile males – usually produced using irradiation. When females in the target population mate with these males, they produce no viable offspring – hopefully crashing population numbers.

The modern twist on this method has been to generate transgenic males that carry a dominant lethal gene that essentially makes them sterile; offspring sired by these males die late in the larval stage, eliminating future generations. This method has been promulgated by the biotech company Oxitec and is currently used in Brazil.

Rather than just killing mosquitoes, a more effective and lasting strategy would be to genetically change them so they can no longer transmit a disease-causing microbe.

The powerful new CRISPR gene editing technique could be used to make transgenes (genetic material from another species) take over a wild population. This method works well in mosquitoes and is potentially a way to “drive” transgenes into populations. CRISPR could help quickly spread a gene that confers resistance to transmission of a virus – what scientists call refractoriness.

But CRISPR has been controversial, especially as applied to human beings, because the transgenes it inserts into an individual can be passed on to its offspring. No doubt using CRISPR to create and release genetically modified mosquitoes into nature would stir up controversy. The U.S. Director of National Intelligence, James Clapper, has gone so far as to dub CRISPR a potential weapon of mass destruction.

But are transgenic technologies necessary to genetically modify mosquito populations?

Examples of successful artificial selection of various traits through the years. In the center is a cartoon of the ‘block’ scientists would like to select for in mosquitoes so they can’t pass on the virus.
Jeff Powell, Author provided

Selective breeding the old-fashioned way

Genetic modification of populations has been going on for centuries with great success. This has occurred for almost all commercially useful plants and animals that people use for food or other products, including cotton and wool. Selective breeding can produce immense changes in populations based on naturally occurring variation within the species.

Artificial selection using this natural variation has proven effective over and over again, especially in the agricultural world. By choosing parents with desirable traits (chickens with increased egg production, sheep with softer wool) for several consecutive generations, a “true breeding” strain can be produced that will always have the desired traits. These may look very different from the ancestor – think of all the breeds of dogs derived from an ancestor wolf.

To date, only limited work of this sort has been done on mosquitoes. But it does show that it’s possible to select for mosquitoes with reduced ability to transmit human pathogens. So rather than introducing transgenes from other species, why not use the genetic variation naturally present in mosquito populations?

Deriving strains of mosquitoes through artificial selection has several advantages over transgenic approaches.

  • All the controversy and potential risks surrounding transgenic organisms (GMOs) are avoided. We’re only talking about increasing the prevalence in the population of the naturally occurring mosquito genes we like.
  • Selected mosquitoes derived directly from the target population would likely be more competitive when released back to their corner of the wild. Because the new refractory strain that can’t transmit the virus carries only genes from the target population, it would be specifically adapted to the local environment. Laboratory manipulations to produce transgenic mosquitoes are known to lower their fitness.
  • By starting with the local mosquito population, scientists could select specifically for refractoriness to the virus strain infecting people at the moment in that locality. For example, there are four different “varieties” of the dengue virus called serotypes. To control the disease, the selected mosquitoes would need to be refractory to the serotype active in that place at that time.
  • It may be possible to select for strains of mosquitoes that are unable to transmit multiple viruses. Because the same Aedes aegypti mosquito species transmits dengue, chikungunya and Zika, people living in places that have this mosquito are simultaneously at risk for all three diseases. While it has not yet been demonstrated, there is no reason to think that careful, well-designed selective breeding couldn’t develop mosquitoes unable to spread all medically relevant viruses.

Fortunately, Ae. aegypti is the easiest mosquito to rear in captivity and has a generation time of about 2.5 weeks. So unlike classical plant and animal breeders dealing with organisms with generations in years, 10 generations of selection of this mosquito would take only months.

Researchers are working out mass rearing techniques for Aedes mosquitoes – their generation time is only 2.5 weeks.
IAEA Imagebank, CC BY-NC-ND

This is not to imply there may not be obstacles in using this approach. Perhaps the most important is that the genes that make it hard for these insects to transmit disease may also make individual insects weaker or less healthy than the target natural population. Eventually the lab-bred mosquitoes and their offspring could be out-competed and fade from the wild population. We might need to continuously release refractory mosquitoes – that is, the ones that aren’t good at transmitting the disease in question – to overcome selection against the desirable refractory genes.

