Archive for the ‘Crispr’ Category

CRISPR may be a cure, but clinical trials may lack volunteers because of black patients' mistrust of biased and unethical medical practices.

"It's like something stabbing you in your bone just like repeatedly. And you can't stop it. And... it's something that makes you really tense, you just can't move... It's been a part of my life since I was six months old."

Twenty-three-year-old Maiya Washington has been living with sickle cell, a life-long "invisible" disease. It's a disorder with no visibly detectable signs and not often talked about.

"Although it is a disease that is invisible, so to speak, we go through a lot, you know, and we go through a lot to be normal to live life to get jobs to go to school. And it's hard. It's really hard," says Washington.

Sickle Cell Disease impacts about100,000 Americans,mostly African Americans. It's a genetic defect that affects red blood cells turning normal round red blood cells into sickle shapes. That shape can cause clumping andblock oxygen and blood flow,which can then lead to a wide range of health issues likestrokes, kidney problems,and organ failure.

"It's really debilitating, honestly," says Washington. "And it's hard because... you need somebody there with you. It's not like you can take yourself to the hospital. Or if you're at home and you're trying to manage the pain by yourself, you still need somebody there with you, because you're not able to do the simple things like go to the restroom by yourself, get yourself some water, fix yourself something to eat"

Currently, the only option to cure sickle cell disease is a bone marrow transplant, also called a stem cell transplant. But it requires a matching donor, which can be difficult to find. But now, the gene-editing tool CRISPR may bypass the need for a match and serve as a cure for most people. Dr. John Tisdale has worked on another Sickle Cell Disease gene therapy at the National Institute of Health.

"And it's all coming from red blood cells with a single misspelling, in their hemoglobin. So we should be able to fix that it's just one base," says Tisdale.

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Here's how it works. First, stem cells are pulled from the patient's body. CRISPR is used to edit the DNA, and the new, edited stem cells are then re-inserted back into the body. The hope is that it will generate healthy red blood cells. Changes to the DNA won't be passed down to future children.

Could this development lead to a cure for sickle cell? A clinical trial using CRISPR can help determine that. So far, testing on 12 people has already begun. Biopharmaceutical company Vertex has partnered with CRISPR Therapeutics and they're looking for morevolunteers for this clinical trial.

Dr. Alexis Thompson, Head of Hematology, Lurie Childrens Hospital of Chicago says, "I certainly am very excited about it as a provider, is that patients will have choices. There is not a one size fits all for sickle cell disease, or really, really for in a community For the first time, there will be multiple, multiple clinical trials that are opening and hopefully those will lead to new treatments... because much of what we learn from sickle cell will be rapidly applicable to other conditions."

But as exciting as the potential for a cure for Sickle Cell Disease is, finding enough volunteers for testing is really difficult. Sure, CRISPR poses risks, including death. But one of the biggest barriers isblack patients' mistrust of the medical community,so much so that clinical trialstend to lack black patient enrollment.This is based on racist treatment both in the past and present.Science writer Usha Lee MacFarlingpoints to unethical medical practices of the past, like the use of Henrietta Lacks' cells without permission and leaving syphilis untreated in hundreds of black men in the Tuskegee experiment.

"I think there's just a huge awareness in the black community of these studies that were, you know, racist, that really treated black women of color and poor women as guinea pigs. It's a very sharp pain And it's definitely affecting people's reluctance, and inability to trust, the largely white medical establishment," says MacFarling.

And today, some black patients say that bias persists in medicine. Because sickle cell patientsvisit the emergency room an average of three times a year, they're often assumed to be addicts for seeking drugs to ease their pain.

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"Doctors [have]...given me an inappropriate amount of medicine. That wasn't helping, that kind of basically looked at me as like, you know, drug-seeking, or just like faking it," says Washington.

In spite of all this, Maiya says she wants to participate in the trial because of how excruciating her pain is.

"I feel as though most people who deal with sickle cell or any kind of disability that alters their quality of life, they're going to be willing to figure out anything to get rid of what they had," says Washington.

In order to build trust between black sickle cell patients and CRISPR researchers, organizations like the American Society of Hematology and the Minority Coalition for Precision Medicine are doing community outreach. That includes even teaming up with churches.

Michael Friend, co-founder of Minority Coalition for Precision Medicine says: "It was kind of very easy to talk to faith-based leaders about sickle cell disease because it's a disease that primarily affects African Americans. And it's a disease that we found prevalent in most churches, and most pastors were familiar with the disease."

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For now, every month Maiya's gets a blood transfusion to ease the pain. Her baby is lucky -- she doesn't have sickle cell, because her father doesn't have the gene.

"I wake up every day and she's there. She's my best friend and I love seeing her watching her grow so far, says Washington.

Most sickle cell patients don't live past their 40's and Maiya does worry about her future.

She says, "it does concern me because I want to be here as long as possible for her. And hoping that, you know, there's something that can come up that can be permanent, you know, as in terms of a cure or medication...just to help us have a longer lifespan and live a better quality of life. Because I do want to be able to see her grow up."

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CRISPR Cure For Sickle Cell May Be Slowed By Black Patients' Mistrust - Newsy

We are at war with the mosquito. A swarming and consuming army of 110tn enemy mosquitoes patrols every inch of the globe except for Antarctica, Iceland and a handful of French Polynesian micro-islands. The biting female warrior of this droning insect population is armed with at least 15 lethal and debilitating biological weapons, to be used against 7.7 billion humans deploying suspect and often self-detrimental defensive capabilities. In fact, our defence budget for personal shields, sprays and other means of deterring her unrelenting raids is $11bn (8.8bn) a year, and rising rapidly. And yet her deadly offensive campaigns and crimes against humanity continue with reckless abandon. While our counterattacks are reducing the number of casualties she perpetrates malaria deaths in particular are declining rapidly the mosquito remains the deadliest hunter of human beings on the planet.

Taking a broad range of estimates into account, since 2000, the average annual number of human deaths caused by the mosquito was around 2 million. Humans came in a distant second at 475,000, followed by snakes (50,000), dogs and sandflies (25,000 each), the tsetse fly, and the assassin or kissing bug (10,000 each). The fierce killers of lore and Hollywood celebrity were much further down our list. The crocodile was ranked 10th, with 1,000 annual deaths. Next on the list were hippos with 500, and elephants and lions with 100 fatalities each. The much-slandered shark and wolf shared 15th position, killing an average of 10 people per annum.

Yet the mosquito does not directly harm anyone. It is the toxic and highly evolved diseases she transmits that cause an endless barrage of desolation and death. Without her, however, these sinister pathogens could not be transferred or vectored to humans, nor could they continue their cyclical contagion. In fact, without her, these diseases would not exist at all.

Our immune systems are finely tuned to our local environments. Mosquitoes do not respect international borders. Marching armies, inquisitive explorers and land-hungry colonists brought new diseases to distant lands, but were also brought to their knees by micro-organisms in the foreign lands they intended to conquer. As the mosquito transformed the landscapes of civilisation, humans were unwittingly required to respond to her universal projection of power. After all, the truth is that, more than any other external participant, the mosquito, as our deadliest predator, drove the events of human history to create our present reality.

It has been one of the most universally recognisable and aggravating sounds on Earth for 190m years the whine of a mosquito. After a long day of walking while camping with your family or friends, you quickly shower, settle into your lawn chair, open an ice-cold beer and exhale a deep, contented sigh. Before you can enjoy your first satisfying swig, however, you hear that all-too-familiar sound, signalling the approach of your soon-to-be tormentors.

It is nearing dusk, her favourite time to feed. Although you heard her droning arrival, she gently lands on your ankle without detection, as she usually bites close to the ground. It is always a female, by the way. She conducts a tender, probing, 10-second reconnaissance, looking for a prime blood vessel. With her backside in the air, she steadies her crosshairs and zeros in with six sophisticated needles. She inserts two serrated mandible cutting blades (much like an electric carving knife, with two blades shifting back and forth), and saws into your skin, while two other retractors open a passage for the proboscis, a hypodermic syringe that emerges from its protective sheath. With this straw she starts to suck out 3-5 mg of your blood, immediately excreting its water while condensing its 20% protein content. All the while, a sixth needle is pumping in saliva that contains an anticoagulant, preventing your blood from clotting at the puncture site. This shortens her feeding time, lessening the likelihood that you feel her penetration and splat her across your ankle. The anticoagulant causes an allergic reaction, leaving an itchy bump as her parting gift. The mosquito bite is an intricate and innovative feeding ritual required for reproduction. She needs your blood to grow and mature her eggs.

Please dont feel singled out. She bites everyone. There is absolutely no truth to the persistent myths that mosquitoes fancy females over males, that they prefer blonds and redheads over those with darker hair, or that the darker or more leathery your skin, the safer you are from her bite. It is true, however, that she does play favourites and feasts on some more than others. Blood type O seems to be the vintage of choice over types A and B, or their blend. People with blood type O get bitten twice as often as those with type A, with type B falling somewhere in between. (Disney/Pixar must have done their homework when portraying a tipsy mosquito ordering a Bloody Mary, O-positive in the 1998 movie A Bugs Life.) Those who have higher natural levels of certain chemicals in their skin, particularly lactic acid, also seem to be more attractive. From these elements, she can analyse which blood type you are. These are the same chemicals that determine an individuals level of skin bacteria and unique body odour. While you may offend others and perhaps yourself, in this case, being pungently rancid is a good thing, for it increases bacterial levels on the skin, which makes you less alluring to mosquitoes except for stinky feet, which emit a bacterium that is a mosquito aphrodisiac. The mosquito is also enticed by deodorants, perfumes, soap and other applied fragrances.

She also has an affinity for beer drinkers. Wearing bright colours is also not a wise choice, since she hunts by both sight and smell the latter depending chiefly on the amount of carbon dioxide exhaled by the potential target. So all your thrashing and huffing and puffing only magnetises mosquitoes and puts you at greater risk. She can smell carbon dioxide from 200 feet away. When you exercise, you emit more carbon dioxide through frequency of breath and output. You also sweat, releasing those appetising chemicals, primarily lactic acid, that invite the mosquitos attention. Lastly, your body temperature rises an easily identifiable heat signature. On average, pregnant women suffer twice as many bites, as they respire 20% more carbon dioxide, and have a marginally elevated body temperature. This is bad news for the mother and the foetus when it comes to infection from Zika and malaria.

Unlike their female counterparts, male mosquitoes do not bite. Their world revolves around two things: nectar and sex. Like other flying insects, when they are ready to mate, male mosquitoes assemble over a prominent feature in the landscape from chimneys to antennas to trees to people. Many of us grumble and flail in frustration as that dogged cloud of bugs droning over our heads shadows us when we walk, refusing to disperse. Take it as a compliment. Male mosquitoes have graced you with the honour of being a swarm marker. Mosquito swarms have been photographed extending 1,000 feet into the air, resembling a tornado funnel cloud. With the cocksure males stubbornly assembled over your head, females will fly into their horde to find a suitable mate. While males will mate frequently in a lifetime, one dose of sperm is all the female needs to produce numerous batches of offspring. She stores the sperm and dispenses them piecemeal for each separate birthing of eggs. Her short moment of passion has provided one of the two necessary components for procreation. The only ingredient missing is your blood.

Back at the campsite, you have just finished your strenuous hike, and proceed to the shower, where you lather up with soap and shampoo. After drying off, you apply body spray and deodorant before finally putting on your bright red-and-blue beachwear.

It is nearing dusk dinnertime for the Anopheles mosquito. You have done everything in your power to lure a famished female of the species. Having just mated in a swarming frenzy of eager male suitors, she willingly takes the bait and makes off with a few drops of your blood a blood meal three times her own body weight. She quickly finds the nearest vertical surface and, with the aid of gravity, continues to evacuate the water from your blood. Using this concentrated blood, she will develop her eggs over the next few days. She then deposits roughly 200 floating eggs on the surface of a small pool of water that has collected on a crushed beer can that was overlooked during cleanup as you and your party headed home. She always lays her eggs in water, although she does not need much. From a pond or stream to a minuscule puddle in the bottom of an old container, used tire or backyard toy, any will suffice.

Our mosquito will continue to bite and lay eggs during her one-to-three-week lifespan. While she can fly up to two miles, she rarely ranges more than 400 metres from her birthplace. Although it takes a few days longer in cool weather, given the high temperatures, her eggs hatch into wiggling, water-bound worms within two or three days. Skimming the water for food, they quickly turn into upside-down, comma-shaped tumbling caterpillars who breathe through two trumpets protruding from their water-exposed buttocks. A few days later, a protective encasement splits and healthy adult mosquitoes take flight, with a new generation of succubus females ready to feed. This maturation to adulthood takes roughly one week.

Bacteria, viruses and parasites, along with worms and fungi, have triggered untold misery, and have commanded the course of human history. Why have these pathogens evolved to exterminate their hosts? If we can set aside our bias for a moment, we can see that these microbes have journeyed through the natural selection voyage just as we have. This is why they still make us sick and are so difficult to eradicate. You may be puzzled: it seems self-defeating and detrimental to kill your host. The disease kills us, yes, but the symptoms of the disease are ways in which the microbe conscripts us to help it spread and reproduce. It is dazzlingly clever, when you stop to think about it. Generally, germs guarantee their contagion and replication prior to killing their hosts. Some, like the salmonella food poisoning bacteria and various worms, wait to be ingested that is, one animal eating another animal.

There is a wide range of waterborne transmitters, including giardia, cholera, typhoid, dysentery and hepatitis. Others, including the common cold, the 24-hour flu and true influenza, are passed on through coughing and sneezing. Some, such as smallpox, are transferred directly or indirectly by lesions, open sores, contaminated objects or coughing. My personal favourites strictly from an evolutionary standpoint, of course are those that covertly ensure their reproduction while we intimately ensure our own. These include the full gamut of microbes that trigger sexually transmitted diseases. Many sinister pathogens are passed from mother to foetus in utero.

Others that germinate typhus, bubonic plague, Chagas and trypanosomiasis (African sleeping sickness) catch a free ride provided by a vector (an organism that transmits disease) such as fleas, mites, flies, ticks and mosquitoes. To maximise their chances of survival, many germs use a combination of more than one method. The diverse collection of symptoms, or modes of transference, assembled by micro-organisms helps them effectively procreate and ensures the existence of their species. These germs fight for their survival just as much as we do, and stay an evolutionary step ahead of us as they continue to morph and shape-shift to circumvent our best means of extermination.

To understand the stealthy, sprawling influence of the mosquito on history and humanity, it is first necessary to appreciate the animal itself, and the diseases it transmits. According to a quotation erroneously attributed to Charles Darwin: It is not the strongest of the species that survives, nor the most intelligent that survives. It is the one that is most adaptable to change. Regardless of the origin of this passage, the mosquito and its diseases most notably malaria parasites are the quintessential example of the point it is making. They are masters of evolutionary adaptation. Mosquitoes can evolve and adapt to their changing environments within a few generations. During the Blitz of 1940-41, for example, as German bombs rained down on London, isolated populations of Culex mosquitoes were confined to the air-raid tunnel shelters of the London Underground, along with the citys resilient citizens. These trapped mosquitoes quickly adapted to feed on mice, rats and humans instead of birds, and are now a species distinct from their above-ground parental ancestors.

What should have taken thousands of years of evolution was accomplished in less than 100. In another 100 years, jokes Richard Jones, former president of the British Entomological and Natural History Society, there may be separate Circle line, Metropolitan line and Jubilee line mosquito species in the tunnels below London.

While the mosquito is miraculously adaptable, it is also a purely narcissistic creature. Unlike other insects, it does not pollinate plants in any meaningful way, or aerate the soil, or ingest waste. Contrary to popular belief, the mosquito does not even serve as an indispensable food source for any other animal. She has no purpose other than to propagate her species, and perhaps to kill humans. As the apex predator throughout our odyssey, it appears that her role in our relationship is to act as a countermeasure against uncontrolled human population growth.

Throughout our existence, the mosquitos toxic twins of malaria and yellow fever have been the prevailing agents of death and historical change, playing the role of antagonists in the protracted chronological war between man and mosquito.

Following that fateful mosquito bite, the miscreant malaria parasite will mutate and reproduce inside your liver for one to two weeks, during which time you will show no symptoms. A toxic army of this new mutated form will then explode out of your liver and invade your bloodstream. The parasites attach to your red blood cells, penetrate the outer defences, and feast on the haemoglobin within. Inside the cell, they undergo another metamorphosis and reproductive cycle. Engorged blood cells eventually burst, spewing both a duplicate form, which marches forward to attack fresh red blood cells and also a new asexual form that relaxedly floats in your bloodstream, waiting for mosquito transportation.

The parasite is a shape-shifter, and it is precisely this genetic flexibility that makes it so difficult to eradicate or suppress with drugs or vaccines. You are now gravely ill with an orderly, clockwork progression of chills followed by a mercury-driving fever that may touch 41C. This full-blown cyclical malarial episode has you in its firm grip, and you are at the mercy of the parasite. Lying prostrate and agonisingly helpless on sweat-soaked sheets, you twitch and fumble, curse and moan. You look down and notice that your spleen and liver are visibly enlarged, your skin has the yellowing patina of jaundice and you vomit sporadically. Your fever will relapse at precise intervals with each new burst and invasion of the parasite from your blood cells. The fever then subsides while the parasite eats and reproduces inside new blood cells.

The parasite uses sophisticated signalling to synchronise its sequencing, and this entire cycle adheres to a very strict schedule. The new asexual form transmits a chemical bite me signal in our blood, significantly boosting the chances of being picked up by a mosquito from an infected human to complete the reproductive cycle. Inside the stomach of the mosquito, these cells mutate once more, into both male and female varieties. They quickly mate, producing threadlike offspring that make their way out of the gut and into the salivary glands of the mosquito. Within the saliva glands, the malaria parasite shrewdly manipulates the mosquito to bite more frequently by suppressing the production of her anticoagulant and thus minimising her blood intake during a single feeding. This forces her to bite more frequently to get her required fill. In doing so, the malaria parasite ensures that it maximises its rate and range of transfer, its procreation and its survival.

Temperature is an important element for both mosquito reproduction and the life cycle of malaria. Given their symbiotic relationship, they are also both climate-sensitive. In colder temperatures, it takes longer for mosquito eggs to mature and hatch. Mosquitoes are also cold-blooded and, unlike mammals, cannot regulate their own body temperatures. They simply cannot survive in environments below 10C. Mosquitoes are generally at their prime health and peak performance in temperatures above 23C. A direct heat of 40C degrees will boil mosquitoes to death. For temperate, non-tropical zones, this means that mosquitoes are seasonal creatures with breeding, hatching and biting taking place from spring through autumn. Although never seeing the outside world, malaria needs to contend with both the short lifespan of the mosquito and temperature conditions to ensure replication. The timeframe of malaria reproduction is dependent on the temperature of the cold-blooded mosquito, which itself is dependent on the temperature outside. The colder the mosquito, the more sluggish malaria reproduction becomes, eventually hitting a threshold. Between 15C and 21C (depending on the type of malaria), the reproductive cycle of the parasite can take up to a month, exceeding the average life span of the mosquito. By then, she is long dead, and brings malaria down with her.

Warmer climates can sustain year-round mosquito populations, promoting endemic circulation of her diseases. Abnormally high temperatures can cause seasonal epidemics of mosquito-borne diseases in regions where they are generally absent or fleeting. Global warming also allows the mosquito and her diseases to broaden their topographical range. As temperatures rise, disease-carrying species, usually confined to southern regions and lower altitudes, creep north and into higher elevations.

Since a breakthrough discovery by a team led by the biochemist Dr Jennifer Doudna at the University of California, Berkeley in 2012, the revolutionary gene-editing innovation known as Crispr has shocked the world and altered our preconceived notions about our planet and our place on it.

The pages of many widely read magazines and journals are currently consumed by the topic of Crispr and mosquitoes. First successfully used in 2013, Crispr is a procedure that snips out a section of DNA sequencing from a gene and replaces it with another desired one, permanently altering a genome, quickly, cheaply, and accurately.

The Bill and Melinda Gates Foundation has been funding research into mosquito-borne diseases since its creation in 2000. In 2016 it made investments in Crispr mosquito research totalling $75m. Our investments in mosquito control, said the foundation, include nontraditional biological and genetic approaches as well as new chemical interventions aimed at depleting or incapacitating disease-transmitting mosquito populations. These genetic approaches include the use of Crispr machinery to eradicate mosquito-borne diseases, most notably malaria.

The strategic goal of the Gates Foundation is the extermination of malaria and other mosquito-borne diseases; it is not to bring the mosquito which is harmless when flying solo, untethered from a hitchhiking micro-organism to the brink of extinction. Of the more than 3,500 mosquito species, only a few hundred are capable of vectoring disease. Prefabricated, genetically modified mosquitoes rendered incapable of harbouring the parasite (a hereditary trait passed down their bloodline) might just end the timeless scourge of malaria. But, as Doudna and the Gates Foundation are aware, gene-swapping technology also has the potential to unleash darker, more sinister genetic blueprints with dangerous possibilities. Crispr research is a global phenomenon, and neither Doudna nor the foundation has a monopoly on its limitless designs, its instruments of implementation or its operational execution.

It has been dubbed the extinction drive, as this is precisely what it can accomplish the extermination of mosquitoes by way of genetic sterilisation. This theory has been floating around the scientific community since the 1960s. Crispr can now put these principles into practice. To be fair, the mosquito altered our DNA in the form of sickle cell and other genetic malarial safeguards; perhaps it is time to return the favour. Male mosquitoes that have been genetically modified with domineering selfish genes using Crispr are released into mosquito zones to breed with females to produce stillborn, infertile or only male offspring. The mosquito would be extinct in one or two generations. With this war-winning weapon, humanity would never again have to fear the bite of a mosquito. We would awaken to a brave new world, one without mosquito-borne disease.

An alternative is simply to make mosquitos harmless, a strategy supported and funded by the Gates Foundation. With gene drive technology, Gates explained in October 2018, essentially, scientists could introduce a gene into a mosquito population that would either suppress the population or prevent it from spreading malaria. For decades, it was difficult to test this idea. But with the discovery of Crispr, the research became a lot easier. And just last month, a team from the research consortium Target Malaria announced that they had completed studies where mosquito populations were fully suppressed. To be clear: the test was only in a series of laboratory cages filled with 600 mosquitoes each. But it is a promising start.

Dr Anthony James, a molecular geneticist at the University of California, Irvine, Crisprd a species of Anopheles mosquito to make it incapable of spreading malaria, by eliminating the parasites as they are processed through the mosquitos salivary gland. We added a small package of genes, explains James, that allows the mosquitoes to function as they always have, except for one slight change they can no longer harbour the malaria parasite.

The Aedes breed is more difficult to tackle, since it transmits a handful of diseases that include yellow fever, Zika, West Nile, chikungunya, Mayaro, dengue and other encephalitides. What you need to do is engineer a gene drive that makes the insects sterile, James said of the Aedes breed. It doesnt make sense to build a mosquito resistant to Zika if it could still transmit dengue and other diseases.

We have valid, although yet unknown, reasons to be careful what we wish for. If we eradicate disease-vectoring mosquito species, would other mosquito species or insects simply fill the ecological niche? What effect would eliminating mosquitoes have on natures biological equilibrium? What would happen if we exterminate species that play an essential but unrecognised role in our ecosystem? We are just beginning to ask these morally fraught and biologically ambiguous questions, and for now, no one really knows the answers.

This is an edited extract from The Mosquito: A Human History of Our Deadliest Predator by Timothy Winegard, published by Text on 26 September and available at guardianbookshop.co.uk

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People v mosquitos: what to do about our biggest killer - The Guardian

The gene drive is one of the latest of advancements in genetic modification of living things. It may also be the most controversial, in a field that has seen more than its fair share of controversy. Traditionally, coverage of genetic modifications in food products has resembled war correspondence:

Side 1Gene drives are a valuable tool for controlling pests and perpetuating beneficial genes through agricultural productsSide 2Gene drives are a dangerous, untested and unnatural genetic changes created to deliberately drive a species to extinction.

Gene drive is a version of gene editinga newer, more precise way to change a DNA (or RNA) sequence, in this case by combining a guide RNA with an enzyme that can make a splice in the exact place where a sequence can be removed, another sequence inserted, or the existing sequence altered. Gene drive takes this to another level, making sure that a new or altered genetic sequence has a greater than 50 percent chance of being inherited. This can be done in a number of ways, some of which already exist in nature, some which are no different than traditional gene editing using CRISPR-Cas9, and others that have triggered a backlash from environmental activist groupsnon-governmental organizations (NGOs) that utilize fear for their own political ends.

So far, this debate has pitted NGOs like Friends of the Earth, Greenpeace and ETC Group (who are opposed to any genetic manipulations in food crops and animals) against scientists, some agricultural companies and even some government regulators (almost all of whom conclude that these products are no more dangerous than those developed through traditional breeding).

The activist effort is part of a long-standing campaign to conflate gene drives, gene editing and traditional transgenics (GMO) as the same technology, with the same scientific certainty (or uncertainty) and risks. In 2016, FOE and others asked for a worldwide moratorium on gene drives:

Gene drives, developed through new gene-editing techniques, are designed to force a particular genetically engineered trait to spread through an entire wild population potentially changing entire species or even causing deliberate extinctions. The statement urges governments to put in place an urgent, global moratorium on the development and release of the new technology, which poses serious and potentially irreversible threats to biodiversity, as well as national sovereignty, peace and food security.

Many times, these activist organizations have claimed the public shares their concerns. Citing survey data, the science community has retorted that most people embrace biotechnology when they recognize that it benefits them directly. But what has been missing from the battle between the pro- and anti-GMO positions is a scientific measure of public opinion on more recent techniques such as gene drive. In 2016, a comprehensive National Academies of Science (NAS) report called for not only continued research on the effectiveness and usefulness of gene drives, but also their ecological risks and engagement with the public. While institutions like FOE and ETC Group objected to the existence of gene drives, they did not represent the opinion of the public.

For the first time, that opinion was actually tested, by researchers at North Carolina State University and the University of Wisconsin. In a paper published in Science Advances, Zack Brown, assistant professor of resource economics at NC State and his colleagues surveyed 1,000 American adults on their opinions of gene drives. What they found, instead of opposition, was support for the technology, with a few caveats:

The survey results could be valuable in this early stage of gene drive (or gene editing, for that matter) development as research could possibly be directed toward designing drive strategies that could incorporate controlsnot an easy thing to do, Brown said in a press release.

This is the right timewhile the technology is still under development and before any release decisions have been madeto gain insights into what the public thinks, what types of information they prioritize from researchers, and who is trusted to carry out this sensitive research, said Michael Jones, a graduate student at NC State and co-author of the published survey results.

Another significant finding in the NC State/Wisconsin study was that Americans surveyed trusted universities and the US Department of Agriculture (USDA) (60 percent) over foreign universities, the US Department of Defense (18 percent) and private companies (16 percent) to research gene drive systems.

The survey did not ask respondents for their trust levels of NGOs like FOE and Greenpeace.

However, in another recently published survey, this one in Current Research in Biotechnology, 113 experts (scientists, government officials, agribusiness professionals) found that gene-edited crops posed little to no risk to society, the economy, human health or the environment. Less than five percent thought the techniques posed a high risk.

The experts, most of whom observed that NGOs generally opposed gene editing, gene drive and any other genetic modification, noted that this opposition is based on speculative risks, those that have no established theory or evidence data. The authors of the study, based at the University of Saskatchewan in Canada, warned that the problem with attempting to reconcile speculative risks with risks grounded in theory and evidence, is that speculative risks can be very fluid and dynamic, changing at will and [frequently] at the whim of eNGO political motives.

These new studies seem to support the idea that consumers are less wary of biotechnology when they know how its being deployed. While public opinion surveys show support with some caveats about taking precautions against accidents and outbreaks, by and large members of the public trust scientists, particularly those in academia and at relevant regulatory agencies to navigate this controversial but promising field of research.

Andrew Porterfield is a writer and editor, and has worked with numerous academic institutions, companies and non-profits in the life sciences.BIO. Follow him on Twitter@AMPorterfield

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Viewpoint: Public supports CRISPR, gene drives to battle infectious disease, plant pestsdespite activist opposition - Genetic Literacy Project

Scientists are reporting the first use of the gene-editing tool CRISPR to try to cure a patients HIV infection by providing blood cells that were altered to resist the AIDS virus.

The gene-editing tool has long been used in research labs, and a Chinese scientist was scorned last year when he revealed he used it on embryos that led to the birth of twin girls. Editing embryos is considered too risky, partly because the DNA changes can pass to future generations.

Wednesdays report in the New England Journal of Medicine, by different Chinese researchers, is the first published account of using CRISPR to treat a disease in an adult, where the DNA changes are confined to that person.

The attempt was successful in some ways but fell short of being an HIV cure.

Still, it shows that gene editing holds promise and seems precise and safe in this patient so far, said Dr. Carl June, a University of Pennsylvania genetics expert who wrote a commentary in the journal.

Thats really good for the field, June said.

Chinese government grants paid for the research, which was done openly with advance notice on a scientific registry and standard informed consent procedures. Some of those steps were missing or questioned in last years embryo work.

There are no ethical concerns on this one, June said.

Gene editing permanently alters DNA, the code of life. CRISPR is a relatively new tool scientists can use to cut DNA at a specific spot.

The new case involves a 27-year-old man with HIV who needed a blood stem cell transplant to treat cancer. Previously, two other men were apparently cured of both diseases by transplants from donors with natural resistance to HIV because they have a gene mutation that prevents HIV from entering cells.

Since donors like this are very rare, the Chinese scientists tried to create similar HIV resistance by editing that gene in blood cells in the lab to try to mimic the mutation.

The transplant put the mans cancer in remission, and the cells that were altered to resist HIV are still working 19 months later. But they comprise only 5 percent to 8 percent of such blood cells, so theyre outnumbered by ones that can still be infected.

They need to approach 90 percent or more, I think, to actually have a chance of curing HIV, June said.

Scientists are testing various ways to make the gene editing more efficient, and our results show the proof of principle for this approach, one study leader, Hongkui Deng of Peking University in Beijing, wrote in an email.

One very encouraging result: multiple tests show that the editing did not have unintended effects on other genes.

One of the concerns is that they could make a Frankenstein cell, that they would hit other genes instead of the intended target, so its good that this did not happen, June said.

China appears to be moving fast on such research and may get treatments approved sooner than the United States, June said. He has financial ties to some gene therapy companies and is leading a different study testing CRISPR to fight cancer in the U.S. Three patients have been treated so far and some results are expected by the end of this year.

Several other U.S. studies have been trying to control HIV by altering patients own blood cells using a different gene-editing tool called zinc finger nucleases. The first such test began a decade ago in the U.S.

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Gene-editing tool shows promise in fight against HIV - The Columbian

For the first time, scientists used the gene editing tool CRISPR-Cas9 in an attempt to treat an HIV patient in China. The patient showed improvements after the procedure and did not experience side effects.

The scientists at Peking University in Beijing utilized CRISPR to delete the gene called CCR5 from stem cells in a donated bone marrow. CCR5 is known for contributing to HIV infection.

A 27-year-old patient diagnosed with AIDS and acute lymphoblastic leukemia received the transplant. Doctors said the new and modified bone marrow should help treat his cancer and eliminate HIV.

"After being edited, the cells -- and the blood cells they produce -- have the ability to resist HIV infection," lead scientist Deng Hongkui told CNN.

The patient went under the knife for the bone marrow transplant in 2017. In early 2019, scientists said the man's acute lymphoblastic leukemia was in complete remission.

The stem cells with the editedCCR5 gene also stayed in his system 19 months after the procedure. The team published the results in The New England Journal of Medicine.

However, the new cells did not completely eliminatethe HIV virus. Scientists said the patient lacked enough amounts of stem cells to treat the disease.

Cells in the transplanted bone marrow carried only 5 percent to 8 percent of the edited CCR5. But it might not be a major problem for future experiments since enhancing the gene editing technique may improve outcomes, the scientists said.

"In the future, further improving the efficiency of gene-editing and optimizing the transplantation procedure should accelerate the transition to clinical applications," Deng said.

The initial study mainly aimed to test the safety and feasibility of using genetically-edited stem cells for AIDS treatment. Deng said one key finding is that the procedure did not cause any negative effect.

He added CRISPR has the potential to end blood-related diseases such as AIDS, sickle anemia, hemophilia and beta thalassemia.

Dengs team is not the first group to explore the use of CRISPR. China has been increasing its investment in the gene editing tool, which led to a number of first time experiments.

In 2016, the government announced biotechnology as part of its new Five-Year Plan. China is the first country to allow the use of CRISPR in humans and for the modification of nonviable human embryos.

CRISPR/Cas9 continues to provide scientists new ways to understand and fight previously untreatable diseases. Pixabay

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Scientists Use Gene Editing To Help HIV Patient Delete Disease In China - Medical Daily

In the quest for the next cancer cure, few researchers bother to look back at the graveyard of failed medicines to figure out what went wrong.

The number of failures is staggering: 97 percent of the time that a new drug is tested in a clinical trial for a particular type of cancer, it never makes it to the market. That means the humans (and animals) who participate in these experiments risk their lives on treatments that end up in the dustbin.

Now, a new study helps explain why the rate of failure is so high: In the case of targeted cancer therapies a relatively new class of oncology drugs the medicines may not actually hit the targets researchers intended.

Targeted therapies in cancer work differently from traditional treatments, like chemotherapy. Theyre supposed to be aimed at the specific genes, proteins, or tissues cancer cells rely on to thrive. (Chemo, on the other hand, generally works on all cells that are rapidly dividing, regardless of whether theyre healthy or cancerous.)

The new study, published in Science Translational Medicine, used CRISPR the latest and most precise gene-editing tool available to examine whether 10 different drugs worked as researchers projected. In every case, the researchers found that they didnt.

When the papers authors removed the genes from the genomes of cancer cells that were supposed to be essential for cancer growth, the cells continued to grow. And when they applied the medicines each targeting one of six genes to the newly removed genes, the drugs killed the cancer cells anyway. In other words, even when the supposed target of the therapies was fully deleted, the drugs worked.

This suggests its possible that a big driver of cancer-drug failures in clinical studies is that the drugs dont actually work as drug developers intended.

I hope this paper will help people see the need to raise to bar in terms of how we choose and validate cancer drug targets, said William George Kaelin, a Harvard University professor of medicine who was not involved in the study.

The study should also be a wake-up call for drug developers: [They] should make sure their drugs stop working if the target protein has been genetically removed, said Nathanael Gray, a Dana-Farber Cancer Institute cancer biologist, who was also independent of the research.

The finding is definitely fascinating. But so is the story of why the researchers decided to run the study in the first place and use the latest gene-editing technology to reanalyze, and possibly debunk, previous findings in cancer clinical studies.

A few years ago, one of the papers authors Jason Sheltzer, a research fellow in cancer biology at Cold Spring Harbor Laboratory and his colleagues became interested in a gene called MELK, which is supposed to serve as a biomarker for aggressive breast cancer in patients with a poor prognosis. In the US, some 270,000 new cases of invasive breast cancer will be diagnosed in 2019, and nearly 42,000 women are likely to die from the disease, according to the American Cancer Society.

The researchers started to tinker with the gene using CRISPR and found they couldnt reproduce many of the previous findings about MELK that had been uncovered using older gene-analyzing technologies, such as RNA interference. Namely, even when MELK was cut out, the breast cancer cells proliferated.

When a drug that was supposed to target MELK for breast cancer entered clinical studies, the researchers decided to use CRISPR again, this time to edit out the gene to see whether the drug still worked. We found the drug continued to kill breast cancer cells, regardless of whether the MELK it was targeting was present in the breast cancer genome, said Sheltzer.

This led Sheltzer and his colleagues to a big question: Had they just studied a uniquely bad cancer drug or did we stumble upon a bigger problem? he recalled. The extremely high failure rate [in cancer clinical trials] made us suspect there might be other instances of poorly designed drugs and poorly researched drug targets being tested in human patients.

Enter the new study. Sheltzer and his co-authors chose 10 drugs and drug targets that, like MELK, were at various stages of clinical development. They focused mainly on targets that had been discovered using RNA interference, again, a once-popular gene-analyzing technology that predated CRISPR. And they suspected that like MELK maybe itd been leading researchers down the wrong path.

In each case, they used CRISPR to cut out genes from the genomes of the cancer cells they were looking at genes thought to be essential for cancer growth. And they found that in every case, the drugs killed the cancer cells even though the gene that was supposed to be driving the cancer had been removed.

We wound up with 10 drugs that are potent anti-cancer agents. So we think that if we can figure out what these drugs actually do, we might be able to discover new cancer targets or we might be able to find patients who are more likely to respond, Sheltzer said.

Its also possible this kind of mistaken target helps explain why drugs fall short as they make their way through more and more rigorous stages of clinical studies.

But there could also be other explanations for the misfired targets. Sheltzer acknowledged that they chose medicines primarily discovered with RNA interference technology. And, Technology is always improving. So a lot of the drugs that are being tested in patients now were discovered and characterized five to 10 years ago. Its possible that targeted therapies, discovered more recently using newer genetic technologies, are more precise.

Both Kaelin and Gray shared a word of caution about the study: The researchers focused on targeted drugs that were already known to be problematic. As Kaelin put it, [They] picked drugs against targets where there was never, in my opinion, strong genetic data to support them. So, perhaps, cancer drugs with better-established targets would work as projected.

But Sheltzer says honing in on poor performers was part of the point of the study. A lot of cancer drugs get into clinical trials based on very weak genetic evidence, and when you carefully evaluate them, the rationale for targeting particular genes evaporates.

Either way, he and his colleagues hope the research inspires more analyses into why so many cancer drugs dont help patients. Research funding agencies are very interested in finding the next cure, Sheltzer argued, and they arent excited about this research into reproducibility and why some drugs fail. If we want to accelerate the quest for effective, new treatments, maybe they should be.

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Cancer treatment: drugs often fail in clinical studies. Heres a reason why. - Vox.com

Cancer Cells Resort to Cannibalism To Survive Chemo

By consuming neighboring cancer cells, some cells have found a way to obtain the energy they need to remain alive and induce relapse after a course of chemotherapy is completed.

Published in:Journal of Cell Biology

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Bone Marrow May Be the Missing Piece of the Fertility Puzzle

Study shows that when an egg is fertilized, stem cells leave the bone marrow and travel via the bloodstream to the uterus, where they help transform the uterine lining for implantation.

Published in: PLOS Biology

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Alzheimer's Risk Gene Targets the Brain's Immune Cells

The most prevalent genetic risk factor of Alzheimer's disease (AD), apolipoprotein E4, impairs the function of human brain immune cells, microglia.

Published in: Stem Cell Reports

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Researchers Use CRISPR to Correct Mutation in Duchenne Muscular Dystrophy Model

Duchenne muscular dystrophy (DMD) is a rare but devastating genetic disorder that causes muscle loss and physical impairments. Researchers at the University of Missouri School of Medicine have shown in a mouse study that the powerful gene editing technique known as CRISPR may provide the means for lifelong correction of the genetic mutation responsible for the disorder.

Published in: Molecular TherapyRead full story

Were the Neanderthals Wiped Out by a Common Childhood Illness?

The path to extinction for Neanderthals may well have been the most common and innocuous of childhood illnesses and the bane of every parent of young children chronic ear infections.

Published in:The Anatomical RecordRead full story

Failed drugs often are left on the shelf to gather dust. But sometimes, drugs can be dusted down and repurposed. In this article, we profile a new effort to bring drugs back from the dead and find solutions to conditions such as multiple sclerosis and chronic pain.

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A number of projects are underway to harness bioprinting to print functional human tissues, the first step to printing an entire organ. In this article, we take a closer look at three of these projects.

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TSE Explores Microplastics Detection Techniques

Astrocytes

Blood vessels and astrocytes in aging rat retina, confocal imaging, 40x. Blood vessels are shown in blue; astrocytes (supportive cells of the nervous system) are mostly in red. As organisms age, changes in astrocytes might contribute to disease and degeneration.

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7 Days in Science September 20, 2019 - Technology Networks

Genomes of living organism like that of humans can be edited with the technique of CRISPR. Things that could only be imagines earlier can be made possible with this technique, such as reversing congenital conditions or killing off viruses. CRISPR has now found another application in which it equips materials to change their properties when nearby there are specific DNA sequences.