And mosquito-borne pathogens themselves evolve. Viruses may mutate to evade any genetically modified mosquito’s block. Any plan to genetically modify mosquito populations needs to have contingency plans in place for when viruses or other pathogens evolve. New strains of mosquitoes can be quickly selected to combat the new version of the virus – no costly transgenic techniques necessary.

Today, plant and animal breeders are increasingly using new gene manipulation techniques to further improve economically important species. But this is only after traditional artificial selection has been taken about as far as it can to improve breeds. Many mosquito biologists are proposing to go directly to the newest fancy transgenic methodologies that have never been shown to actually work in natural populations of mosquitoes. They are skipping over a proven, cheaper and less controversial approach that should at least be given a shot.

The Conversation

Jeffrey Powell, Professor, Yale University

This article was originally published on The Conversation. Read the original article.

Your Body Can Now Be Run With Computer Programming

Scientists have found a new way through which the cells of our body can be controlled through a proprietary programming language, which could help you from falling prey to diseases. This latest innovation comes from a group of biological engineers at MIT, who have developed a programming language capable of designing complex DNA functions that can further be put in a human being’s cell.

How does it work?

Commenting on the functionality of the latest innovation, Christopher Voigt, a biological engineering professor at MIT revealed that it was more of text-based language used to program a computer. Similarly, the program is then compiled into a DNA sequence, which is then inputted into the cell, and its circuit runs within the cell.

How did they do it?

Verilog, a hardware description language has been used by researchers to make this a reality. Sensors that can be programmed into DNA sequences have been used with specially designed computing elements.

The interesting part lies in the way the program works. The DNA sequences are first programmed into a cell to create a circuit. The customizable sensors then detect the amount of glucose, oxygen, and temperature. What wonders science and technology today can put together is completely inspiring.

Ancient DNA reveals how Europeans developed light skin and lactose tolerance

Food intolerance is often dismissed as a modern invention and a “first-world problem”. However, a study analysing the genomes of 101 Bronze-Age Eurasians reveals that around 90% were lactose intolerant.

The research also sheds light on how modern Europeans came to look the way they do – and that these various traits may originate in different ancient populations. Blue eyes, it suggests, could come from hunter gatherers in Mesolithic Europe (10,000 to 5,000 BC), while other characteristics arrived later with newcomers from the East.

About 40,000 years ago, after modern humans spread from Africa, one group moved north and came to populate Europe as well as north, west and central Asia. Today their descendants are still there and are recognisable by some very distinctive characteristics. They have light skin, a range of eye and hair colours and nearly all can happily drink milk.

However, exactly when and where these characteristics came together has been anyone’s guess. Until now.

Clash of cultures

Throughout history, there has been a pattern of cultures rising, evolving and being superseded. Greek, Roman and Byzantine cultures each famously had their 15 minutes as top dog. And archaeologists have defined a succession of less familiar cultures that rose and fell before that, during the Bronze Age. So far it has been difficult to work out which of these cultures gave rise to which – and eventually to today’s populations.

The Bronze Age (around 3,000–1,000 BC) was a time of major advances, and whenever one culture developed a particularly advantageous set of technologies, they become able to support a larger population and to dominate their neighbours. The study found that the geographical distributions of genetic variations at the beginning of the Bronze Age looked very different to today’s, but by the end it looked pretty similar, suggesting a level of migration and replacement of peoples not seen in western Eurasia since.

One people that was particularly important in the spread of both early Bronze-Age technologies and genetics were the Yamnaya. With a package of technologies including the horse and the wheel, they exploded out of the Russian and Ukrainian Steppe into Europe, where they met the local Neolithic farmers.

Yamnaya skull
Natalia Shishlina.

By comparing DNA from various Bronze-Age European cultures to that of both Yamnaya and the Neolithic farmers, researchers found that most had a mixture of the two backgrounds. However the proportions varied, with the Corded Ware people of northern Europe having the highest proportion of Yamnaya ancestry.

And it appears that the Yamnaya also moved east. The Afanasievo culture of the Altai-Sayan region in central Asia seemed to be genetically indistinguishable from the Yamnaya, suggesting a colonisation with little or no interbreeding with pre-existing populations.

Mutations traced

So how have traits that were rare or non-existent in our African ancestors come to be so common in western Eurasia?

The DNA of several hunter gatherers living in Europe long before the Bronze Age was also tested. It showed that they probably had a combination of features quite striking to the modern eye: dark skin with blue eyes.

The blue eyes of these people – and of the many modern Europeans who have them – are thanks to a specific mutation near a gene called OCA2. As none of the Yamnaya samples have this mutation, it seems likely that modern Europeans owe this trait to their ancestry from these European hunter gatherers of the Mesolithic (10,000-5,000 BC).

Reconstruction of a Yamnaya person from the Caspian steppe in Russia about 5,000-4,800 BC.
Alexey Nechvaloda

Two mutations responsible for light skin, however, tell quite a different story. Both seem to have been rare in the Mesolithic, but present in a large majority by the Bronze Age (3,000 years later), both in Europe and the steppe. As both areas received a significant influx of Middle Eastern farmers during this time, one might speculate that the mutations arose in the Middle East. They were probably then driven to high levels by natural selection, as they allowed the production of sufficient vitamin D further north despite relatively little sunlight, and/or better suited people to the new diet associated with farming.

Another trait that is nearly universal in modern Europeans (but not around the world) is the ability to digest the lactose in milk into adulthood. As cattle and other livestock have been farmed in western Eurasia since long before, one might expect such a mutation to already be widespread by the Bronze Age. However the study revealed that the mutation was found in around 10% of their Bronze Age samples.

Interestingly, the cultures with the most individuals with this mutation were the Yamnaya and their descendents. These results suggest that the mutation may have originated on the steppe and entered Europe with the Yamnaya. A combination of natural selection working on this advantageous trait and the advantageous Yamnaya culture passed down alongside it could then have helped it spread, although this process still had far to go during the bronze age.

This significant study has left us with a much more detailed picture of Bronze Age Europeans: they had the light skin and range of eye colours we know today. And although most would have got terrible belly ache from drinking milk, the seeds for future lactose tolerance were sown and growing.

The Conversation

Daniel Zadik is Postdoctoral researcher in genetics at University of Leicester.

This article was originally published on The Conversation.
Read the original article.

Genetically modified human embryo raises ethical concerns

It all started with a rumour. Then just six weeks ago, a warning rang out in the scientific journal Nature, expressing “grave concerns regarding the ethical and safety implications” of creating the world’s first genetically-modified human embryo.

Then last week, a Chinese group from Sun Yat-sen University, reported that they had, in fact, done it: they had created the first genetically-modified human embryo.

They reported that, in a world first, they had taken “human tripronuclear embryos”, and altered mutant DNA that causes the human disease β-thalassemia, which is life-threatening and affects 100,000 people worldwide.

But one person’s stern warning is another’s delight. The promise of technologies like this – to cure diseases like cystic fibrosis or Huntington’s, or even to remove the BRCA mutation, which dramatically increases a woman’s risk of dying from breast or ovarian cancer – have been exciting biologists for years.

Cut and paste

So what exactly did the Chinese researchers do? And why has it caused such an uproar?

First, the experiments were performed on human embryos. The researchers collected non-viable embryos from IVF clinics. Then they used this non-viability argument as the ethical justification for performing the work. Scientists know that the embryos were not capable of resulting in a human life, because they were tripronuclear. That means one egg had been fertilised by two sperm, a biological situation we know cannot result a live baby.

Into these embryos, the scientists injected “molecular scissors”, known as the CRISPR/Cas9 system, which can target a specific segment of DNA.

In this case, they targeted the HBB gene, which causes β-thalassemia. They then cut out the disease-causing region and replaced it, almost as simply as you may cut and paste in a word-processing document.

But it wasn’t quite that clean and simple. The researchers reported “off target effects” and “mosaicism”. This means the editing sometimes occurred at the wrong place in the DNA and that it wasn’t occurring in all embryos equally. There were many mistakes, which they could not have predicted.