The research behind this technology was done by a team of scientists from MIT and Harvard who also developed multiple types of devices using the technology. These include an electronic circuit that reacts to DNA cues, a microfluidic device with a DNA sensor that activates a valve to open and close and also gels that release drugs. The idea is to deliver therapies, perform diagnostics, and many impossible tasks up till now; by the interaction between human body and a whole set of new smart materials.

With the help of proteins known as Cas enzymes, DNA can be cut by scientists in specific locations by using CRISPR. A single- stranded DNA was used by the scientists in this new research as a structural component or a control mechanism. This gave smart biological functionality to whatever material it is in.

They developed a polyethylene glycol gel containing DNA bound to encapsulated drug. Acrylamide gel with the DNA was also created by the team. An electronic circuit with another gel was also created by the team with idea of advancing the technique where the result was conductive when the DNA strands within it are intact. One of the next things the team is working on is to find a way to use the technology to deliver engineered bacteria to help treat conditions that are gastrointestinal.

Gaurang Tayloris an MD/MBA candidate at the Johns Hopkins School of Medicine and Harvard Business School. He contributes regularly to CardioSource World News and Emergency Physicians Monthly. He is interested in developing scalable, tech-based solutions for medicine and education. He loves to share his knowledge and recent trends in the Healthcare Department by posting various articles. He has experience in medical device pathways and is passionate about understanding the human body.

Mail: gaurang.taylor@storyoffuture.com

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Biological Cues | CRISPR-Responsive Materials - Story of Future

DURHAM Biomedical engineers at Duke University have developed a method to address failures in a promising anti-cancer drug, bringing together tools from genome engineering, protein engineering and biomaterials science to improve the efficacy, accuracy and longevity of certain cancer therapies.

Using a combination of CRISPR-based targeting, a protein depot that allows for sustained release of the drug and a highly potent binding system, the team showed that their new strategy could overcome three critical problems that limit the efficacy of many cancer drugs their limited potency, their quick elimination from the body, and the ability of cancer cells to develop resistance to the drug.

The research appeared online Sept. 4 in the journal Science Advances.

More than 20 years ago, researchers discovered that the protein drug TRAIL, short for TNF-related apoptosis-inducing ligand, could effectively kill cancer cells without harming healthy cells at least, in the lab. TRAIL works by binding to specific protein receptors on cancer cells, ominously called death receptors, sending a signal that causes the cells to self-destruct. Although initial experiments showed the drug worked in a variety of cancer cell lines, including melanoma, lymphoma, pancreatic, prostate, lung, colon and breast cancer, TRAIL and similar drugs surprised researchers by showing limited success in clinical trials.

After more study, scientists pinpointed three reasons why the promising drug failed: TRAIL wasnt potent enough, the drug was being cleared from the body too quickly and some cancer cells were resistant to the therapy.

Duke engineers improve CRISPR genome editing with biomedical tails

Using a combination of three tools a highly potent protein drug, a depot that allows for sustained release of the drug, and CRISPR/Cas9 based gene editing to pinpoint the cause of resistance to the drug the Duke team, which included Mandana Manzari, a recent PhD graduate,Ashutosh Chilkoti, the chair of Duke biomedical engineering, andKris Wood, an assistant professor of pharmacology and cancer biology, demonstrated that their new strategy could provide a solution to these problems and give protein-based anti-cancer biologics like TRAIL that failed in the clinic a second chance.

The real significance of this research for me is the true cross-disciplinary nature of it, said Manzari, first author on the paper and now a post-doctoral researcher at the Memorial Sloan Kettering Cancer Center in New York. This is really the first example Ive seen where were bringing in pharmacology, drug delivery, and genomics to pinpoint the exact circumstances that cause a biologic to fail and then develop solutions.

The first step of the process involved addressing TRAILs limited potency. Typically, cells have multiple death receptors, but a specific receptor called death receptor 5 (DR5) is more prevalent in certain cancer cells. TRAIL, a three-part protein, binds to DR5 and links three death receptors together, sending a signal for cells to self-destruct. TRAIL can also bind to other death receptors and decoy receptors on normal cells. A more potent drug would be specific for a given death receptor, like DR5 that is present on cancer cells, and link together larger numbers of the receptor on a cell surface to send a stronger death signal to the cancer cell.

Manzari produced a highly potent, six-part death receptor agonist (DRA) that could bind six death receptors together and indude a much stronger self-destruct signal.

Next, the team examined how to prevent the super-potent death receptor agonist from being cleared from the body too quickly. They genetically fused the DRA to a temperature-responsive protein called elastin-like polypeptide (ELP), which forms a gel-like depot within a room-temperature solution. After the solution is injected under the skin, it dissolves, releasing the DRA over a longer period of time.

Duke researchers: Single CRISPR treatment provides long-term benefits in mice

Finally, Chilkoti and Manzari partnered with Kris Wood to better understand what caused certain cells to resist death by TRAIL or death receptor agonist (DRA). The team systematically disabled various genes in the cancer cells using CRISPR/Cas9 until they could deduce which were responsible for TRAIL or DRA resistance. Then they selected drugs to target the proteins produced by those genes and paired them with the DRA slow-release depot.

This work opens another exciting avenue for targeting a critical cell death pathway in cancer, an area of increasing interest in the translational cancer therapeutics community, Wood said.

When we figured out the genes that drive resistance, we were able to map them to commercially-available drugs that could specifically target the proteins that come from those genes, said Manzari. It basically gave us a platform to figure out what drugs we can combine with the DRA in cases where this drug or other protein drugs dont work well to nip that resistance in the bud.

With their triple-whammy tool, the team was able to effectively overcome intrinsic resistance, repress tumor growth and extend survival in mice that were implanted with colorectal cancers from human patients that are highly resistant to treatment with TRAIL.

Now, the researchers are considering how they could apply this method to other protein and small-molecule drugs that face similar barriers that limit their effectiveness.

I think the thing that really sets this approach apart is designing each piece of the platform rationally to address a specific problem and bringing them all together holistically to solve three critical problems that limit not just TRAIL, but many new cancer therapies, Chilkoti said.

Typically the protein engineering is one platform, the ELP strategy is one platform and the genomic screen strategy is its own platform, Manzari said. This is a good example of true synergy of engineering, pharmacology, genomics and materials. People always talk about bringing those together, and this is a clear example of that.

(C) Duke University

This research was funded by the National Institutes of Health (5R01EB007025-08, 5R01EB000188-12, 5R01GM061232-16, R01CA207083, and 5T32GM007105) and the Duke University BME/DCI Collaborative Grant.

CITATION: Genomically Informed Small Molecule Drugs Overcome Resistance to a Sustained Release Formulation of an Engineered Death Receptor Agonist in Patient-Derived Tumor Models, Mandana T. Manzari, Grace R. Anderson, Kevin H. Lin, Ryan S. Soderquist, Merve Cakir, Mitchell Zhang, Chandler E. Moore, Rachel N. Skelton, Mareva Fevre, Xinghai Li, Joseph J. Bellucci, Suzanne E. Wardell, Simone A. Costa, Kris C. Wood, Ashutosh Chilkoti. Science Advances, 2019. DOI 10.1126/sciadv.aaw9162

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Duke researchers utilize gene editing to improve cancer drugs' performance - WRAL Tech Wire

(more about Power Words)

applicationA particular use or function of something.

base (in genetics) A shortened version of the term nucleobase. These bases are building blocks of DNA and RNA molecules.

biologyThe study of living things. The scientists who study them are known as biologists.

Cas9An enzyme that geneticists are now using to help edit genes. It can cut through DNA, allowing it to fix broken genes, splice in new ones or disable certain genes. Cas9 is shepherded to the place it is supposed to make cuts by CRISPRs, a type of genetic guides. The Cas9 enzyme came from bacteria. When viruses invade a bacterium, this enzyme can chop up the germs DNA, making it harmless.

cellThe smallest structural and functional unit of an organism. Typically too small to see with the naked eye, it consists of watery fluid surrounded by a membrane or wall. Animals are made of anywhere from thousands to trillions of cells, depending on their size. Some organisms, such as yeasts, molds, bacteria and some algae, are composed of only one cell.

chemicalA substance formed from two or more atoms that unite (become bonded together) in a fixed proportion and structure. For example, water is a chemical made of two hydrogen atoms bonded to one oxygen atom. Its chemical symbol is H2O.

CRISPRAn abbreviation pronounced crisper for the term clustered regularly interspaced short palindromic repeats. These are pieces of RNA, an information-carrying molecule. They are copied from the genetic material of viruses that infect bacteria. When a bacterium encounters a virus that it was previously exposed to, it produces an RNA copy of the CRISPR that contains that virus genetic information. The RNA then guides an enzyme, called Cas9, to cut up the virus and make it harmless. Scientists are now building their own versions of CRISPR RNAs. These lab-made RNAs guide the enzyme to cut specific genes in other organisms. Scientists use them, like a genetic scissors, to edit or alter specific genes so that they can then study how the gene works, repair damage to broken genes, insert new genes or disable harmful ones.

developmental(in biology) An adjective that refers to the changes an organism undergoes from conception through adulthood. Those changes often involve chemistry, size and sometimes even shape.

DNA(short for deoxyribonucleic acid) A long, double-stranded and spiral-shaped molecule inside most living cells that carries genetic instructions. It is built on a backbone of phosphorus, oxygen, and carbon atoms. In all living things, from plants and animals to microbes, these instructions tell cells which molecules to make.

engineeringThe field of research that uses math and science to solve practical problems.

fieldAn area of study, as in: Her field of research was biology. Also a term to describe a real-world environment in which some research is conducted, such as at sea, in a forest, on a mountaintop or on a city street. It is the opposite of an artificial setting, such as a research laboratory.

fluorescentCapable of absorbing and reemitting light. That reemitted light is known as a fluorescence.

gene(adj. genetic) A segment of DNA that codes, or holds instructions, for producing a protein. Offspring inherit genes from their parents. Genes influence how an organism looks and behaves.

genomeThe complete set of genes or genetic material in a cell or an organism. The study of this genetic inheritance housed within cells is known as genomics.

muscleA type of tissue used to produce movement by contracting its cells, known as muscle fibers. Muscle is rich in a protein, which is why predatory species seek prey containing lots of this tissue.

mutation(v. mutate) Some change that occurs to a gene in an organisms DNA. Some mutations occur naturally. Others can be triggered by outside factors, such as pollution, radiation, medicines or something in the diet. A gene with this change is referred to as a mutant.

nucleusPlural is nuclei. (in biology) A dense structure present in many cells. Typically a single rounded structure encased within a membrane, the nucleus contains the genetic information.

organ(in biology) Various parts of an organism that perform one or more particular functions. For instance, an ovary is an organ that makes eggs, the brain is an organ that interprets nerve signals and a plants roots are organs that take in nutrients and moisture.

palindrome (adj. palindromic) A word, a name or a phrase that has the same ordering of letters when read forwards or backwards. For instance, dad and mom are both palindromes.

proteinCompoundmade from one or more long chains of amino acids. Proteins are an essential part of all living organisms. They form the basis of living cells, muscle and tissues; they also do the work inside of cells. The hemoglobin in blood and the antibodies that attempt to fight infections are among the better-known, stand-alone proteins. Medicines frequently work by latching onto proteins.

RNAA molecule that helps read the genetic information contained in DNA. A cells molecular machinery reads DNA to create RNA, and then reads RNA to create proteins.

tag(in biology) To attach some rugged band or package of instruments onto an animal. Sometimes the tag is used to give each individual a unique identification number. Once attached to the leg, ear or other part of the body of a critter, it can effectively become the animals name. In some instances, a tag can collect information from the environment around the animal as well. This helps scientists understand both the environment and the animals role within it.

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Explainer: How CRISPR works | Science News for Students

Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811 (1998)

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Root, D. E., Hacohen, N., Hahn, W. C., Lander, E. S. & Sabatini, D. M. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat. Methods 3, 715719 (2006)

Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635637 (2003)

Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat. Methods 6, 331338 (2009)

Shmakov, S. et al. Diversity and evolution of class 2 CRISPRCas systems. Nat. Rev. Microbiol. 15, 169182 (2017)

Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPRCas systems. Mol. Cell 60, 385397 (2015)

Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016)

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RNA targeting with CRISPRCas13 | Nature

Discovery of CRISPR and its function 1993 - 2005 Francisco Mojica, University of Alicante, Spain

Francisco Mojica was the first researcher to characterize what is now called a CRISPR locus, reported in 1993. He worked on them throughout the 1990s, and in 2000, he recognized that what had been reported as disparate repeat sequences actually shared a common set of features, now known to be hallmarks of CRISPR sequences (he coined the term CRISPR through correspondence with Ruud Jansen, who first used the term in print in 2002). In 2005 he reported that these sequences matched snippets from the genomes of bacteriophage (Mojica et al., 2005). This finding led him to hypothesize, correctly, that CRISPR is an adaptive immune system. Another group, working independently, published similar findings around this same time (Pourcel et al., 2005)

Discovery of Cas9 and PAMMay, 2005 Alexander Bolotin, French National Institute for Agricultural Research (INRA)

Bolotin was studying the bacteria Streptococcus thermophilus, which had just been sequenced, revealing an unusual CRISPR locus (Bolotin et al., 2005). Although the CRISPR array was similar to previously reported systems, it lacked some of the known cas genes and instead contained novel cas genes, including one encoding a large protein they predicted to have nuclease activity, which is now known as Cas9. Furthermore, they noted that the spacers, which have homology to viral genes, all share a common sequence at one end. This sequence, the protospacer adjacent motif (PAM), is required for target recognition.

Hypothetical scheme of adaptive immunityMarch, 2006 Eugene Koonin, US National Center for Biotechnology Information, NIH

Koonin was studying clusters of orthologous groups of proteins by computational analysis and proposed a hypothetical scheme for CRISPR cascades as bacterial immune system based on inserts homologous to phage DNA in the natural spacer array, abandoning previous hypothesis that the Cas proteins might comprise a novel DNA repair system.(Makarova et al., 2006)

Experimental demonstration of adaptive immunityMarch, 2007 Philippe Horvath, Danisco France SAS

S. thermophilus is widely used in the dairy industry to make yogurt and cheese, and scientists at Danisco wanted to explore how it responds to phage attack, a common problem in industrial yogurt making. Horvath and colleagues showed experimentally that CRISPR systems are indeed an adaptive immune system: they integrate new phage DNA into the CRISPR array, which allows them to fight off the next wave of attacking phage (Barrangou et al., 2007). Furthermore, they showed that Cas9 is likely the only protein required for interference, the process by which the CRISPR system inactivates invading phage, details of which were not yet known.

Spacer sequences are transcribed into guide RNAsAugust, 2008 John van der Oost, University of Wageningen, Netherlands

Scientists soon began to fill in some of the details on exactly how CRISPR-Cas systems interfere with invading phage. The first piece of critical information came from John van der Oost and colleagues who showed that in E-scherichia coli, spacer sequences, which are derived from phage, are transcribed into small RNAs, termed CRISPR RNAs (crRNAs), that guide Cas proteins to the target DNA (Brouns et al., 2008).

CRISPR acts on DNA targets December, 2008 Luciano Marraffini and Erik Sontheimer, Northwestern University, Illinois

The next key piece in understanding the mechanism of interference came from Marraffini and Sontheimer, who elegantly demonstrated that the target molecule is DNA, not RNA (Marraffini and Sontheimer, 2008). This was somewhat surprising, as many people had considered CRISPR to be a parallel to eukaryotic RNAi silencing mechanisms, which target RNA. Marraffini and Sontheimer explicitly noted in their paper that this system could be a powerful tool if it could be transferred to non-bacterial systems. (It should be noted, however, that a different type of CRISPR system can target RNA (Hale et al., 2009)).

Cas9 cleaves target DNADecember, 2010 Sylvain Moineau, University of Laval, Quebec City, Canada

Moineau and colleagues demonstrated that CRISPR-Cas9 creates double-stranded breaks in target DNA at precise positions, 3 nucleotides upstream of the PAM (Garneau et al., 2010). They also confirmed that Cas9 is the only protein required for cleavage in the CRISPR-Cas9 system. This is a distinguishing feature of Type II CRISPR systems, in which interference is mediated by a single large protein (here Cas9) in conjunction with crRNAs.

Discovery of tracrRNA for Cas9 systemMarch, 2011 Emmanuelle Charpentier, Umea University, Sweden and University of Vienna, Austria

The final piece to the puzzle in the mechanism of natural CRISPR-Cas9-guided interference came from the group of Emmanuelle Charpentier. They performed small RNA sequencing on Streptococcus pyogenes, which has a Cas9-containing CRISPR-Cas system. They discovered that in addition to the crRNA, a second small RNA exists, which they called trans-activating CRISPR RNA (tracrRNA) (Deltcheva et al., 2011). They showed that tracrRNA forms a duplex with crRNA, and that it is this duplex that guides Cas9 to its targets.

CRISPR systems can function heterologously in other species July, 2011 Virginijus Siksnys, Vilnius University, Lithuania

Siksnys and colleagues cloned the entire CRISPR-Cas locus from S. thermophilus (a Type II system) and expressed it in E. coli (which does not contain a Type II system), where they demonstrated that it was capable of providing plasmid resistance (Sapranauskas et al., 2011). This suggested that CRISPR systems are self-contained units and verified that all of the required components of the Type II system were known.

Biochemical characterization of Cas9-mediated cleavageSeptember, 2012 Virginijus Siksnys, Vilnius University, Lithuania

Taking advantage of their heterologous system, Siksnys and his team purified Cas9 in complex with crRNA from the E. coli strain engineered to carry the S. thermophilus CRISPR locus and undertook a series of biochemical experiments to mechanistically characterize Cas9s mode of action (Gasiunas et al., 2012).They verified the cleavage site and the requirement for the PAM, and using point mutations, they showed that the RuvC domain cleaves the non-complementary strand while the HNH domain cleaves the complementary site. They also noted that the crRNA could be trimmed down to a 20-nt stretch sufficient for efficient cleavage. Most impressively, they showed that they could reprogram Cas9 to target a site of their choosing by changing the sequence of the crRNA.

June, 2012 Charpentier and Jennifer Doudna, University of California, Berkeley

Similar findings as those in Gasiunas et al. were reported at almost the same time by Emmanuelle Charpentier in collaboration with Jennifer Doudna at the University of California, Berkeley (Jinek et al., 2012). Charpentier and Doudna also reported that the crRNA and the tracrRNA could be fused together to create a single, synthetic guide, further simplifying the system. (Although published in June 2012, this paper was submitted after Gasiunas et al.)

CRISPR-Cas9 harnessed for genome editingJanuary, 2013 Feng Zhang, Broad Institute of MIT and Harvard, McGovern Institute for Brain Research at MIT, Massachusetts

Zhang, who had previously worked on other genome editing systems such as TALENs, was first to successfully adapt CRISPR-Cas9 for genome editing in eukaryotic cells (Cong et al., 2013). Zhang and his team engineered two different Cas9 orthologs (from S. thermophilus and S. pyogenes) and demonstrated targeted genome cleavage in human and mouse cells. They also showed that the system (i) could be programmed to target multiple genomic loci, and (ii) could drive homology-directed repair. Researchers from George Churchs lab at Harvard University reported similar findings in the same issue of Science (Mali et al., 2013).

Citations

Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 17091712.

Bolotin, A., Quinquis, B., Sorokin, A.,and Ehrlich, S.D. (2005). Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 25512561.

Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V., van der Oost, J. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960-964.

Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819823.

Deltcheva, E., Chylinski, K., Sharma, C.M., Gonzales, K., Chao, Y., Pirzada, Z.A., Eckert, M.R., Vogel, J., and Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602607.

Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012). Cas9crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Pnas 109, E2579E2586.

Hale, C.R., Zhao, P., Olson, S., Duff, M.O., Graveley, B.R., Wells, L., Terns, R.M., and Terns, M.P. (2009). RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex. Cell 139, 945956.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816821.

Makarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I., Koonin, E.V. (2006). A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biology Direct 2006, 1:7.

Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., and Church, G.M. (2013). RNA-guided human genome engineering via Cas9. Science 339, 823826.

Marraffini, L.A., and Sontheimer, E.J. (2008). CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 18431845.