Made to order?

This raises at least two issues. The first is the ethical issue surrounding the use of human embryos for scientific research, and associated concerns around creating designer babies. The second is the fact that this editing went so wrong in so many embryos.

Without total control of the DNA editing process, the outcome for a baby born from a technology like this one is completely unknown.

This unpredictability and uncertainty means the promise of eliminating certain diseases by editing the DNA of embryos is likely to be a very long way off. There is also the issue of testing whether the technology is safe.

The notion of testing the technology on a live human baby is problematic indeed. Should a scientific research ethics committee ever agree to let this research be performed?

Fortunately, in Australia, all research performed on human embryos is tightly regulated by the NHMRC, which prohibits human cloning as well as many other technologies, and enforces strong penalties for non-compliance. This means that, for the foreseeable future, this type of research is very unlikely in Australia.

While the scientific world is divided as to the possibilities for this technology in embryos, including the reality of preventing or curing disease, there is consensus that this research must proceed with extreme caution.

The Conversation

This article was originally published on The Conversation.
Read the original article.

Gene-editing technique has been used on human embryos

In what may be a memorably controversial and groundbreaking new research paper, scientists from China describe in detail the way in which they were successful at manipulating the genomes, or genetic blueprints of human embryos for what is the first time in human history, reintroducing all new ethical concerns about what just may be the next frontier for science.

The story was first reported by Nature News this Wednesday, and their paper was originally published by a little known online journal called Protein and Cell.

In their paper, Junjiu Huang, who is a gene-function researcher from Sun Yat-sen University of Guangzhou, and his colleagues describe the way in which they edited the genomes from embryos they received from a fertility clinic.

The embryos were described as non-viable in the paper, ones that would not be capable of resulting in a live birth because a genetic replication error resulted in them containing an extra set of chromosomes from being fertilized by two different sperm.

The researchers “attempted to modify the gene responsible for beta-thalassaemia, a potentially fatal blood disorder, using a gene-editing technique known as CRISPR/Cas9,” according to the report from Nature News.
“The researchers say that their results reveal serious obstacles to using the method in medical applications.”
The researchers injected the CRISPR into 86 different embryos and then they waited another 48 hours for the molecules that would replace any missing DNA to begin their work.

71 of the embryos survived, and 54 out of that number were then tested.

Researchers learned that just 28 of the embryos had been “successfully spliced, and that just a small fraction of these successes contained the necessary replacement genetic material,” read the report.
“If you want to do it in normal embryos, you need to be close to 100 percent,” Huang said in a statement to Nature News.

“That’s why we stopped. We still think it’s too immature.”

What was more concerning, however, is that there were a “surprising number” of unintended mutations that occurred during the process, and accelerating at a speed that was far higher than anything seen in earlier gene-editing studies which used either mice or adult human cells.

Such mutations, moving at an unchecked speed, could be harmful, and they are one of the primary reasons for why people in the scientific community are expressly concerned. It’s a worry that grew when rumors of Huang’s research team began to circulate at the beginning of the year.

“It underlines what we said before: we need to pause this research and make sure we have a broad based discussion about which direction we’re going here,” said Edward Lanphier, president of Sangamo BioSciences in Richmond, California, in an interview with Nature News.

While the gene editing technique has shown some unprecedented success, there is the question of what effect rapid rates of mutation may have – bringing to light some potential disorders that the scientific community is not yet aware of.

George Daley, who is a stem-cell biologist at Harvard Medical School of Boston, Massachusetts, was careful in his praise of the research, describing it as “a landmark, as well as a cautionary tale.”

“Their study should be a stern warning to any practitioner who thinks the technology is ready for testing to eradicate disease genes,” he said to Nature News.

More studies may be coming to light soon. So far, there are rumors of at least four other Chinese research teams also actively working on human embryos, according to the report.

James Sullivan
James Sullivan is the assistant editor of Brain World Magazine and a contributor to Truth Is Cool and OMNI Reboot. He can usually be found on TVTropes or RationalWiki when not exploiting life and science stories for another blog article.