Mojica, F.J.M., D ez-Villase or, C.S., Garc a-Mart nez, J.S., and Soria, E. (2005). Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements. J Mol Evol 60, 174182.

Pourcel, C., Salvignol, G., and Vergnaud, G. (2005). CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653663.

Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P., and Siksnys, V. (2011). The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucl. Acids Res. 39, gkr606gkr9282.

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CRISPR Timeline | Broad Institute

-Enrollment ongoing in Phase 1/2 clinical trials of CTX001 for patients with severe hemoglobinopathies-

-IND and CTA approved for CTX110, wholly-owned allogeneic CAR-T cell therapy targeting CD19+ malignancies-

-On track to initiate Phase 1/2 clinical trial for CTX110 in 1H 2019-

-$437.5 million in cash as of March 31, 2019-

ZUG, Switzerland and CAMBRIDGE, Mass., April 29, 2019 (GLOBE NEWSWIRE) -- CRISPR Therapeutics(CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today reported financial results for the first quarter ended March 31, 2019.

This past quarter, we began an important new period for CRISPR Therapeutics with the treatment of the first patient in our clinical trial for CTX001 in hemoglobinopathies, said Samarth Kulkarni, Ph.D., Chief Executive Officer of CRISPR Therapeutics. This is a significant landmark for the Company and we continue to enroll patients in our trials for both beta thalassemia and sickle cell disease. With the acceptance of our IND and CTA for CTX110, we look forward to the initiation of our clinical trials for our allogeneic CAR-T programs in the near-term and hope to bring other CAR-T programs to the clinic in the next six to twelve months.

Recent Highlights and Outlook

First Quarter 2019 Financial Results

About CRISPR TherapeuticsCRISPR Therapeutics is a leading gene editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR/Cas9 platform. CRISPR/Cas9 is a revolutionary gene editing technology that allows for precise, directed changes to genomic DNA. CRISPR Therapeutics has established a portfolio of therapeutic programs across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine and rare diseases. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic collaborations with leading companies including Bayer AG, Vertex Pharmaceuticals and ViaCyte, Inc. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Cambridge, Massachusetts, and business offices in London, United Kingdom. For more information, please visit http://www.crisprtx.com.

CRISPR Forward-Looking StatementThis press release may contain a number of forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, as amended, including statements regarding CRISPR Therapeutics expectations about any or all of the following: (i) clinical trials (including, without limitation, the timing of filing of clinical trial applications and INDs, any approvals thereof and the timing of commencement of clinical trials), development timelines and discussions with regulatory authorities related to product candidates under development by CRISPR Therapeutics and its collaborators; (ii) the number of patients that will be evaluated, the anticipated date by which enrollment will be completed and the data that will be generated by ongoing and planned clinical trials, and the ability to use that data for the design and initiation of further clinical trials; (iii) the scope and timing of ongoing and potential future clinical trials; (iv) the intellectual property coverage and positions of CRISPR Therapeutics, its licensors and third parties; (v) the sufficiency of CRISPR Therapeutics cash resources; and (vi) the therapeutic value, development, and commercial potential of CRISPR/Cas9 gene editing technologies and therapies. Without limiting the foregoing, the words believes, anticipates, plans, expects and similar expressions are intended to identify forward-looking statements. You are cautioned that forward-looking statements are inherently uncertain. Although CRISPR Therapeutics believes that such statements are based on reasonable assumptions within the bounds of its knowledge of its business and operations, forward-looking statements are neither promises nor guarantees and they are necessarily subject to a high degree of uncertainty and risk. Actual performance and results may differ materially from those projected or suggested in the forward-looking statements due to various risks and uncertainties. These risks and uncertainties include, among others: the outcomes for each CRISPR Therapeutics planned clinical trials and studies may not be favorable; that one or more of CRISPR Therapeutics internal or external product candidate programs will not proceed as planned for technical, scientific or commercial reasons; that future competitive or other market factors may adversely affect the commercial potential for CRISPR Therapeutics product candidates; uncertainties inherent in the initiation and completion of preclinical studies for CRISPR Therapeutics product candidates; availability and timing of results from preclinical studies; whether results from a preclinical trial will be predictive of future results of the future trials; uncertainties about regulatory approvals to conduct trials or to market products; uncertainties regarding the intellectual property protection for CRISPR Therapeutics technology and intellectual property belonging to third parties; and those risks and uncertainties described under the heading "Risk Factors" in CRISPR Therapeutics most recent annual report on Form 10-K, and in any other subsequent filings made by CRISPR Therapeutics with the U.S. Securities and Exchange Commission, which are available on the SEC's website at http://www.sec.gov. Existing and prospective investors are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date they are made. CRISPR Therapeutics disclaims any obligation or undertaking to update or revise any forward-looking statements contained in this press release, other than to the extent required by law.

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Gene editing method

CRISPR gene editing is a method by which the genomes of living organisms may be edited. It is based on a simplified version of the bacterial CRISPR/Cas (CRISPR-Cas9) antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.[1] The Cas9-gRNA complex corresponds with the CAS III CRISPR-RNA complex in the accompanying diagram.

While genomic editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proved to be inefficient and impractical to implement on a larger scale. Genomic editing leads to irreversible changes to the gene. Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of DNA to introduce the modification by one of two methods. Knock-in mutations, facilitated via Homology Directed Repair (HDR), is the traditional pathway of targeted genomic editing approaches.[2] This allows for the introduction of targeted DNA damage and repair. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template.[2] This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for a repair to commence. Knock-out mutations caused by Cas9/CRISPR results in the repair of the double-strand break by means of NHEJ (Non-Homologous End Joining). NHEJ can often result in random deletions or insertions at the repair site disrupting or altering gene functionality. Therefore, genomic engineering by CRISPR-Cas9 allows researchers the ability to generate targeted random gene disruption.

Because of this, the precision of genomic editing is a great concern. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing is now a reality. Cas9 allows for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRna guide strands.[3] Cas9 derived from Streptococcus pyogenes bacteria has facilitated the targeted genomic modification in eukaryotic cells. The ease with which researchers can insert Cas9 and template RNA in order to silence or cause point mutations on specific loci has proved invaluable to the quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. A newly engineered variant of the Cas9 nuclease has been developed that significantly reduces off-target manipulation. Called spCas9-HF1 (Streptococcus pyogenes Cas9 High Fidelity 1), it has a success rate of modification in vivo of 85% and undetectable off-target manipulations as measured by genome wide break capture and targeted sequencing methods used to measure total genomic changes.[4][5]

CRISPR-Cas genome editing techniques have many potential applications, including medicine and crop seed enhancement. The use of CRISPR-Cas9-gRNA complex for genome editing[6] was the AAAS's choice for breakthrough of the year in 2015.[7] Bioethical concerns have been raised about the prospect of using CRISPR for germline editing.[8]

In the early 2000s, researchers developed zinc finger nucleases (ZFNs), synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the creation of a custom protein for each targeted DNA sequence, which is a more difficult and time-consuming process than that for guide RNAs. CRISPRs are much easier to design because the process requires making only a short RNA sequence.[9]

Whereas RNA interference (RNAi) does not fully suppress gene function, CRISPR, ZFNs and TALENs provide full irreversible gene knockout.[10] CRISPR can also target several DNA sites simultaneously by simply introducing different gRNAs. In addition, CRISPR costs are relatively low.[10][11][12]

CRISPR-Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, crRNA, tracrRNA along with an optional section of DNA repair template that is utilized in either non-homologous end joining (NHEJ) or homology directed repair (HDR).

CRISPR-Cas9 often employs a plasmid to transfect the target cells.[13] The main components of this plasmid are displayed in the image and listed in the table. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the cell's DNA. The crRNA must bind only where editing is desired. The repair template is designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.

Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA).[14] This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.

CRISPR-Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the PAM. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array.[13] A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently synthesized.[15][16]

The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. However this is not too limiting as it is a short sequence and nonspecific (e.g. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs).[13]

Once these have been assembled into a plasmid and transfected into cells the Cas9 protein with the help of the crRNA finds the correct sequence in the host cell's DNA and depending on the Cas9 variant creates a single or double strand break in the DNA.[17]

Properly spaced single strand breaks in the host DNA can trigger homology directed repair, which is less error prone than the non-homologous end joining that typically follows a double strand break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9 induced DNA break.[13] The goal is for the cell's HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells.

Many online tools are available to aid in designing effective sgRNA sequences.[18][19]

Delivery of Cas9, sgRNA, and associated complexes into cells can occur via viral and non-viral systems. Electroporation of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells.[20] Chemical transfection techniques utilizing lipids have also been used to introduce sgRNA in complex with Cas9 into cells.[21] Hard-to-transfect cells (e.g. stem cells, neurons, and hematopoietic cells) require more efficient delivery systems such as those based on lentivirus (LVs), adenovirus (AdV) and adeno-associated virus (AAV).[22][23]

Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules.[24][25][26] These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation,[27][28] or fusing similar light responsive domains with two constructs of split-Cas9,[29][30] or by incorporating caged unnatural amino acids into Cas9,[31] or by modifying the guide RNAs with photocleavable complements for genome editing.[32]

Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of 4-hydroxytamoxifen (4-HT),[24] 4-HT responsive intein-linked Cas9s[33] or a Cas9 that is 4-HT responsive when fused to four ERT2 domains.[34] Intein-inducible split-Cas9 allows dimerization of Cas9 fragments[35] and Rapamycin-inducible split-Cas9 system developed by fusing two constructs of split Cas9 with FRB and FKBP fragments.[36] Furthermore, other studies have shown to induce transcription of Cas9 with a small molecule, doxycycline.[37][38] Small molecules can also be used to improve Homology Directed Repair (HDR),[39] often by inhibiting the Non-Homologous End Joining (NHEJ) pathway.[40] These systems allow conditional control of CRISPR activity for improved precision, efficiency and spatiotemporal control.

Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. Cas9 can be easily introduced into the target cells via plasmid transfection along with sgRNA in order to model the spread of diseases and the cell's response and defense to infection.[41] The ability of Cas9 to be introduced in vivo allows for the creation of more accurate models of gene function, mutation effects, all while avoiding the off-target mutations typically observed with older methods of genetic engineering. The CRISPR and Cas9 revolution in genomic modeling doesn't only extend to mammals. Traditional genomic models such as Drosophila melanogaster, one of the first model species, have seen further refinement in their resolution with the use of Cas9.[41] Cas9 uses cell-specific promoters allowing a controlled use of the Cas9. Cas9 is an accurate method of treating diseases due to the targeting of the Cas9 enzyme only affecting certain cell types. The cells undergoing the Cas9 therapy can also be removed and reintroduced to provide amplified effects of the therapy.[42]

CRISPR-Cas9 can be used to edit the DNA of organisms in vivo and entire chromosomes can be eliminated from an organism at any point in its development. Chromosomes that have been deleted in vivo are the Y chromosomes and X chromosomes of adult lab mice and human chromosomes 14 and 21, in embryonic stem cell lines and aneuploid mice respectively. This method might be useful for treating genetic aneuploid diseases such as Down Syndrome and intersex disorders.[43]

Successful in vivo genome editing using CRISPR-Cas9 has been shown in several model organisms, such as Escherichia coli,[44] Saccharomyces cerevisiae,[45] Candida albicans,[46] Caenorhadbitis elegans,[47] Arabidopsis,[48] Danio rerio,[49] Mus musculus.[50][51] Successes have been achieved in the study of basic biology, in the creation of disease models,[47] and in the experimental treatment of disease models.[52]

Concerns have been raised that off-target effects (editing of genes besides the ones intended) may obscure the results of a CRISPR gene editing experiment (the observed phenotypic change may not be due to modifying the target gene, but some other gene). Modifications to CRISPR have been made to minimize the possibility of off-target effects. In addition, orthogonal CRISPR experiments are recommended to confirm the results of a gene editing experiment.[53][54]

CRISPR simplifies creation of animals for research that mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the germline level to create animals where the gene is changed everywhere, or it may be targeted at non-germline cells.[55][56][57]

CRISPR can be utilized to create human cellular models of disease. For instance, applied to human pluripotent stem cells CRISPR introduced targeted mutations in genes relevant to polycystic kidney disease (PKD) and focal segmental glomerulosclerosis (FSGS).[58] These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids that exhibited disease-specific phenotypes. Kidney organoids from stem cells with PKD mutations formed large, translucent cyst structures from kidney tubules. The cysts were capable of reaching macroscopic dimensions, up to one centimeter in diameter.[59] Kidney organoids with mutations in a gene linked to FSGS developed junctional defects between podocytes, the filtering cells affected in that disease. This was traced to the inability of podocytes ability to form microvilli between adjacent cells.[60] Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications.[58]

A similar approach was taken to model long QT syndrome in cardiomyocytes derived from pluripotent stem cells.[61] These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.

CRISPR-Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause.[62] Its ability to modify specific DNA sequences makes it a tool with potential to fix disease-causing mutations. Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases,[63] including cancer,[64] beta-thalassemia,[65] sickle cell disease,[66] hemophilia,[67] cystic fibrosis,[68] Duchenne's muscular dystrophy,[69] Huntington's,[70][71] and heart disease.[72] CRISPR may have applications in tissue engineering and regenerative medicine, such as by creating human blood vessels that lack expression of MHC class II proteins, which often cause transplant rejection.[73]

CRISPR-Cas-based "RNA-guided nucleases" can be used to target virulence factors, genes encoding antibiotic resistance and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations.[74][75] Recent studies suggested a correlation between the interfering of the CRISPR-Cas locus and acquisition of antibiotic resistance[76] This system provides protection of bacteria against invading foreign DNA, such as transposons, bacteriophages and plasmids. This system was shown to be a strong selective pressure for the acquisition of antibiotic resistance and virulence factor in bacterial pathogens.[76]

Therapies based on CRISPRCas3 gene editing technology delivered by engineered bacteriophages could be used to destroy targeted DNA in pathogens. [77] Cas3 is more destructive than the better known Cas9[78][79]

Research suggests that CRISPR is an effective way to limit replication of multiple herpesviruses. It was able to eradicate viral DNA in the case of Epstein-Barr virus (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for immunocompromised patients of viral invaders, or preventing cold sore outbreaks and recurrent eye infections by blocking HSV-1 reactivation. As of August2016[update], these were awaiting testing.[80]

CRISPR may revive the concept of transplanting animal organs into people. Retroviruses present in animal genomes could harm transplant recipients. In 2015, a team eliminated 62 copies of a retrovirus's DNA from the pig genome in a kidney epithelial cell.[81] Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using CRISPR for the first time.[82]

As of 2016[update] CRISPR had been studied in animal models and cancer cell lines, to learn if it can be used to repair or thwart mutated genes that cause cancer.[83]

The first clinical trial involving CRISPR started in 2016. It involved removing immune cells from people with lung cancer, using CRISPR to edit out the gene expressed PD-1, then administrating the altered cells back to the same person. 20 other trials were under way or nearly ready, mostly in China, as of 2017[update].[64]

In 2016, the United States Food and Drug Administration (FDA) approved a clinical trial in which CRISPR would be used to alter T cells extracted from people with different kinds of cancer and then administer those engineered T cells back to the same people.[84]

Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity.[81] Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription. These precisely placed modifications may then be used to regulate the effects on gene expressions and DNA dynamics after the inhibition of certain genome sequences within DNA. Within the past few years, epigenetic marks in different human cells have been closely researched and certain patterns within the marks have been found to correlate with everything ranging from tumor growth to brain activity.[6] Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription.[85] Cas9 is an effective way of targeting and silencing specific genes at the DNA level.[86] In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene.[87]

Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.[87]

In 2016, researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit RNA. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled Crispr genes. They considered the fusobacteria Leptotrichia shahii. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense.[88]

Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses. HIV and poliovirus are such viruses. Bacteria with C2c2 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing.[88]

CRISPR-Cas systems can also be employed for editing of micro-RNA and long-noncoding RNA genes in plants.[89]

Gene drives may provide a powerful tool to restore balance of ecosystems by eliminating invasive species. Concerns regarding efficacy, unintended consequences in the target species as well as non-target species have been raised particularly in the potential for accidental release from laboratories into the wild. Scientists have proposed several safeguards for ensuring the containment of experimental gene drives including molecular, reproductive, and ecological.[90] Many recommend that immunization and reversal drives be developed in tandem with gene drives in order to overwrite their effects if necessary.[91] There remains consensus that long-term effects must be studied more thoroughly particularly in the potential for ecological disruption that cannot be corrected with reversal drives.[92]

Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant off-target effects as seen with restriction enzymes. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries.[93]

As of November2013[update], SAGE Labs (part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models.[94] By 2015[update], Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits.[95]

As of December2014[update], patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools.[96] As companies ramp up financing, doubts as to whether CRISPR can be quickly monetized were raised.[97] In February 2017 the US Patent Office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR-Cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California.[98][99][100]Shortly after, University of California filed an appeal of this ruling.[101][102]

In March 2017, the European Patent Office (EPO) announced its intention to allow broad claims for editing all kinds of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna,[103][104] and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed.[103] As of August2017[update] the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad.[105]

As of March 2015, multiple groups had announced ongoing research to learn how they one day might apply CRISPR to human embryos, including labs in the US, China, and the UK, as well as US biotechnology company OvaScience.[106] Scientists, including a CRISPR co-discoverer, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations".[107][108] These scientists support further low-level research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans.[109]

In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes beta thalassemia, a lethal heritable disorder.[110][111] The study had previously been rejected by both Nature and Science in part because of ethical concerns.[112] The experiments resulted in successfully changing only some of the intended genes, and had off-target effects on other genes. The researchers stated that CRISPR is not ready for clinical application in reproductive medicine.[112] In April 2016, Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR - this time to alter the CCR5 gene to make the embryo HIV resistant.[113]

In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of David Baltimore. Members of national scientific academies of America, Britain and China discussed the ethics of germline modification. They agreed to support basic and clinical research under certain legal and ethical guidelines. A specific distinction was made between somatic cells, where the effects of edits are limited to a single individual, versus germline cells, where genome changes could be inherited by descendants. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene/environment interactions) and culturally (e.g. Social Darwinism). Altering of gametocytes and embryos to generate inheritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries.[114]

In November 2018, Jiankui He announced that he had edited two human embryos, to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.[115] An international group of scientists called for a global moratorium on genetically editing human embryos.[116]

Policy regulations for the CRISPR-Cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify human embryos by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days.[117]

The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the Agriculture Risk Protection Act of 2000 gives the USDA the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment and economy of the US. The act regulates any genetically modified organism that utilizes the genome of a predefined "plant pest" or any plant not previously categorized.[118] In 2015, Yinong Yang successfully deactivated 16 specific genes in the white button mushroom, to make them non-browning. Since he had not added any foreign-species (transgenic) DNA to his organism, the mushroom could not be regulated by the USDA under Section 340.2.[119] Yang's white button mushroom was the first organism genetically modified with the CRISPR-Cas9 protein system to pass US regulation.[120] In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US National Academies of Sciences, Engineering and Medicine, special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next five years and any new regulations that might be needed as a result.[121] The FDA in 2017 proposed a rule that would classify genetic engineering modifications to animals as "animal drugs", subjecting them to strict regulation if offered for sale, and reducing the ability for individuals and small businesses to make them profitably.[122][123]

In China, where social conditions sharply contrast with the west, genetic diseases carry a heavy stigma.[124] This leaves China with fewer policy barriers to the use of this technology.[125][126]

In 2012, and 2013, CRISPR was a runner-up in Science Magazine's Breakthrough of the Year award. In 2015, it was the winner of that award.[81] CRISPR was named as one of MIT Technology Review's 10 breakthrough technologies in 2014 and 2016.[127][128] In 2016, Jennifer Doudna, Emmanuelle Charpentier, along with Rudolph Barrangou, Philippe Horvath, and Feng Zhang won the Gairdner International award. In 2017, Jennifer Doudna and Emmanuelle Charpentier were awarded the Japan Prize for their revolutionary invention of CRISPR-Cas9 in Tokyo, Japan. In 2016, Emmanuelle Charpentier, Jennifer Doudna, and Feng Zhang won the Tang Prize in Biopharmaceutical Science.[129]

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CRISPR gene editing - Wikipedia

While ethicists debate the applications of blockbuster gene-editing tool Crispr in human healthcare, an inventor of the tool believes it has a more immediate application: improving our food.

"I think in the next five years the most profound thing we'll see in terms of Crispr's effects on people's everyday lives will be in the agricultural sector," Jennifer Doudna, the University of California Berkeley geneticist who unearthed Crispr in early experiments with bacteria in 2012, told Business Insider.

Crispr has dozens of potential uses, from treating diseases like sickle cell to certain inherited forms of blindness. The tool recently made headlines when a scientist in China reportedly used it to edit the DNA of a pair of twin baby girls.

Then there are Crispr's practical applications the kinds of things we might expect to see in places like grocery stores and farmers' fields within a decade, according to Doudna.

Crispr's appeal in food is straightforward: it's cheaper and easier than traditional breeding methods, including those that are used to make genetically modified crops (also known as GMOs) currently. It's also much more precise. Where traditional breeding methods hack away at a crop's genome with a dull blade, tools like Crispr slice and reshape with scalpel-like precision.

Want a mushroom that doesn't brown? A corn crop that yields more food per acre? Both already exist, though they haven't yet made it to consumers' plates. What about a strawberry with a longer shelf life or tomatoes that do a better job of staying on the vine?

"I think all of those things are coming relatively quickly," Doudna said.

Read more: The 10 people transforming healthcare

Work on Crispr'd produce has been ongoing for about half a decade, but it's only recently that US regulators have created a viable path for Crispr'd products to come to market.

Back in 2016, researchers at Penn State used Crispr to make mushrooms that don't brown. Last spring, gene-editing startup Pairwise scored $125 million from agricultural giant Monsanto to work on Crispr'd produce with the goal of getting it in grocery stores within the decade. A month later, Stefan Jansson, the chief of the plant physiology department at Sweden's Umea University, grew and ate the world's first Crispr'd kale.

More recently, several Silicon Valley startups have been experimenting with using Crispr to make lab-grown meat.

Read more: Startups backed by celebrities like Bill Gates are using Crispr to make meat without farms

Memphis Meats, a startup with backing from notable figures like Bill Gates and Richard Branson that has made real chicken strips and meatball prototypes from animal cells (and without killing any animals), is using the tool. So is New Age Meats, another San Francisco-based startup that aims to create real meat without slaughter.

Last spring, the US Department of Agriculture issued a new ruling on crops that exempts many Crispr-modified crops from the oversight that accompanies traditional GMOs. So long as the edited DNA in those crops could also have been created using traditional breeding techniques, the Crispr'd goods are not subject to those additional regulatory steps, according to the agency.

"With this approach, USDA seeks to allow innovation when there is no risk present," secretary of agriculture Sonny Perdue said in a statement. Genome editing tools like Crispr, he added, "will help farmers do what we aspire to do at USDA: do right and feed everyone."

Read more: A controversial technology could save us from starvation if we let it

Although several researchers and scientists have cheered the decision, many anti-GMO activists have not been pleased.

Despite the pushback, Doudna believes that Crispr'd food could help dispel some of the fear around GMOs and increase awareness about the role of science in agriculture.

"I hope this brings that discussion into a realm where we can talk about it in a logical way," she said. "Isn't it better to have technology that allows for precise manipulation of a plant genome, rather than relying on random changes?"

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Jennifer Doudna: We will eat the first Crispr'd food In 5 ...

William Peranteau is the guy parents call when theyve received the kind of bad news that sinks stomachs and wrenches hearts. Sometimes its a shadow on an ultrasound or a few base pairs out of place on a prenatal genetic test, revealing that an unborn child has a life-threatening developmental defect. Pediatric surgeons like Peranteau, who works at Childrens Hospital of Philadelphia, usually cant try to fix these abnormalities until their patients leave their mothers bodies behind. And by then it might be too late.

Its with the memory of the families he couldnt help in the back of his mind that Peranteau has joined a small group of scientists trying to bring the fast-moving field of gene editing to the womb. Such editing in humans is a long way off, but a spate of recent advances in mouse studies highlight its potential advantages over other methods of using Crispr to snip away diseases. Parents confronted with an in utero diagnosis are often faced with only two options: terminate the pregnancy or prepare to care for a child who may require multiple invasive surgeries over the course of their lifetime just to survive. Prenatal gene editing may offer a third potential path. What we see as the future is a minimally invasive way of treating these abnormalities at their genetic origin instead, says Peranteau.

To prove out this vision, Peranteau and colleagues at the University of Pennsylvania injected Crispr editing components, encoded in a virus, into the placentas of pregnant mice whose unborn pups were afflicted with a lethal lung-disease-causing mutation. When the fetuses breathed in the amniotic fluid they also inhaled the Crispr bits, which went to work editing the DNA inside their rapidly dividing alveolar progenitor cells. These cells give rise to many types of cells that line the lungsincluding ones that secrete a sticky substance that keeps the lungs from collapsing every time you breathe. Mutations to proteins that make up this secretion are a major source of congenital respiratory conditions. All of the mice with the mutation died within a few hours of birth. Of those edited with Crispr, about a quarter survived. The results were published in todays issue of Science Translational Medicine.

Its the second proof of concept from the group of scientists in the past year. In October, they published a paper describing a slightly different procedure to edit mutations that lead to a lethal metabolic disorder. By changing a single base pair in the liver cells of prenatal mice, Peranteaus team was able to rescue nearly all of the mouse pups. Other recent successes include unborn mice cured of a blood disorder called beta-thalassemia following a prenatal injection of Crispr, carried out last year by a team at Yale and Carnegie Mellon.

Though the field is still in its infancy, its pioneers believe that many of the problems Crispr-based therapies have to contend withlike reaching enough of the right cells and evading the human immune systemcan be solved by treating patients while they are still in the womb.

If youre trying to edit cells in an adult organ, theyre not proliferating, so you have to reach a lot of them to have any impact, says Edward Morrisey, a cardiologist at the University of Pennsylvania, who coauthored the latest study. Fetuses, on the other hand, are still developing, which means their cells are in a state of rapid division as they grow into new tissues. The earlier in life you can edit, the more those genetic changes will multiply and propagate through developing organs. Morriseys mice might have only been born with the genetic edit in about 20 percent of their lung cells, but 13 weeks later, the correction had spread to the entire surface of the lung. Theyve actually outcompeted the nonedited cells, because those cells are very sick, says Morissey.

For lung diseases in particular, this represents a huge advantage. As soon as a baby leaves the watery world of the womb, its lung cells start secreting a barrier of mucus mixed with surfactant, to keep any dust or viruses or other foreign objects, including Crispr components, from reaching those tissues. A developing fetus also has a less aggressive immune system than a human whos been exposed to the outside world. So its less likely to mount an attack on Crispr components, which do, after all, originate in the bacterial kingdom.

Now, you might be thinking, if editing earlier is better, why not edit an embryo right after its been fertilized, when its only a cell or two old? But this technique, known as germline editing (you might remember it from last years Chinese Crispr baby scandal), is a much more complicated ethical endeavor. Editing at that stage would pass on any changes to every cell, including the ones that would go on to make sperm or eggs. This kind of editing is effectively banned in the United States, following a directive from Congress to the US Food and Drug Administration to not allow any clinical trials involving genetically modified human embryos. (The ban, which has to be renewed annually, was most recently reaffirmed in February of 2019). The other thing though, is that getting an accurate diagnosis when an embryo is only a few cells old can be tricky. Waiting long enough to get a visual on a fetus along with other vital signs can provide important clues as to the severity of the condition. It gets us right in that sweet spot to treat a disease at the very beginning, basically as soon as its diagnosed, says Peranteau.

But there are still safety issues to resolve. For one thing, in utero editing involves two patients, not just one. In the process of curing a child, this technique would potentially expose a healthy bystanderthe motherto a treatment that provides no potential benefit and only potential risks, including dangerous immune reactions. And because the editing is taking place inside her reproductive tract, some wayward Crispr components might wend their way up her fallopian tubes and into her ovaries, potentially making changes to other, unfertilized eggs. A lot more science will need to be done to better assess these risks. To give you an idea of how long these things can take, consider that in utero gene therapyan older approach that entails replacing a defective gene with a functioning one using a viruswas first proposed back in the mid-1990s following a series of positive proof of concept studies in mice. Today, only a single clinical trial is in progress.

This is not a panacea for curing every genetic disease thats out there, says Peranteau. But he believes that a Crispr approach will be able to piggyback on the work of the gene therapy field, and may offer a new way forward for at least some of his patients. At some point in the futurenot tomorrow or the next day, years from nowI think in utero editing would provide hope for families that today have none.

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Crispr Gene Editing Is Coming for the Womb | WIRED

CRISPR gene-editing technology allows scientists to make highly precise modifications to DNA. The technology is now starting to be used in human trials to treat several diseases in the U.S. Molekuul/Getty Images/Science Photo Library hide caption

CRISPR gene-editing technology allows scientists to make highly precise modifications to DNA. The technology is now starting to be used in human trials to treat several diseases in the U.S.

The powerful gene-editing technique called CRISPR has been in the news a lot. And not all the news has been good: A Chinese scientist stunned the world last year when he announced he had used CRISPR to create genetically modified babies.

But scientists have long hoped CRISPR a technology that allows scientists to make very precise modifications to DNA could eventually help cure many diseases. And now scientists are taking tangible first steps to make that dream a reality.

For example, NPR has learned that a U.S. CRISPR study that had been approved for cancer at the University of Pennsylvania in Philadelphia has finally started. A university spokesman on Monday confirmed for the first time that two patients had been treated using CRISPR.

One patient had multiple myeloma, and one had sarcoma. Both had relapsed after undergoing standard treatment.

The revelation comes as several other human trials of CRISPR are starting or are set to start in the U.S., Canada and Europe to test CRISPR's efficacy in treating various diseases.

"2019 is the year when the training wheels come off and the world gets to see what CRISPR can really do for the world in the most positive sense," says Fyodor Urnov, a gene-editing scientist at the Altius Institute for Biomedical Sciences in Seattle and the University of California, Berkeley.

Here are highlights of the year ahead in CRISPR research, and answers to common questions about the technology.

What is CRISPR exactly?

CRISPR is a new kind of genetic engineering that gives scientists the power to edit DNA much more easily than ever. Researchers think CRISPR could revolutionize how they prevent and treat many diseases. CRISPR could, for example, enable scientists to repair genetic defects or use genetically modified human cells as therapies.

Traditional gene therapy uses viruses to insert new genes into cells to try to treat diseases. CRISPR treatments largely avoid the use of viruses, which have caused some safety problems in the past. Instead they directly make changes in the DNA, using targeted molecular tools. The technique has been compared to the cut and paste function in a word processing program it allows scientists to remove or modify specific genes causing a problem.

Is this the same technique that caused a recent scandal when a scientist in China edited the genes of two human embryos?

There's an important difference between the medical studies under discussion here and what the Chinese scientist, He Jiankui, did. He used CRISPR to edit genes in human embryos. That means the changes he made would be passed down for generations to come. And he did it before most scientists think it was safe to try. In fact, there have been calls for a moratorium on gene-editing of heritable traits.

For medical treatments, modifications are only being made in the DNA of individual patients. So this gene-editing doesn't raise dystopian fears about re-engineering the human race. And there's been a lot of careful preparation for these studies to avoid unintended consequences.

So what's happening now with new or planned trials?

We've finally reached the moment when CRISPR is moving out of the lab and into the clinic around the world.

Until now, only a relatively small number of studies have tried to use CRISPR to treat disease. And almost all of those studies have been in China, and have been aimed at treating various forms of cancer.

There's now a clinical trial underway at the University of Pennsylvania using CRISPR for cancer treatment. It involves removing immune system cells from patients, genetically modifying them in the lab and infusing the modified cells back into the body.

The hope is the modified cells will target and destroy cancer cells. No other information has been released about how well it might be working. The study was approved to eventually treat 18 patients.

"Findings from this research study will be shared at an appropriate time via medical meeting presentation or peer-reviewed publication," a university spokesperson wrote in an email to NPR.

But beyond the cancer study, researchers in Europe, the United States and Canada are launching at least half a dozen carefully designed studies aimed at using CRISPR to treat a variety of diseases.

What other diseases are they testing treatments for?Two trials sponsored by CRISPR Therapeutics of Cambridge, Mass., and Vertex Pharmaceuticals of Boston are designed to treat genetic blood disorders. One is for sickle cell disease, and another is a similar genetic condition called beta thalassemia.

In fact, the first beta thalassemia patient was recently treated in Germany. More patients may soon get their blood cells edited using CRISPR at that hospital and a second clinic in Germany, followed by patients at medical centers in Toronto, London and possibly elsewhere.

The first sickle disease patients could soon start getting the DNA in their blood cells edited in this country in Nashville, Tenn., San Antonio and New York.

And yet another study, sponsored by Editas Medicine of Cambridge, Mass., will try to treat an inherited form of blindness known as Leber congenital amaurosis.

That study is noteworthy because it would be the first time scientists try using CRISPR to edit genes while they are inside the human body. The other studies involve removing cells from patients, editing the DNA in those cells in the lab and then infusing the modified cells back into patients' bodies.

Finally, several more U.S. cancer studies may also start this year in Texas, New York and elsewhere to try to treat tumors by genetically modifying immune system cells.

What can go wrong with CRISPR? Are there any concerns?

Whenever scientists try something new and powerful, it always raises fears that something could go wrong. The early days of gene therapy were scarred by major setbacks, such as the case of Jesse Gelsinger, who died after an adverse reaction to a treatment.

The big concern about CRISPR is that the editing could go awry, causing unintended changes in DNA that could cause health problems.

There's also some concern about this new wave of studies because they are the first to get approved without going through an extra layer of scrutiny by the National Institutes of Health. That occurred because the NIH and FDA changed their policy, saying only some studies would require that extra layer of review.

"Every human on the planet should hope that this technology works. But it might work. It might not. It's unknown," says Laurie Zoloth, a bioethicist at the University of Chicago. "This is an experiment. So you do need exquisite layers of care. And you need to really think in advance with a careful ethical review how you do this sort of work."

The researchers conducting the studies say they have conducted careful preliminary research, and their studies have gone through extensive scientific and ethical review.

When might we know whether any of these experimental CRISPR treatments are working?

All of these studies are very preliminary and are primarily aimed at first testing whether this is safe. That said, they are also looking for clues to whether they might be helping patients. So there could be at least a hint about that later this year. But it will be many years before any CRISPR treatment could become widely available.

Continued here:
CRISPR Research Moves Out Of Labs And Into Clinics Around The ...

It's not certain how effective the treatment has been, and you won't find out for a while when the trial has been cleared to treat a total of 18 patients. You won't hear more about it until there's been a presentation or a peer-reviewed paper, the university said. Other trials, such as ones for blood disorders in the Boston area, have yet to get underway.

No matter what, any practical uses could take a long time. There are widespread concerns that CRISPR editing could have unanticipated effects, and scientists have yet to try editing cells while they're still in the body (a blindness trial in Cambridge, MA may be the first instance). There's also the not-so-small matter of ethical questions. Chinese scientist He Jiankui raised alarm bells when he said he edited genes in human embryos -- politicians and the scientific community will likely want to address practices like that before you can simply assume that CRISPR is an option.

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CRISPR gene editing has been used on humans in the US

Like any self-respecting farmer, Zachary Lippman was grumbling about the weather. Stout, with close-cropped hair and beard, Lippman was standing in a greenhouse in the middle of Long Island, surrounded by a profusion of rambunctiously bushy plants. Dont get me started, he said, referring to the late and inclement spring. It was a Tuesday in mid-April, but a chance of snow had been in the forecast, and a chilly wind blew across the island. Not the sort of weather that conjures thoughts of summer tomatoes. But Lippman was thinking ahead to sometime around Memorial Day, when thousands of carefully nurtured tomato plants would make the move from the greenhouse to Long Island loam. He hoped the weather would finally turn.

Although he worked on a farm as a teenager and has a romantic attachment to the soil, Lippman isnt a farmer. Hes a plant biologist at Cold Spring Harbor Laboratory in New York with an expertise in genetics and development. And these greenhouse plants arent ordinary tomatoes.

After introducing me to his constant companion, Charlie (a slobberingly gregarious Labrador-Rottweiler mix), Lippman walked me through hundreds of plants, coddled by 80-degree daytime temperatures and 40 to 60 percent humidity, and goaded into 14 hours of daily photosynthetic labor by high-pressure sodium lights overhead. Some were seedlings that had barely unfurled their first embryonic leaves; others had just begun to flash their telltale yellow flowers, harbingers of the fruit to come; still others were just about ripe, beginning to sag with the weight of maturing red fruit.

What makes this greenhouse differentwhat makes it arguably an epicenter of a revolution in plant biology that may forever change not just the future of the tomato but the future of many cropsis that 90 percent of the tomato plants in the building had been genetically altered using the wizardly new gene-editing tool known as Crispr/Cas-9. Lippman and Joyce Van Eck, his longtime collaborator at the Boyce Thompson Institute in Ithaca, New York, are part of a small army of researchers using gene editing to turn the tomato into the laboratory mouse of plant science. In this greenhouse, Crispr is a verb, every plant is an experiment, and mutant isnt a dirty word.

Lippman walked to the rear of the building and pointed out a variety of tomato known as Large Fruited Fresh Marketone of the commercial varieties that turn up in supermarkets, not farmers markets. This particular plant, about two months old, bowed with big, nearly ripe fruit. It was, Lippman explained, a mutant called jointless. Most tomato varieties have a swollen knuckle of tissue (or joint) on the stem, just above where the fruit forms; when the tomato is ready, it tells itself, as Lippman put it, OK, Im ripetime to fall, and the cells in the joint receive a signal to die, letting go of the tomato. That is natures way of spreading tomato seeds, but the joint has been a thorny problem for agricultural production, because it leaves a residual stem that pokes holes in mechanically harvested fruit. Jointless tomatoes, whose stems can be plucked clean, have been bred and grown commercially, but often with unwanted side effects; these gene-edited versions avoid the unintended consequences of traditional breeding. We can now use Crispr to go in and directly target that gene for the molecular scissors to cut, which leads to a mutation, Lippman said. Voil: the jointless trait in any variety you want.

We moved on to several examples of Physalis pruinosa, a relative of the tomatillo that produces a small, succulent fruit called a ground cherry. The plant has never been domesticated, and Lippman referred to the wild version as a monstrosity: tall, unkempt, and stingy, bestowing a single measly fruit per shoot. Next to it stood a Physalis plant after scientists had induced a mutation called self-pruning. It was half as tall, much less bushy, and boasted half a dozen fruits per shoot. Lippman plucked a ground cherry off one of the mutated plants and offered it to me.

Smell it first, he entreated. Enjoy the smell. It was exotic and faintly tropical. I popped it in my mouth and bit into a complex burst of flavor. Like all its cousin tomatoes, the taste was a mystical, time-lapse blur of sugar and acidity, embellished by the whiff of volatile compounds that found my nose and rounded out the flavor.

You just ate an edited plant, Lippman said with a smile. But dont be too nervous.

Zach Lippman in a Cold Spring Harbor test field of tomato plants edited to produce more fruit.

Dolly Faibyshev

A gene-edited tomato plant.

Dolly Faibyshev

Like the majority of scientists, Lippman regards genetically modified plants as safe to eat. But his mischievous smile acknowledged that not everyone views the technology as innocuous. There is a lot of nervousness about genetic tinkering with food plants. Genetically modified (GM) transgenic crops such as corn and soybeans have infiltrated processed foods, animal feed, and biofuels for many years, and the battle over them has long divided the public in the US and overseas. The Crispr revolution is reinventing, if not reigniting, that debate. Most of the plants that have been gene-edited to date have been created by knocking out genes (that is, mutating them), not by introducing genes from unrelated species, as first-generation genetic modification generally didrousing cries of Frankenfoods and fears of environmental contamination. Precisely because its subtraction rather than addition, scientists argue that this form of gene editing mimics the process of agriculturally induced mutations that characterizes traditional plant breeding. This distinction may not assuage critics, but it has apparently persuaded federal regulators; gene-edited soybean and potato crops are already in the ground, and last March the US Department of Agriculture declared that crops developed with gene-edited mutations are indistinguishable from those produced by traditional breeding and do not require regulatory oversight.