Spider Venom and the Search for Safer Pain Meds

Some of the most poisonous animals on the planet are found down under. Australian researchers retrieved exciting new data when taking a closer look at spider venom. Biosynthesized chemicals designed to be highly reactive with other organisms could inspire new drugs and, eventually, an entire new class of painkillers.

It can be defensive but the function of spider venom is often to incapacitate or kill prey. University of Queensland academics released their findings in The British Journal of Pharmacology, after they isolated seven unique peptides found in certain spider venoms that can block the molecules that allow pain-sensitive nerve pathways to communicate with the brain. One of the pepetides originated in the physiology of a Borneo orange-fringed tarantula. That peptide possessed the correct chemical structure, combined with a stability and effectiveness to become a non-opiate painkiller.

15% of all adults are in chronic pain, according a study published in 2012 Journal of Pain. Most readers are already aware of the danger of addiction and lagging effetiveness of opiate drugs like morphine, hydrocodone, oxycodone. The medical community is hungry for a change in available medications. Opiates are all derivatives or inspired by opium plants which have been tried and tested for centuries. Venomous spiders are difficult to study but the motivation for new drugs has loosened funding with the help of promising finds like this one.

“Spider venom acts in a different way to standard painkillers,” ~ Dr. Jennifer Smith, research officer @ University of Queensland’s Institute for Molecular Bioscience.

While cessation from pain might in itself create an addictive reaction, this venom is promising, according to Dr. Smith, because it blocks the channel through which the pain would even reach the brain. Opiates merely block the widespread opioid receptors in actual brain cells, deep within and in the surrounding nerve tissue of the brain itself.

What’s the mechanism of action for this spider-drug? Some people are born with a rare genetic defect that renders them unable to feel pain. Geneticists identified the human gene responsible, known as SCN9A. Dr. Smith hopes the peptide will enable the cells of a human without the defect to shut down part of the DNA that manifests this immunity to pain.

There could be other breakthroughs in medicine and chemistry. The findings are awesome in the Australian project but those researchers only documented findings of roughly 200 out of 45,000 known species of spider.  Out of those 200, 40% contained peptides that interacted with the way pain channels communicate. The next step would be to test the painkillers on animals.

“We’ve got a massive library of different venoms from different spider species and we’re branching out into other arachnids: scorpions, centipedes and even assassin bugs,” said Dr. Smith.


Jonathan Howard
Jonathan is a freelance writer living in Brooklyn, NY

Chromosome errors cause many pregnancies to end before they are even detected

Given the fact that there are seven billion people on Earth, one might conclude that human beings are pretty good at reproducing. But fewer than 30% of all fertilization events result in successful pregnancy, even for young, fertile couples.

The remaining 70% of conceptions result in pregnancy loss, with most of these losses occurring before the mother misses a menstrual period. This means that many pregnancies begin and end before the mother even notices. These early pregnancy losses are one reason why it generally takes several months for couples to achieve a successful pregnancy. But why is pregnancy loss so common?

The pregnancy loss iceberg.
Larsen et al. BMC Medicine 2013 11:154

Current evidence suggests that both the process of egg formation (this is called meiosis) in the mother’s ovaries and the initial embryonic cell divisions (called mitosis) just after fertilization are extremely error-prone, producing embryos with too many or too few chromosomes.

What happens after fertilization?

During fertilization, the sperm and egg fuse so that the resulting embryo will have 23 chromosomes inherited from the father and 23 chromosomes inherited from the mother. If all goes well, the subsequent cell divisions in the embryo (called mitotic divisions) simply replicate this 46-chromosome set as new cells are formed.

An example of aneuploidy formation: a cell with mis-segregating chromosomes.
Stefano Santaguida and Angelika Amon, MIT

Chromosomes contain genes, the blueprints for human development. When processes go awry in meiosis or mitosis, chromosomes can go into the wrong cell or get lost completely, drastically altering this blueprint. The resulting cell will not possess the standard 46-chromosome set – an imbalance that is the defining feature of aneuploidy. This means that many genes will either be missing or present in extra copies, placing cells under stress.