Huge questions vex the future of foodhow to feed 9 billion mouths, how to farm in an era of unprecedented climate uncertainty, how to create more resilient and nutritious foods for a public wary of the new technology. Plant scientists are already using Crispr and related technologies to reshape food crops in dramatic waysediting wheat to reduce gluten, editing soybeans to produce a healthier oil, editing corn to produce higher yields, editing potatoes to store better (and not throw off a carcinogen when cooked). In both industrial and academic labs, new editing tools are being developed that will have a profound impact on the foods all of us eat. Yet this newfound power to transform food traits coincides with a moment when the agriculture business has consolidated into essentially three mega-conglomerates. Those companies have the money to put this new technology to use. The question is: What use will they put it toward?

Soybeans, potatoes, and corn melt invisibly into the food chain, but tomatoes add a big red exclamation point to the current debate. Perhaps no food crop is more emblematic of what is at stakeagriculturally, biologically, culturally, and perhaps even in homegrown foodie waysthan the tomato: queen of the farmers market, jewel of the backyard garden, alpha vegetable of locavores everywhere. Lippmans greenhouse reveals just some of the ways gene editing is already altering the tomatohe has plants that flower earlier, that are oblivious to daylight cues, that prune themselves into smaller footprints, that can be genetically programmed to space out the position of fruits on the stem like an accordion.

For people who love to eat or grow tomatoes (I do both), the arrival of Crispr provokes both cynicism and giddy hope about the future of our favorite vegetable. Cynicism because most of the practical scientific efforts would perpetuate the dreary taste of commercially produced tomatoes. In one sense, this is simply the latest in a century-long conquest of the produce aisles by the desires of food growers, who prize greater yield at lesser cost, over the desires of consumers, who cherish taste and nutrition. (Harry Klee, a tomato expert at the University of Florida, says that the perfect tomato for industry is one that exactly matches the size of a McDonalds hamburger.) Hope because there is something intriguing about using new technology to preserve the ravishing, sweet acidic burst of an heirloom tomato in a hardier, disease-resistant plantan heirloom-plus, if you will.

After Lippman walked me through his garden of man-made mutations, I couldnt resist asking if the heirlooms I struggle to grow every year might also benefit from Crisprs scissors.

Were not doing any editing of heirlooms, Lippman said. Not yet. But its in the works. They could benefit from a little bit of tweaking.

Tomatoes are coddled and goaded into photosynthetic labor in a greenhouse at the Boyce Thompson Institute.

Dolly Faibyshev

This is a story about tomatoes, of course. But it is also, like all agricultural stories, about mutationsnatural mutations and man-made mutations, invisibly insidious mutations and overtly grotesque mutations, mutations that were created earlier this year at Cold Spring Harbor Laboratory and mutations that may have occurred 10,000 years ago, like the ones that transformed Solanum pimpinellifolium from a scraggly perennial weed producing pea-sized fruit along the Pacific coastal margins of Peru and Ecuador to those beautiful big-lobed heirlooms in your backyard. Our cultural thesaurus has reduced the word mutant to a term of derision, but if you think mutation is a dirty word, you should probably stop readingand probably stop eating plant-based food too. The foundational principle of plant breeding is to take advantage of genetic modification, whether the mutation is caused by sunlight or x-rays or Crispr. As Klee puts it, there isnt a single crop that I know of in your produce aisle that is not drastically modified from what is out there in the wild.

Every backyard gardener is a connoisseur, witting or otherwise, of mutation. The intense, thin-skinned freshness of Brandywines, the apricot glow of Jaune Flamme, the green standoffish shoulders of Black Krims, and my personal favorite, Rose de Berne, with its blush of color and amazing tasteall those heirlooms are the product of long-ago, hand-me-down mutations.

Every spring, almost inevitably during March Madness (this year, during Villanova-Michigan), I get down on the floor with a bunch of peat pots and starter soil and clumsily press seeds of all of the above varieties into virgin dirt. My wife wonders why I cant buy seedlings at the market like everyone else, but Ive never outgrown the childlike thrill of watching an itty-bitty snippet of plant DNA, encased in the stiff callus of a seed coat, unfurl into a 5-foot-tall plant that yields its sublime bounty. Gardenersthe original DIY biologistsall know this thrill. And so does Lippman. Thats how he got into gene-editing tomatoes in the first place.

If you think mutation is a dirty word, you should probably stop reading. And probably stop eating plant-based food too.

Lippman grew up in Milford, Connecticut; his father was an English teacher and his mother worked in health care. Among his earliest memories is visiting a nearby farm with his father when he was 6 or 7 years old and picking up leftover Halloween pumpkins and gourdswith their mind-blowing shapes and colorsthat littered the field.

That pumpkin field was part of Robert Treat Farm, and when he was 13, Lippman began working summers there, cultivating his fascination with plants. By the time he graduated from high school in 1996, he had decided to pursue plant breeding and genetics, first at Cornell University and then at Cold Spring Harbor, where he got his PhD and is now a Howard Hughes Medical Institute investigator.

Lippmans office is a shrine to the tomato: On his walls are old tomato-can labels and antique postcards of implausibly gigantic tomatoes, and thousands of little brown envelopes containing seeds, each marked by year and variety, are stacked on his desk, in old seed boxes, in wooden trays and plastic cabinets against the wall. The most telling relic is just behind the door: a large framed reproduction from a 16th-century book by Pietro Andrea Mattioli, believed to be the earliest color depiction of the tomato following the Spanish conquest of the Americas. To a geneticist like Lippman, the Mattioli print is especially significant because it is early evidence that pre-Columbian cultures knew a beneficial tomato mutation when they saw onethey had already converted the nubbin of wild fruit into a large, multiple-lobed golden beefsteak.

Up until the 1930s, agricultural scientists essentially relied on the same techniques as the original tomato farmers in Central America: Be patient enough to wait for nature to produce a useful mutation, be smart enough to recognize that desirable trait (bigger fruit, for example), and be clever enough to create a new variety with that trait by selecting the mutant strains and propagating them. Put another way, agriculture has always been about unnatural selectionhuman choice privileging certain mutations while discarding others. Biologists sped up this process around the time of World War II by deliberately inducing random mutations in seeds with the use of chemicals, x-rays, and other forms of radiation. But even so, the process was slow. Selective breeding of a desirable trait could easily take a decade.

This all began to change in 2012, an annus mirabilis for the tomato. In May of that year, plant geneticists completed the Tomato Genome Projectthe entire DNA sequence of the tomato plant, all 900 million base pairs on 12 chromosomes. Then, in June, a group led by Jennifer Doudna at UC Berkeley published the first report on the new gene-editing technique known as Crispr, followed soon after by a group at the Broad Institute of MIT and Harvard. The fruit of those two converging streams of researchand, yes, botanically speaking, tomato is a fruitwas a race among scientists to see if the new technique worked in plants.

As soon as word of Crispr broke, Lippman wondered, Can we do it in tomato? And if we can, lets move. Moving fast meant doing an experiment on a tomato gene that would prove the efficacy of Crispr without too much delay. Which gene did Lippman and Van Eck target? Not one that would improve the size or shape of the fruitthat would take too long, and Van Eck was impatient. I dont want to have to put it in the greenhouse and wait for it to grow, she told Lippman. I want to be able to see something in the petri plate. So they picked a gene that was of zero economic significance and less-than-zero consumer appeal. It was a weird gene that, when mutated, produced disfigured tomato leaves that looked like needles. The mutant version was called wiry.

A research field at Cold Spring Harbor with some 8,000 gene-edited plants.

Dolly Faibyshev

Seeds are stored in boxes, and then planted.

Dolly Faibyshev

The wiry mutation was so obscure that Van Eck had to dig up a paper from 1928 that described it for the first time to know what shed be looking for. Each Crispr-directed mutation requires a customized, genetically engineered tool called a constructa so-called guide RNA to target the right tomato gene and an enzyme riding shotgun to cut the plant DNA at precisely that spot. In this case, Lippman designed the construct to target the wiry gene and cut it; the mutation is not created by Crispr per se but by the plant when it attempts to repair the wound. Van Eck used a bacterium that is very good at infecting plants to carry the Crispr mutation tool inside tomato cells. Once mutated, these cells were spread onto petri plates where they began to develop into plants. Van Eck still had to wait about two months before the tomato cells developed into seedlings and sprouted leaves, but it was worth the wait.

I still remember when I saw the first leaves coming up, she recalls. The leaves were radializedcurled up into needlelike shapes. Omigod, it worked! she cried, and raced down the hallways of the institute to tell anyone who would listen. I was thrilled because, you know, when does something work the first time?

Not only had they demonstrated that Crispr could produce a heritable trait change in a fruit crop, they also had their answer in two months rather than a year. They knew that the same basic process could theoretically be used to edit, with exquisite precision and unprecedented speed, any gene in any food crop.

As soon as they knew it worked, Lippman and Van Eck began Crispring every trait theyd wanted to study for the past 15 years. One of them was jointless. For 60 years, researchers had been trying to solve the problem of the joint on the tomato plants stem. Large-scale farming of tomatoesCalifornia alone produces more than 10 million tons each yearrequires mechanical harvesting, and those stabbing stems of jointed tomatoes make the task harder and more wasteful. Lippman, who studies plant architecture, knew that many jointless tomato plants produce excessive branching and lower yields. He discovered that this unintended consequence was the result of traditional breeding: When breeders favored the jointless mutation, they unwittingly produced unwanted branching as well because of a complex interaction between jointless and another ancient mutation. Traditional breeding produced another side effectabnormally shaped tomatoesbecause the process of selecting the jointless trait dragged along a chunk of DNA with an unwanted mutation. (This phenomenon is known as linkage drag.)

If Lippman could Crispr his way to the jointless mutation without dragging along the deleterious effects related to traditional breeding, it would offer a significant advance for growers. He and Van Eck had to wait longer than they had for the needle-nosed leaves of wiry, but by March 2016, Lippman had jointless tomatoes growing in his greenhouse. They published the work in the journal Cell in the spring of 2017, and Lippman shared the gene-editing tool with Klee at the University of Florida. Last March, Klee and his team planted a plot of gene-edited jointless mutants, in a commercial variety called Florida 8059, in a test field north of Gainesville.

Joyce Van Eck saw curled leaves on a tiny tomato plant growing on a petri plate and knew that the Crispr experiment was a success.

Dolly Faibyshev

Quick reality check: Despite the hype about the gene-editing revolution, the past couple of years have revealed limitations as well as successes. Scientists will tell you Crispr is great at knocking out a gene. But using it to insert a new gene and, as many popular accounts suggest, rewrite the germline of man, beast, or plant? Not so easy. Crispr is not the be-all and end-all, says Dan Voytas of the University of Minnesota, one of the pioneers of agricultural gene editing. Moreover, genomes are complex, even in plants. Just as a dozen knobs on a stereo console can shape the overall sound of a single song, multiple genetic elements can control the effect of a single gene.

That daunting complexity inspired Lippmans lab to pursue a clever riff on gene editing. I remember having a sticky note here, Lippman says, pointing to his keyboard. The note simply read: Promoter CRISPR.

In plants as well as animals (and humans), there is part of the DNA that lies outside the protein-encoding segment of the gene and essentially regulates its output. This upstream patch of regulatory DNA is called the promoter, and it sets different levels of outputvolume, if you willfor specific genes, from a little to a lot. What if, Lippmans group asked, you could use Crispr to, in effect, adjust the volume of a particular gene, turning it up or down like a stereo knob, by mutating the promoter in different places?

The Long Island greenhouse is now full of examples of what happens. As they reported in Cell last October, Daniel Rodrguez-Leal and colleagues in Lippmans lab showed that, by mutating the promoter of the self-pruning gene in different places, they could adjust its output like a dimmer switch, producing subtle but important changes. By using Crispr to create varying doses of a gene, Lippman says, scientists can now find better versions of plants than nature ever provided.

But better for whom? One of Lippmans pet phrases is sweet spotthat point of genetic balance where desirable traits for agriculture can be improved without sacrificing essential features like flavor or shape. Now we can start to think about taking some of our best tomato varieties, and if they can flower faster, you can start to grow them in more northern latitudes, where the summers are shorter, he says. We can begin to imagine new crops, or new versions of existing crops, for urban agriculture, like tiered cropping that they have in these abandoned warehouses ... Adapt the plant so that its more compact, flowers faster, gives you a nice-sized fruit with a decent yield, in a very compressed growth setting, with the equivalent of protective agriculturegreenhouse conditionsbut with LED lights. Because every plant gene comes with its own promoter, this genetic tuning, as Lippman puts it, could apply to virtually any vegetable crop.

The sad reality is that industry is not really committed to making a better-tasting tomato.

Tuning is just one of many ways biologists are remaking the tomato. Last year, researchers at the Sainsbury Laboratory in England gene-edited a tomato variety called Moneymaker to be resistant to powdery mildew, and a Japanese research group recently created tomatoes without seeds. On the day in May that I set my first heirloom seedlings into the ground, I happened to have a Skype conversation with two plant biologists in Brazil who have taken the gene editing of tomatoes to a whole new level. In collaboration with the Voytas lab at the University of Minnesota, Agustin Zsgn of the University of Viosa and Lzaro Peres of the University of So Paulo claim to have, in essence, reverse-engineered the weedlike wild tomato believed to be the forerunner of all cultivated varieties. (They havent published this work to date, but have discussed it at meetings.) Rather than tweak a domesticated variety of tomato, they went back to square onethe wild plantand used Crispr to knock out a handful of genes all at once. The result? Where the wild plant was sprawling and weedy, the gene-edited tomato was compact and bushy; where the ancestral plant had pea-sized fruit, the gene-edited version had reasonably plump, cherry-sized tomatoes. The edited fruit also contained more lycopene, an important antioxidant, than any other known variety of tomato. The process is called de novo domestication.

We didnt go from pea-sized to beefsteak, but we went from pea-sized to cherry-sized, said Zsgn of this first attempt. And how did the tomatoes taste? They taste great! Peres insisted. In a similar vein, Lippman and Van Eck are domesticating the wild ground cherry in the hope that it can join blueberries and strawberries as one of the basic berry crops.

What makes the de novo approach so intriguing is that it takes advantage of all the accumulated botanical wisdom of a wild plant. Over tens of thousands of years of evolution, a wild species acquires traits of hardiness and resilience, such as resistance to disease and stress. Domestication eliminated some of those traits. Since those resistance traits typically involve a suite of genes, Peres says, they would be extremely difficult to introduce into domesticated tomatoes, via Crispr or any other technology. And the approach can exploit other extreme traits. Peres wants to domesticate a wild species from the Galapagos, which can tolerate extreme environmental conditions such as high salinity and droughttraits that might enhance food security in a future with enormous climate fluctuations.

Rising temperatures. Changing growing seasons. A rising global population. The environmental toll of herbicide overuse. What if gene editing, for example, could favor disease-resistance genes that would reduce pesticide use? Lippman asks. Thats not just feeding the world, thats protecting the planet.

Lippman, outside a tomato greenhouse: Ive eaten many gene-edited tomatoes, yeah.

Dolly Faibyshev

All this new plant scienceknocking out genes, fiddling with the volume knob of promoters, de novo domesticationis wonderfully creative and happening very fast. But sooner or later, the other shoe drops in the conversation. Will consumers want to eat these tomatoes? Are Crispr vegetables and grains simply new GMOs, as a number of environmental groups maintain, or are gene-edited plants intrinsically different? This is the beginning of the new conversation, Lippman says.

The old conversation was acrimonious and emotional. The initial GM foods that Monsanto introduced in the 1990s were transgenic, meaning that biologists used genetic engineering to introduce foreign DNA, from an unrelated species, into the plant. Gene editing is much more analogous to older forms of mutagenesis such as irradiation and chemicals, though much less scattershot. Rather than creating random mutations, Crispr targets specific genes. (Editing that misses its mark is possible, though Lippman hasnt detected any in his work.) That is why plant scientists have been so eager to use it, and why the USDA regards gene-edited knockouts as similar to earlier mutagens and thus not requiring special regulation. (In the case of knocking in, or adding, a gene to crop plants, the USDA has indicated it will assess on a case-by-case basis.) Some European countries have banned GMOs, and the European Union has yet to issue a final judgment on gene-edited plants.

Although multiple studies have failed to show that GMOs pose a threat to human health, public doubts persista Pew Research Center survey in 2016 indicated that 39 percent of Americans believe that genetically modified foods are less healthy than non-GMOs, and in his household, Lippman admits, his wife initially preferred not to eat his gene-edited tomatoes.

The domesticated tomato possesses thousands, perhaps millions, of spontaneous mutations that helped turn a forlorn, ground-hugging weed into the most popular American garden plant. Now scientists are using gene editing to create these mutations and optimize the plants.

self-pruningThis mutation affects the plants size, shape, and compactness and alone can change the wild, sprawling shrub into the orderly, compact crop familiar to gardeners.

self-pruning 5gThis affects the tomatos day-length sensitivity apparatus, allowing it to be grown during shorter summers at northern latitudes.

fasciatedThis mutation affects the plants size, shape, and compactness and alone can change the wild, sprawling shrub into the orderly, compact crop familiar to gardeners.

jointless-2This mutation eliminates a break point in the middle of the stem, just above the fruit, facilitating mechanical harvesting.

lycopene beta-cyclaseThis mutation increases the fruits lycopene, the chemical that gives the tomato its red color.

There are other reasons that genetically altered foods continue to arouse suspicion. Monsantos early GMO effort used a revolutionary technology not to make healthier or more environmentally sustainable foods but to confer resistance in soybean and corn to the companys proprietary herbicide, Roundup. The companys aggressive promotion of such a self-serving first product was considered a public relations disaster.

Big agribusinesses are now positioning themselves to take advantage of gene editing. A recent rash of mergers has created three giant multinationals in global agriculture: Bayer (which completed its acquisition of Monsanto this year), DowDuPont (following Duponts earlier merger with Dow Chemical), and Syngenta (which was acquired last year by the huge Chinese gene-editing company ChemChina). The intellectual property issues are possibly more complex than plant genetics. Both the Broad Institute and DuPont Pioneer hold basic Crispr patents that apply to agriculture, and the two entities teamed up last fall to jointly negotiate licenses for farming applications (all three giant agribusinesses have licensed the technology). According to agricultural sources, the right to use Crispr for commercial agriculture requires an upfront fee, annual royalty payments on sales, and other conditions. (The Broad Institute did not discuss licensing terms, except to say that it is not involved in product development.)

This is where gene editing bumps up against the harsh economics of agriculture. Academic scientists can conduct basic research with Crispr without paying a licensing fee. But thats as far as it goes. I cant develop products and start to sell them, Lippman says. Commercial development requires payment of a licensing feea cost more easily borne by deep-pocketed agricultural companies.

There are some smaller biotechs seeking to maneuver around the giant companies and the intellectual property obstacles. Calyxt, a Minnesota-based firm cofounded by Voytas, has already received USDA approval to grow several crops using an earlier and more-difficult-to-use gene-editing technology known as TALENs. Lippman consults for a Massachusetts startup called Inari. Benson Hill Biosystems, based in St. Louis, has been working on improving plant productivity using a patented set of new gene-editing scissors the company calls Crispr 3.0. But CEO Matthew Crisp (yes, thats his name) claims innovation is being stifled by an intellectual property landscape that is very murky. Benson Hills partners and prospective licensees, he says, have complained that commercial rights to Crispr gene-editing technology are too expensive, too cumbersome, or too uncertain. The discovery of new gene-editing enzymes and other innovations may complicate the patent landscape even more. As one source put it, Its a mess. And its only going to get worse.

Thats why theres a lot of attention focused on a new startup called Pairwise Plants, in which Monsanto has teamed up with several Crispr pioneers from the Broad Institute. Recent statements to Bloomberg by company CEO Tom Adams, a former vice president of Monsanto, stressing new crops that are really beneficial to people, raised some eyebrows. You know, its not Monsanto language, Voytas noted. And the Monsanto pedigree has some plant biologists concerned. The question will be: They have enormous baggage in terms of consumer acceptance, Lippman says. And if they botch it, theyre going to ruin it for everybody else. Everyone is sort of holding their breath.

Trays for germinating tomato seeds.

Dolly Faibyshev

Physalis pruinosa plants at the Boyce Thompson Institute in Ithaca.