Embryos with many aneuploid cells rarely survive. Trisomy 21, the genetic cause of Down syndrome, is one of the rare forms of aneuploidy in which the baby can survive to live birth. The vast majority of embryos affected with other aneuploidies perish in early development.

What causes aneuploidy?

Aneuploidy is associated with maternal age. Female meiotic errors (these are errors in the eggs themselves) increase from a frequency of less than 20% in mothers younger than 30 years old to greater than 60% in mothers older than 45. Errors in sperm, called paternal meiotic errors, are comparatively rare, affecting fewer than 5% of sperm cells.

But age isn’t the only factor influencing aneuploidy. Our recent work in collaboration with the genetic testing company Natera, published in Science, suggests that risk is also influenced by a common genetic variant in the mother’s genome.

Embryonic cell division.
Henry Vandyke Carter via Wikimedia Commons

Even when the egg and sperm are normal, aneuploidies often arise after fertilization, during the first three embryonic cell divisions. These initial cell divisions of the embryo are controlled by maternal machinery pre-loaded into the egg.

Unlike meiotic errors in the egg, mitotic errors do not increase with age, but affect all age groups.

A maternal genetic variant influences aneuploidy risk

Using data from in vitro fertilized (IVF) embryos screened by our collaborators at Natera, we found that mothers with a particular genetic variant on chromosome 4 tend to produce embryos with more mitotic aneuploidies – the aneuploidies that arise during post-fertilization cell division.

This effect was observed for mothers of all ages and from diverse ethnic backgrounds. This genetic variant is surprisingly common; approximately half of all people carry at least one copy of this risk variant.

The most likely suspect is a gene called Polo-like kinase 4 (PLK4), which is known to be a master regulator of the centrosome cycle. The centrosome is molecular machine that is responsible for proper cell division and distribution of chromosomes.

We estimated that each copy of the risk variant increases the rate of aneuploidy by about 3%, regardless of the mother’s age. Having two copies doubles this risk. This increased risk could be especially important for older mothers who are already more prone to aneuploidy. It is likely that there are other genetic variants that contribute to aneuploidy risk to a lesser degree, and further work will be required to determine if this is the case.

Because of the established link between aneuploidy and pregnancy loss, we hypothesized that the aneuploidy risk variant might also affect embryo survival. We found that mothers with the high-risk genotypes had fewer embryos available for testing, suggesting that their embryos are less likely to survive very early developmental stages due to aneuploidy.

Given these results, it seems like this genetic variant could influence the average time it takes to achieve successful pregnancy, an idea that we are hoping to investigate further.

A signature of natural selection: comparison to the Neanderthals

Normally, natural selection weeds out damaging variation, reducing it to very low frequency. But the aneuploidy risk variant is very common. Hoping to learn more about the evolutionary history of this variant, we compared human genomes to Neanderthals and Denisovans, our ancient hominin relatives.

Comparison of a modern human skull and Neanderthal skull in the Cleveland Museum of Natural History.
hairymuseummatt (original photo), DrMikeBaxter (derivative work) via Wikimedia Commons, CC BY

Despite the fact that the harmful genetic variant is relatively common in humans, it was absent in these close relatives, meaning that it likely rose rapidly in frequency in an ancestral population of humans. If this is true, it means that this version of this gene was actually somehow beneficial (and maybe still is) while simultaneously being harmful in the context of early development.

So what could possibly have been the benefit?

We aren’t sure at this point, but we speculate that for ancient humans, there might have been a benefit to having a reduced probability of successful pregnancy per intercourse. Maybe the benefit had to do with infanticide – men may be less likely to kill a baby if there is a chance it is their child, and not that of a rival. Likewise, lower probability of pregnancy per intercourse might encourage repeated mating with the same female, fostering pair bonding and paternal investment. This hypothesis was first proposed by Alexander and Noonan in 1979 to help explain the human-specific trait of concealed ovulation and continuous sexual receptivity – women do not externally signal or limit intercourse to the fertile portion of their cycles as do some other primates.

Another idea is based on the fact that PLK4 is often mutated in human cancers. Could there be a beneficial effect of the risk variant in the context of cancer? PLK4 plays yet another role in testes development. Could the aneuploidy risk variant have a beneficial effect in this context?