Dolly Faibyshev

Heres a simpler question: What about flavor? When I asked Harry Klee if he had tasted any of the jointless 8059 tomatoes hes growing, he laughed and said he hadnt bothered. We know that Florida 8059 by itself doesnt really have too much taste to begin with. A better-tasting tomato always plays second fiddle to market economics. The majority of tomatoes grown in Florida, for example, go to the food service industryto the McDonalds and Subways, Klee says. The sad reality, Klee says, is that industry is not really committed to making a better-tasting tomato. Klee loves to talk about tastehe heads a group that identified about two dozen genetic regions related to exceptional tomato flavor. We know exactly how to give you a sweeter tomato that will taste better, he says. But those tomatoes are not as economically attractive to producers. The growers wont accept it.

What about consumers? Would they accept a gene-edited tomato if it tasted better? Or, to put it in a slightly more idiosyncratic way, would it be botanical blasphemy to gene-edit an heirloom?

In his tour of the greenhouse, Lippman paused at one point to express good-natured scorn for heirlooms. They are terrific tomatoes, he admits, but pretty crappy producers. From personal experience, I can confirm that heirlooms are finicky plants and stingy producers, with lousy immune systemsmost of all, theyre heartbreakers, at least in the backyard. They start out like Usain Bolt in the 100 meters and end up looking spent, shriveled, hobbled by all manner of wilts and fungi and pests, leaves drooping like brown funereal crepe. It was tempting to think of using the new genetic tools to improve them. Klee is very anxious to introduce gene editing into the home garden. He thinks gardeners like me might be the place to make the argument that gene-edited tomatoes are not GMOs.

What if I could give you a Brandywine that had high lycopene, longer shelf life, and was a more compact plant? Klee asked me. I could do all of those today, with knocking out genes and genome editing. And I could give you something that was virtually identical to Brandywine that was half as tall and had fruit that didnt soften in less than a day, and were deep, deep red with high lycopene. I mean, would you grow that?

Absolutely! I told him.

I think most people would grow that, he replied. I think this could be a huge opportunity to educate home gardeners in what plant breeding is all about.

Not everyone would agree with Klee (or me). Voytas, a pioneer of gene editing in plants, chuckled when I asked him about a gene-edited heirloom. You know, part of it is, theyre heirloom, he said. The name inherently suggests this is something of value from the past. Not something new and techy. More to the point, he reminded me of the sort of outrageous licensing fees for the gene-editing technology. So your heirloom tomato idea would never be financially lucrative enough to pay the requisite licensing fees.

The bottom line: Gene-edited tomatoes are probably on their way to the market. But tomatoes with better flavor? Probably not going to happen anytime soon.

In early June, Zach Lippman went back to being a farmer. On what initially seemed like a sunny day, he and a dozen coworkers got their hands dirty transplanting some 8,000 gene-edited tomato plants into an outdoor field on the grounds of Cold Spring Harbor Laboratory. There were lots of the familiar mutantsjointless, self-pruning, daylight insensitivity. (The outdoor planting required prior approval from the USDA.) Plant em deep! he cried, as the crew raced to get the tomato seedlings in the ground under suddenly darkening skies.

The ultimate fate of the gene-edited tomato is as unpredictable as the weather, but the fate of these particular tomatoes is less of a mystery. Lippman often takes them home. Ive eaten many gene-edited tomatoes, yeah, he laughs. (Not surprisingly, he finds absolutely nothing different about them.) Theyre not GMO, he insists. Its just that youre left with what would be equivalent to a natural mutation. So why not eat it? Its one of just thousands or millions of mutations that may or may not affect the health of the plant and were still eating them!

Stephen S. Hall is the author of six books and teaches science writing at New York University.

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We are in the midst of a gene-editing revolution.

For four decades, scientists have tinkered with our genes. Since the 1970s, they've experimentally switched them on and off, uncovering their functions; mapped their location within our genome; and even inserted or deleted them in animals, plants and human beings.

And in November 2018, aChinese scientist claimedto have created the world's first genetically modified human beings.

Though scientists have made great inroads into understanding human genetics, editing our genes has remained a complex process requiring imprecise, expensive technology, years of expertise and just a little luck, too.

In 2012, a pair of scientists developed a new tool to modify genes, reshaping the entire field of gene-editing forever: CRISPR. Often described as "a pair of molecular scissors," CRISPR is widely considered the most precise, most cost-effective and quickest way to edit genes. Its potential applications are far-reaching, affecting conservation, agriculture, drug development and how we might fight genetic diseases. It could even alter the entire gene pool of a species.

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The field of CRISPR research is still remarkably young, yet we've already seen how it might be used to fight HIV infection, combat invasive species and destroy antibiotic-resistant bacteria. Many unknowns remain, however, including how CRISPR might damage DNA, leading to pathologies such as cancer.

Such a monumental leap in genetic engineering is full of complexities that ask big, often philosophical questions about science, ethics, how we conduct research and the future of humanity itself. With the confirmation that two human embryos were modified using CRISPR and carried to term, those questions have come sharply into focus. The future of gene-editing seemingly arrived overnight.

But what exactly is CRISPR and what are the outstanding concerns about such a powerful tool?

Let's break it all down.

CRISPR has the potential to be used in editing human embryos to create "designer babies."

Few predicted how important CRISPR would become for gene editing upon its discovery 30 years ago.

As early as 1987, researchers at Osaka University studying the function of Escherichia coli genes first noticed a set of short, repeated DNA sequences, but they didn't understand the significance.

Six years later, another microbiologist, Francisco Mojica, noted the sequences in a different single-celled organism, Haloferax mediterranei. The sequences kept appearing in other microbes and in 2002, the unusual DNA structures were given a name: Clustered regularly interspaced short palindromic repeats.

CRISPR.

Studying the sequences more intensely revealed that CRISPR forms an integral part of the "immune system" in bacteria, allowing them to fight off invading viruses. When a virus enters the bacteria, it fights back by cutting up the virus' DNA. This kills the virus and the bacteria stores some of the leftover DNA.

The leftover DNA is like a fingerprint, stored in the CRISPR database. If invaded again, the bacteria produce an enzyme called Cas9 that acts like a fingerprint scanner. Cas9 uses the CRISPR database to match the stored fingerprints with those of the new invader. If it can find a match, Cas9 is able to chop up the invading DNA.

Nature often provides great templates for technological advances. For instance, the nose of a Japanese bullet train is modeled on the kingfisher's beak because the latter is expertly "designed" by evolution to minimize noise as the bird dives into a stream to catch fish.

In a similar way, CRISPR/Cas9's ability to efficiently locate specific genetic sequences, and cut them, inspired a team of scientists to ask whether that ability could be mimicked for other purposes.

The answer would change gene editing forever.

In 2012, pioneering scientists Jennifer Doudna, from UC Berkeley, and Emmanuelle Charpentier, at Umea University Sweden, showed CRISPR could be hijacked and modified. Essentially, they'd turned CRISPR from a bacterial defense mechanism into a DNA-seeking missile strapped to a pair of molecular scissors. Their modified CRISPR system worked marvelously well, finding and cutting any gene they chose.

An illustration of the CRISPR-Cas9 gene editing complex. The Cas9 nuclease protein (white and green) uses a guide RNA (red) sequence to cut DNA (blue) at a complementary site.

Several research groups followed up on the original work, showing that the process was possible in yeast and cultured mouse and human cells.

The floodgates opened, and CRISPR research, which had long been the domain of molecular microbiologists, skyrocketed. The number of articles referencing CRISPR in preeminent research journal Nature has increased by over 6,000 percent between 2012 and 2018.

While other gene-editing tools are still in use, CRISPR provides a gigantic leap because of its precision and reliability. It's really good at finding genes and making accurate cuts. That allows genes to be cut out with ease, but it also provides an opportunity to paste new genes into the gap. Previous gene-editing tools could do this, too, but not with the ease that CRISPR can.

Another huge advantage CRISPR has over alternative gene-editing techniques is its expense. While previous techniques might cost a laboratory upward of $500 to edit a single gene, a CRISPR kit can do the same thing for under $100.

The CRISPR/Cas9 system has been adapted to enable gene editing in organisms including yeast, fungi, rice, tobacco, zebrafish, mice, dogs, rabbits, frogs, monkeys, mosquitoes and, of course, humans -- so its potential applications are enormous.

For research scientists, CRISPR is a tool that provides better, faster tinkering with genes, allowing them to create models of disease in human cell lines and mouse models with much higher proficiency. With better models of say, cancer, researchers are able to fully understand the pathology and how it develops, and that could lead to improved treatment options.

One particular leap in cancer therapy options is the genetic modification of T cells, a type of white blood cell that's critical for the human immune system. A Chinese clinical trial extracted T cells from patients, used CRISPR to delete a gene that usually acts as an immune system brake, and then reintroduced them into the patients in an effort to combat lung cancer. And that's just one of the many trials underway using CRISPR edited cells to fight particular types of cancer.

Beyond cancer, CRISPR has the potential to treat diseases caused by a mutation in a single gene, such as sickle cell anemia or Duchenne muscular dystrophy. Correcting a defective gene is known as gene therapy, and CRISPR is potentially the most powerful way to perform it. Using mouse models, researchers have demonstrated the efficacy of such treatments but human gene therapies using CRISPR remain untested.

Mosquitoes will be targeted using CRISPR gene drives, which could potentially drive malaria-carrying species to extinction.

Then there are CRISPR gene drives, which use CRISPR to guarantee a genetic trait will be passed from parent to offspring -- essentially rewriting the rules of inheritance. Guaranteeing certain genes will spread through a population provides an unprecedented opportunity to tackle mosquito-borne diseases such as malaria, enabling scientists to create infertile mosquitoes in the lab and release them in the wild to crash the population -- or even render a species extinct. CNET published an extensive report of their proposed use and the ethical concerns that surround them in February 2019.

And CRISPR's potential benefits don't end there. The tool opens up new ways of creating antimicrobials to combat rising levels of antibiotic resistance, targeted manipulation of agricultural crops such as wheat to make them hardier or more nutritious, and, potentially, the ability to design human beings, gene by gene.

CRISPR may be the most precise way to cut DNA we've yet discovered, but it's not always perfect.

One of the chief barriers to getting CRISPR effectively working in humans is the risk of "off-target effects." When CRISPR is tasked with hunting down a gene, it sometimes finds genes that look very similar to its target and cuts them, too.

An unintended cut may cause mutations in other genes, leading to pathologies such as cancer, or it may have no effect at all -- but with safety a major concern, scientists will need to ensure CRISPR acts only on the gene it's intended to impact. This work has already begun, and several teams of researchers have tinkered with CRISPR/Cas9 to increase its specificity.

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To date, CRISPR work in humans has been confined to cells that don't pass on their genome to the next generation. But gene editing can also be used to edit embryos and thus, change the human gene pool. In 2015, an expert panel of CRISPR scientists suggested that such editing -- known as germline editing -- would be irresponsible until consensus can be reached on safety, efficacy, regulation and social concerns.

Still, research into germline editing has been occurring for several years. In 2017, scientists in the UK edited human embryos for the first time, and researchers in the US used CRISPR to correct a defective gene that causes heart disease. The ability to edit embryos begins to raise ethical concerns about so-called designer babies, wherein scientists may select beneficial genes to increase physical fitness, intelligence or muscle strength, creeping into the controversial waters of eugenics.

That particular future is likely a long way off -- but the era of editing the human genome has already begun.

On Nov. 25, 2018, Chinese scientist Jiankui He said he had created the world's first CRISPR babies. By using CRISPR, He was able to delete a gene known as CCR5. The modified embryos resulted in the birth of twin girls, known by the pseudonyms Lulu and Nana.

The scientific community widely condemned the research, criticizing He's lack of transparency and asking whether there was an unmet medical need for the two girls to receive such a modification. In the wake of the research, several high-profile researchers involved with CRISPR's creation even suggested a global moratorium on using the tool for germline editing.

Few would argue that He's work highlights a need for stricter regulatory controls and effective oversight of clinical trials in which embryos are edited. While He maintains his own experiment was concerned with improving the health of the twin girls by making them HIV-resistant, the experiment was deemed reckless and ethically wrong and the potential consequences overlooked. Recent research suggests that the deletion He created in the CCR5 gene may affect brain activity, after a study in mice showed that blocking CC5 improves cognition and recovery from stroke.

In January 2019, the Chinese government said that He acted both unlawfully and unethicallyand would face charges. He was later dismissed by his university.

Jiankui He claimed to have created the world's first gene-edited babies.

The most recent International Summit for Human Genome Editing, in November 2018, concluded, as it did in 2015, "the scientific understanding and technical requirements for clinical practice remain too uncertain and the risks too great to permit clinical trials of germline editing at the time."

He's work, which remains unpublished, heralds the first clinical trial and birth of genetically modified human beings -- which means, whether it was the intention or not, a new era for CRISPR has begun.

As the revolution surges forward, the greatest challenges will continue to be effective oversight and regulation of the technology, the technical hurdles that science must overcome to ensure it is precise and safe, and managing the larger societal concerns of tinkering with the stuff that makes usus.

CRISPR continues to make headlines as scientists refine its specificity and turn it toward myriad genetic diseases. On Feb. 4, researchers at UC Berkeley, including CRISPR pioneer Jennifer Douda, revealed that another enzyme, CasX, could be used to edit genes in place of Cas9.

The scientists identified CasX in a ground-dwelling bacteria not normally present in humans, which means our immune systems are less likely to rebel against it. Because it's smaller and potentially more specific than Cas9, it can clip genes with greater success and less chance of any negative effects.

Then, on Feb. 18, scientists at UC San Francisco revealedthey had used CRISPR to make stem cells "invisible" to the immune system. Stem cells are able to mature into adult cells of any tissue, so they have been proposed as a way to repair damaged organs. However, the immune system typically tries to annihilate any foreign invader and stem cells are seen as such. CRISPR has enabled the stem cells to evade the immune system so they can get to work at healing.

Only a day later, researchers at the Salk Institute for Biological Sciencespublished in Nature Medicine their findings on a CRISPR therapy for Hutchinson-Gilford progeria, a disease associated with rapid aging. The disease is caused by a genetic mutation that results in a buildup of abnormal proteins, ultimately leading to premature cell death. A single dose of CRISPR/Cas9 was shown to suppress the disease in a mouse model, paving the way for further exploration of CRISPR's therapeutic potential.

And still more CRISPR success stories continue to roll in. On Feb. 25, CRISPR Therapeutics, a company co-founded by CRISPR visionary Emmanuelle Charpentier, announced thatthe first human patients had been infused with a CRISPR/Cas9 drug to treat the disease beta-thalassemia. The illness is caused by a genetic mutation that results in red blood cells being unable to create the oxygen-transport molecule haemoglobin. To combat this, the CRISPR Therapeutics team takes stem cells from a patient, edits them with CRISPR/Cas9 outside the body to increase haemoglobin production and then transfuses them back into the bloodstream. The company plans to use a similar approach to treating the blood disease known as sickle cell anemia.

CRISPR research is advancing at a rapid pace, and it can be hard to keep up. In only seven years, CRISPR went from an evolutionary adaptation in bacteria to a gene-editing tool that created the very first genetically modified human beings. We've already seen CRISPR transform the entire field of molecular biology and that effect has rippled across the biological and medical fields.

First published, Jan. 23, 2019.Update, on Feb. 28 5 a.m. PT: Adds recent advances section

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CRISPR gene editing explained: What is it and how does it ...

Alt-R CRISPR-Cas9 System

Simple delivery of ribonucleoprotein complexes (crRNA:tracrRNA:Cas9 or sgRNA:Cas9).

CRISPR-Cas9 genome editing methods use a Cas9 endonuclease to generate double-stranded breaks in DNA. Cas9 endonuclease requires a CRISPR RNA (crRNA) to specify the DNA target sequence, and the crRNA must be combined with the transactivating crRNA (tracrRNA) to activate the endonuclease and create a functional editing ribonucleoprotein complex (Figure 1A). In an alternative approach, the crRNA and tracrRNA can be delivered as a single RNA oligonucleotide (Figure 1B). After cleavage, DNA is then repaired by non-homologous end-joining (NHEJ) or homology-directed recombination (HDR), resulting in a modified sequence. Alt-R CRISPR-Cas9 reagents and kits provide essential, optimized tools needed to use this pathway for genome editing research.

Option 1: Alt-R CRISPR-Cas9 crRNA:tracrRNA

Alt-R CRISPR-Cas9 crRNA

Alt-R CRISPR-Cas9 tracrRNA

The Alt-R CRISPR-Cas9 System offers two options for generating synthetic guide RNAs. The two-part system pairs an optimized, shortened universal tracrRNA oligonucleotide (67 nt) with an optimized, shortened, target-specific crRNA oligonucleotide (36 nt) for improved targeting of Cas9 to dsDNA targets (Figure 2). The single guide RNA (sgRNA) option combines the crRNA and tracrRNA segments into one long RNA molecule, reducing the number of components and simplifying the CRISPR workflow.

While delivering Cas9 nuclease as part of an RNP is the preferred method, the Alt-R CRISPR-Cas9 System is also compatible with S. pyogenes Cas9 from any source, including cells that stably express S. pyogenes Cas9 endonuclease, or when Cas9 is introduced as a DNA or mRNA construct.

All Alt-R CRISPR-Cas9 crRNAs are 3536 nt RNA oligos containing the 19 or 20 nt target-specific protospacer region, along with the 16 nt tracrRNA fusion domain. We recommend 20 nt protospacers for most applications. crRNAs must be duplexed with Alt-R CRISPR-Cas9 tracrRNA before RNP complex formation.

Alt-R CRISPR-Cas9 crRNAs are synthesized with proprietary chemical modifications, which protect the crRNA from degradation by cellular RNases and further improve on-target editing performance. When using 2-part gRNAs under highly challenging conditions (e.g., high nuclease environments or with Cas9 mRNA), use Alt-R CRISPR-Cas9 crRNA XT, which have additional chemical modifications for the highest level of stability and performance.

We guarantee* our predesigned guide RNAs targeting human, mouse, rat, zebrafish, or nematode genes. For other species, you may use our proprietary algorithms to design custom guide RNAs. If you have protospacer designs of your own or from publications, use our design checker tool to assess their on- and off-targeting potential before ordering guide RNAs that are synthesized using our Alt-R guide RNA modifications.

The 67 nt Alt-R tracrRNA is much shorter than the classical 89 bases of the natural S. pyogenes tracrRNA. We find that shortening the tracrRNA increases on-target performance. Alt-R CRISPR tracrRNA also contains proprietary chemical modifications that confer increased nuclease resistance.

Alt-R CRISPR-Cas9 tracrRNA labeled with ATTO 550 (ATTO-TEC) provide the same function as their unlabeled counterparts. However, the fluorescent dye allows you to monitor transfection or electroporation efficiency during preliminary experiments to optimize transfection conditions in your cell types (Figure 3).

Labeled tracrRNAs can also help concentrate transfected cells via FACS (fluorescence-activated cell sorting) analysis, which can simplify your screening process for cells with CRISPR events. (For more information and tips on using Alt-R CRISPR-Cas9 tracrRNA ATTO 550, see the application note.)

Alt-R CRISPR tracrRNA orders include Nuclease-Free Duplex Buffer for forming the complex between crRNA and tracrRNA oligos. Alt-R tracrRNA can be ordered in larger scale and paired with all of your target specific crRNAs, allowing for an easy and a cost-effective means of studying many CRISPR sites.

Alt-R CRISPR-Cas sgRNA

Alt-R CRISPR-Cas9 sgRNAs are long RNA oligonucleotides (99100 bases) containing the target-specific crRNA region and the Cas9-interacting tracrRNA region within a single molecule (i.e., 1920 base protospacer region and 80-base universal sgRNA region). Like other Alt-R RNAs, it contains chemical modifications to stabilize the RNA, increasing resistance to nuclease activity. For challenging conditions (e.g., high nuclease environments or with Cas9 mRNA), sgRNAs may provide increased potency.

The Alt-R S.p. Cas9 Nuclease V3 enzyme is a high purity, recombinant S. pyogenes Cas9. The enzymes include nuclear localization sequences (NLSs) and C-terminal 6-His tags. The S. pyogenes Cas9 enzyme must be combined with a gRNA to produce a functional, target-specific editing complex. For the best editing, combine the Alt-R S.p. Cas9 Nuclease V3 enzyme with the optimized Alt-R CRISPR gRNA in equimolar amounts.