We are hoping that additional data and future research can shed more light on this signal of human-specific adaptation. Is it real or simply an artifact of chance events in human evolution? Did our ancestors have lower rates of aneuploidy? What about Neanderthals, Denisovans, and living non-human primates? And perhaps the most basic question: why is human aneuploidy so common? Armed with modern genomic technologies, we can continue to chip away at these questions to understand not only the medical aspects of aneuploidy risk, but also the broader evolutionary basis of this intriguing trait.

The Conversation

This article was originally published on The Conversation.
Read the original article.

Why is CRISPR the Science Buzzword of Early 2015?

CRISPR isn’t just the cutting edge of genetic modification – it is re-framing our understanding of evolution.

 What is CRISPR?
CRISPR is a DNA sequence that can do something most other genes can’t. It changes based on the experience of the cell it’s written in.  It works because of a natural ability for cells to rewrite their own genetic code, first discovered in 1987. The name CRISPR was coined in 2002, and it stands for “clustered regularly interspaced short palindromic repeats”. They function as a method of inserting recognizable DNA of questionable or dangerous viruses into DNA strands so that the offspring of the cell can recognize what its ancestors have encountered and defeated in the past. By inserting a CRISPR-associated protein into a cell along with a piece of RNA code the cell didn’t write, DNA can be edited.A 2012 breakthrough  involved, in part, the work of Dr. Jennifer A. Doudna. Doudna and the rest of the team at UC Berkley were the first to edit human DNA using CRISPR.  Recently, in March 2015, she warned this new genome-editing technique comes with dangers and ethical quandaries, as new tech often does. Dr. Doudna in a NYT article, she called for a planet-wide moratorium on human DNA editing, to allow humanity time to better understand the complicated subset of issues we all now face.
CRISPR-related tech insn’t only about editing human genes, though. It affects cloning and the reactivation of otherwise extinct species. It isn’t immediately clear what purpose this type of species revival would have without acknowledging the scary, rapidly increasing list of animals that are going extinct because of human activity. Understanding and utilizing species revival could allow humans to undo or reverse some of our environmental wrongs. The technique may be able to revive the long lost wooly mammoth by editing existing elephant DNA to match the mammoth‘s, for instance. Mammoths likely died out due to an inability to adapt to natural climate change which caused lower temperatures in their era, and are a non-politically controversial choice but the implications for future environmentalism are promising.
Each year, mosquitoes are responsible for the largest planetary human death toll. Editing DNA with CRISPR bio-techniques could help control or even wipe out malaria someday. The goal of this controversial tech is to make the mosquito’s immune system susceptible to malaria or make decisions about their breeding based on how susceptible they are to carrying the disease. The controversy around this approach to pest and disease control involves the relatively young research behind Horizontal Gene Transfer, where DNA is passed from one organism to an unrelated species. A gene that interferes with the ability of mosquitoes to reproduce could end up unintentionally cause other organisms to have trouble reproducing. This info is based on the work of , ,
Even more controversial are the startups claiming they can create new life forms, and own the publishing rights. Austen Heinz’ firm is called Cambrian Genomics which grows genetically-controlled and edited plants. The most amazing example is the creation of a rose species that literally glows in the dark. Cambrian is collaborating with the rose’s designer, a company called Glowing Plant, whose projects were eventually banned from kickstarter for violating a rule about owning lifeforms. Eventually, Heinz wants to let customers request and create creatures:
The final example in an ongoing list of 2015 breakthroughs involving CRISPR is this CRISPR-mediated direct mutation of cancer genes in the mouse liver might be able to combat cancer. It’s the second cancer-related breakthrough in 2015 that affects the immune system, the first was on Cosmos about a week back: Accidental Discovery Could Turn Cancer Cells Into Cancer-Attacking Immune Cells.

Other Related articles:

Pre-Darwinian Theory of Heredity Wasn’t Too Far Off

Wooly Mammoth Poised to be the First De-Extincted Animal, Son~!


Jonathan Howard
Jonathan is a freelance writer living in Brooklyn, NY