The Alt-R S.p. HiFi Cas9 Nuclease V3 offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events. This Cas9 variant also preserves the high level of editing efficiency expected from a Cas9 nuclease, maintaining 90100% on-target editing activity at most sites. For applications that are sensitive to off-target events, combining the Alt-R S.p. HiFi Cas9 Nuclease V3 with optimized Alt-R CRISPR-Cas9 gRNA (crRNA:tracrRNA) is highly recommended.

Cas9 nickases allow specific cutting of only one strand at the DNA target site. Cuts to both strands of DNA are accomplished by using either Alt-R S.p. Cas9 D10A Nickase V3 or Alt-R S.p. Cas9 H840A Nickase V3, with 2 gRNAs that target two neighboring Cas9 sites, one on either strand of the target region. This functionally increases the length of the recognition sequence from 20 to 40 bases. For more information about using Cas9 nickases, see the application note.

Alt-R S.p. dCas9 Protein V3 has mutations that result in the loss of nuclease activity. This protein can form RNP complexes with Alt-R gRNAs and bind to the target region specified by the gRNA without cutting the DNA.

In some cases, transfection of RNP or the creation of stably transfected cells is not possible. In those applications, AltR S.p. Cas9 Expression Plasmid is designed to provide expression of Cas9 endonuclease under CMV promoter control. Note that the plasmid contains no eukaryotic selectable marker, making expression of S.p. Cas9 transient. The Alt-R CRISPR-Cas9 System Plasmid User Guide provides instructions for using this plasmid.

Optional controls for human, mouse, and rat are available for the 2-part Alt-R CRISPR-Cas9 System.

We recommend using the appropriate Alt-R CRISPR-Cas9 Control Kit for studies in human, mouse, or rat cells. The control kits include an Alt-R CRISPR HPRT Positive Control crRNA targeting the HPRT (hypoxanthine phosphoribosyltransferase) gene and a computationally validated Alt-R CRISPR-Cas9 Negative Control crRNA. The kit also includes the Alt-R CRISPR-Cas9 tracrRNA for complexing with the crRNA controls, Nuclease-Free Duplex Buffer, and validated PCR primers for amplifying the targeted HPRT region in the selected organism. The inclusion of the PCR assay makes the kits ideal for verification of HPRT modification using the Alt-R Genome Editing Detection Kit.

Alt-R control kit components can also be ordered individually.

For information about sgRNA controls, contact applicationsupport@idtdna.com.

If you are studying primary or hard-to-transfect cells, electroporation is often a viable alternative to lipid-based transfection in CRISPR experiments. The Alt-R Cas9 Electroporation Enhancer is a Cas9-specific carrier DNA that is optimized to work with the Amaxa Nucleofector device (Lonza) and Neon System (Thermo Fisher) to increase transfection efficiency and thereby increase genome editing efficiency (Figure 4).

Alt-R HDR Enhancer is a small molecule compound that increases homology-directed repair. Alt-R HDR Enhancer exhibits its activity in multiple cell lines, including both adherent and suspension cell lines. Its activity is independent of the enzyme employed; for example, it can be used either with Alt-R S.p. Cas9 Nuclease V3 or Alt-R A.s. Cas12a (Cpf1) Nuclease V3.This versatile reagent is also compatible with electroporation and lipofection methods.

Use this kit to detect on-target genome editing and estimate genome editing efficiency in CRISPR experiments. Learn more >>

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Start genome editing with CRISPR-Cas9 | idtdna.com

Bassik Human CRISPR Knockout Library 101926 101934 Knockout Human Bassik 3rd 10 Varies Bassik Mouse CRISPR Knockout Library 1000000121 1000000130 Knockout Mouse Bassik 3rd 10 Varies Activity-optimized genome-wide library Discontinued Knockout Human Sabatini and Lander 3rd 10 178,896 Activity-optimized genome-wide library 1000000100 Knockout Human Sabatini and Lander 3rd 10 187,535 Broad GPP genome-wide Brunello 73179 (1 plasmid)73178 (2 plasmid) Knockout Human Doench and Root 3rd 4 76,441 Broad GPP genome-wide Brie 73632 (1 plasmid)73633 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 78,637 Broad GPP kinome Brunello 75314, 75315 (1 plasmid)75312, 75313(2 plasmid) Knockout Human Doench and Root 3rd 4 3,052 Broad GPP kinome Brie 75317 (1 plasmid)75316 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 2,852 Broad GPP activation Calabrese p65-HSF 92379 (Set A)92380 (Set B) Activation Human Doench and Root 3rd 36 56,762 (Set A)56,476 (Set B) Broad GPP activation Caprano p65-HSF 92383 (Set A)92384 (Set B) Activation Mouse Doench and Root 3rd 36 67,187 (Set A)66,889 (Set B) Broad GPP inhibition Dolcetto 92385 (Set A)92386 (Set B) Inhibition Human Doench and Root 3rd 36 57,050 (Set A)57,011 (Set B) Broad GPP inhibition Dolomiti 104090 (Set A)104091 (Set B) Inhibition Mouse Doench and Root 3rd 36 67,366 (Set A)67,194 (Set B) Cas13a/C2c2 Protospacer flanking site (PFS) Library 79153 Knockout E. coli Zhang N/A N/A - The protospacers contained in the library represent all 4096 (46) combinations of 6 nucleotides. N/A CRiNCL - Human CRISPRi Non-coding Libraries 86538 86550 Inhibition Human Weissman 3rd 10 Varies CRISPR/Cas9-assisted Removal of Mitochondrial DNA (CARM) Library 82480 Knockout Mouse Xie N/A N/A 395 CRISPRa Discontinued Activation Human Weissman 3rd 10 198,810 CRISPRa-v2 839781000000091 Activation Human Weissman 3rd 510 104,540209,080 CRISPRa-v2 839961000000093 Activation Mouse Weissman 3rd 510 107,105214,210 CRISPRi Discontinued Inhibition Human Weissman 3rd 10 206,421 CRISPRi-v2 839691000000090 Inhibition Human Weissman 3rd 510 104,535209,070 CRISPRi-v2 839871000000092 Inhibition Mouse Weissman 3rd 510 107,415214,830 Enriched subpools (kinase, nuclear, ribosomal, cell cycle) 51043 51048 Knockout Human Sabatini and Lander 3rd 10 Varies Focused Ras Synthetic Lethal Human CRISPR Knockout Library 92352 Knockout Human Sabatini and Lander 3rd 50 6,661 hCRISPRa-v2 subpooled libraries 83980 83986 Activation Human Weissman 3rd 5 Varies hCRISPRi-v2 subpooled libraries 83971 83977 Inhibition Human Weissman 3rd 5 Varies mCRISPRa-v2 subpooled libraries 83998 84004 Activation Mouse Weissman 3rd 5 Varies mCRISPRi-v2 subpooled libraries 83989 83995 Inhibition Mouse Weissman 3rd 5 Varies Human CRISPR Knockout Library 1000000132 Knockout Human X.S. Liu 3rd 10 185,634 Human GeCKO v2 1000000048 (1 plasmid)1000000049 (2 plasmid) Knockout Human Zhang 3rd 6 123,411 Human genome-wide library v1 69763 Knockout Human Wu 3rd 4 77,406 Human improved genome-wide library v1 67989 Knockout Human Yusa 3rd 5 90,709 Human CRISPR lncRNA Activation Pooled Library 1000000106 Activation Human Zhang 3rd 10 96,458 Human CRISPR Metabolic Gene Knockout Library 110066 Knockout Human Sabatini 3rd 10 30,290 Human miRNA CRISPR Knockout Library 112200 Knockout Human Lin 3rd 4-5 8,382 Human Paired-guide RNA (pgRNA) Library for Long Non-coding RNAs (lncRNAs) 89640 Knockout Human Wei 3rd Varies 12,472 pairs Mouse GeCKO v2 1000000052 (1 plasmid)1000000053(2 plasmid) Knockout Mouse Zhang 3rd 6 130,209 Mouse genome-wide library v1 Discontinued Knockout Mouse Yusa 3rd 5 87,897 Mouse improved genome-wide library v2 67988 Knockout Mouse Yusa 3rd 5 90,230 Oxford Fly 64750 Knockout D. melanogaster Liu N/A 3 40,279 Perturb-seq Guide Barcodes (GBC) 85968 Barcode Human Weissman 3rd N/A N/A SAM v1 - 3 plasmid system 1000000057 (Zeocin)1000000074 (Puromycin) Activation Human Zhang 3rd 3 70,290 SAM v1 - 3 plasmid system 1000000075 (Puromycin) Activation Mouse Zhang 3rd 3 69,716 SAM v2 - 2 plasmid system 1000000078 (Blasticidin) Activation Human Zhang 3rd 3 70,290 Toronto KnockOut - Version 1 1000000069 Knockout Human Moffat 3rd 12 176,500 Toronto KnockOut - Version 3 90294 Knockout Human Moffat 3rd 4 70,948 Toxoplasma Knockout 80636 Knockout T. gondii Lourido N/A 10 8,158 Two plasmid human activity-optimized genome-wide library 1000000095 Knockout Human Sabatini and Lander 3rd 10 187,536 Two plasmid mouse activity-optimized genome-wide library 1000000096 Knockout Mouse Sabatini and Lander 3rd 10 188,509

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Addgene: CRISPR Pooled gRNA Libraries

The past year hasn't been easy for Sangamo Therapeutics (NASDAQ:SGMO), CRISPR Therapeutics (NASDAQ:CRSP), and their respective shareholders. The market has cleaved billions from their market values even though they hardly did anything.

Both are going to have some important clinical trial readouts in 2019 that could send their share prices screaming toward the moon again. Let's stack these biotechs side by side to see which has a better chance of coming out on top.

Image source: Getty Images.

There's no disputing the value of CRISPR-based gene editing in the world of academia, but the jury's still out on whether CRISPR is a useful drug discovery tool. Although investors are ready to leap to broad conclusions, we're going to receive the verdict in tiny baby steps.

In partnership with Vertex Pharmaceuticals (NASDAQ:VRTX), CRISPR Therapeutics treated its first patient with CTX001 in late February. This experimental gene therapy involves editing a patient's stem cells outside their body with CRISPR/cas9 technology before reintroducing the cells to patients who need help producing functional hemoglobin.

CTX001 edits stem cells in a way that allows production of fetal hemoglobin to begin again. That should help patients with transfusion-dependent thalassemia and severe sickle-cell disease to reduce their reliance on frequent blood transfusions. Frequent transfusions are inconvenient, painful, and so expensive that a one-time cure could be a big hit for patients and insurers.

The plan is to treat two thalassemia patients first, then wait for safety data before putting anyone else at risk. If CTX001 can get these patients to safely produce enough fetal hemoglobin to make transfusions unnecessary, CRISPR's stock will soar and trigger milestone payments from Vertex.

CRISPR Therapeutics also plans to begin clinical studies with two cellular cancer therapies that it still owns outright. Once the ball gets rolling with more clinical trials, the bills could start piling up.

By the time patients begin showing meaningful data, CRISPR will probably be ready for a cash injection. The company finished 2018 with $456 million in cash after losing $165 million last year. CRISPR is codeveloping the drug with Vertex, which could get expensive. CRISPR spent $114 million last year on research and development before a single patient had been dosed.

Image source: Getty Images.

Year after year, Sangamo publishes a study that proves its zinc-finger nuclease (ZFN) technology is superior to all other gene editing methods in one way or another. Before you get carried away by the possibilities, it's important to remember that this biotech has been going at it since the mid-1990s and it still hasn't sent a single new drug application to the FDA.

Sadly, the company's recent attempts with ZFN-based candidates haven't been too successful. Sangamo was able to show us that SB-913 successfully inserted a gene that should help patients with mucopolysaccharidosis type II (MPS II) produce iduronate-2-sulfatase (IDS), an important digestive enzyme they can't make on their own.

Sangamo detected a small amount of IDS production from just one patient who received the highest dose during a dose-determination study. Investigators noticed signs of possible liver damage from this patient, which means Sangamo probably won't be able to use a higher, possibly effective dosage in a larger study.

After a couple of fruitless decades with earlier versions, Sangamo plans on releasing next-generation ZFN treatments before the end of the year. Until we see data that says they work, you probably shouldn't pin any significant value to this company's ZFN-based pipeline.

The only reason to buy Sangamo is its proprietary synthetic liver-specific gene promoter that's also a part of SB-525, a candidate for the treatment of hemophilia A. Pfizer (NYSE:PFE) has agreed to take the reins for SB-525 if an ongoing trial hits the right mark.

It turns out that the hard part about gene editing isn't the editing, it's getting the target cells to do something with the new gene once it's been pasted into place. Pfizer's interested in Sangamo's synthetic liver-specific promoter because it looks like it did the trick for hemophilia A patients.

Hemophilia patients need regular infusions of blood-clotting factors that they can't produce themselves or they risk an uncontrolled bleeding event. During the first dose-ranging study with SB-525, patients produced more of the clotting factor than anyone imagined possible.

Sangamo promised to give us an interim look at the follow-up Alta study with SB-525 last December but decided at the last moment to hold these cards close to the vest. Instead, the complete results will be released sometime in 2019.

Image source: Getty Images.

At recent prices, Sangamo's market cap is just $935 million, so another success for SB-525 could send its stock price through the roof. Pfizer's already on the hook for development expenses, and Sangamo is entitled to milestones and royalty payments that the big pharma could avoid by simply acquiring Sangamo.

Right now CRISPR Therapeutics' market cap is at $1.8 billion, which is a lot for a company without any human proof-of-concept data yet. If the first two patients don't report amazing results, the stock will receive an awful beating.It's probably best to cheer for CRISPR from the sidelines, and put the better buy, Sangamo, in a diverse portfolio.

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Better Buy: CRISPR Therapeutics vs. Sangamo Therapeutics

In the early days of gene editing, biologists had a molecular tool kit that was somewhat akin to a printing press. Which is to say, altering DNA was a messy, labor-intensive process of loading genes onto viruses bound for target cells. It involved more than a fair amount of finger-crossing. Today, scientists have the genetic equivalent of Microsoft Word, and they are beginning to edit DNA almost as easily as software engineers modify code. The precipitating event? Call it the Great Crispr Quake of 2012.

If youre asking, whats Crispr? the short answer is that its a revolutionary new class of molecular tools that scientists can use to precisely target and cut any kind of genetic material. Crispr systems are the fastest, easiest, and cheapest methods scientists have ever had to manipulate the code of life in any organism on Earth, humans included.

The long answer is that Crispr stands for Clustered Regularly Interspaced Palindromic Repeats. Crispr systems consist of a protein with sequence-snipping capabilities and a genetic GPS guide. Such systems naturally evolved across the bacterial kingdom as a way to remember and defend against invading viruses. But researchers recently discovered they could repurpose that primordial immune system to precisely alter genomes, setting off a billion-dollar boom in DNA hacking.

Every industry is throwing mad money at Crisprpharma, agriculture, energy, materials manufacturing, you name it. Even the weed guys want in. Companies are using it to make cancer-curing medicines, climate-change-fighting crops, biofuel-oozing algae, and self-terminating mosquitoes. Academic researchers have almost universally adopted Crispr to more deeply understand the biology of their model organisms. Supporting this biohacking bonanza is an increasingly crowded Crispr backend supply chain; businesses building gene-editor design tools and shipping synthetic guide RNAs or pre-Crisprd cell lines to these companies doors. So far, though, very few Crispr-enhanced products have made it into the hands of actual consumers. In their place, hyperbolic headlines have bugled societys greatest hopes and fears for the technology, from saving near-extinct species to igniting a superbaby arms race.

Crispr isnt going to end disease or hunger or climate change any time soon. Maybe it never will. Nor is it about to deliver designer children or commit genetic genocide. (Though its never too early to start talking about the ethical dilemmas such a powerful technology could pose.) Crispr is, however, already beginning to reshape the physical world around us in much less radical ways, one base pair at a time.

It all started with yogurt. To make it, dairy producers have long employed the help of Streptococcus thermophilus, a bacteria that gobbles up the lactose in milk and poops out lactic acid. It wasnt until 2005, though, that a young microbiologist named Rodolphe Barrangou discovered that S. thermophilus contained odd chunks of repeating DNA sequencesCrisprsand that those sequences were keeping it safe from the viruses that attack it and result in spoilage. (If the thermophilus is gone, nastier bacteria can move in and feed off the lactose, ruining the product.)

Before long, DuPont bought the Danish company that Barrangou worked for and began using Crispr to protect all of its yogurt and cheese cultures. Since DuPont owns about 50 percent of the global dairy culture market, that means youve probably already eaten Crispr-optimized cheese on your pizza.

Viruses work by turning your cells into little factories for their DNA. A Crispr-based test could pick out that foreign DNA from just a drop of blood, spit, or urine and tell you in minutes if youve got the Zika virus, dengue, or yellow fever circulating in your body.

Every year, fungi wipe out a third of all crops. Crispr panels tuned to identify the worst offenders could help farmers save their harvests before the blight sets in.

Thanks to overuse, the worlds antibiotic arsenal is losing its effectiveness. New Crispr-based drugs that only target bad bugs would leave your microbiome intact and help fight antibiotic resistance.

All the while, gene sequencing costs were plummeting and research scientists around the world were assembling the genomes of bacteria. As they did, they found Crisprs everywheremore than half of the bacterial kingdom turned out to have them. Oftentimes those sequences were flanked by a set of genes coding for a class of strand-cutting enzymes called endonucleases. Scientists suspected they were involved in this primitive immune system, but how exactly?

The key insight came from a particularly nasty bugthe one that causes strep throat. Its Crispr system made two RNA sequences that attached to a clam-shaped endonuclease called Cas9. Like a genetic GPS, those sequences directed the enzyme to a strand of DNA complementary to the RNA sequences. When it got there, Cas9 changed shape, grabbing the DNA and slicing it in two. The molecular biologists who made this discoveryJennifer Doudna and Emmanuelle Charpentierpublished their work on bacteria in Science in 2012. But not before patenting the technology as a tool for genetic engineering. If you just switch out the RNA guide, you can send Cas9 anywhereto the gene that causes Huntingtons disease, say, and snip it out. Crispr, they realized, would be a molecular biologists warp drive.

Six months later, a molecular biologist at the Broad Institute of MIT and Harvard named Feng Zhang published a paper in Science showing how Crispr-Cas9 could edit human cells too. In fact, with the right genetic guides, you can Crispr pretty much anything. That meant it might be put to work on next-generation medicines that could do things like erase genetic defects and supercharge the bodys natural defenses against cancer. And that meant big money.

Perhaps predictably, a patent battle ensuedone that is still going on today. Crisprs early pioneers founded three companies with exclusive licenses to exploit Crispr/Cas9 to cure human diseases; the first clinical trials are expected to begin in the US in 2018. Uncertainty over who will ultimately own the technology has done little to slow the appetite for all things Crispr. If anything, it has unleashed a flood of interest in developing competing and adjacent tools that promise to further refine and expand Crisprs already ample potential.

For now, Crispr is still a biologist's buzzword. But just as computers evolved from a nerdy, niche tool for math geeks to a ubiquitous, invisible extension of our own bodies, so Crispr will one day weave seamlessly into the fabric of our physical reality. It will simply be the way to solve a problem, if that problem is remotely biological in nature.

Take industrial fermentation for example. With the help of old-school genetic engineering techniques, scientists have already reprogrammed microbes like E. Coli and brewers yeast into factories that can make everything from insulin to ethanol. Crispr will rapidly enlarge the catalog of designer chemicals, molecules, and materials that biorefineries can produce. Self-healing concrete? Fire-resistant, plant-based building materials lighter than aluminum? Fully biodegradable plastics? Crispr not only makes all these possible, it makes it possible to produce them at scale.

But we wont get there with the tools weve currently got. Which is why researchers are now racing to chart the full expanses of the Crispr universe. At this moment theyre scouring the globe for obscure bacteria to sequence, and theyre tinkering with the systems that have already been discovered. Theyre filing patents on every promising new nuclease they come across, adding to a list that is sure to expand in the coming decade. Each new enzyme will not only advance Crisprs gene editing powers, but extend its capabilities far beyond DNA manipulation. You see, slicing and dicing isnt the only interesting thing to do to DNA. Tricked out new Crispr systems could temporarily toggle genes on and off or surveil the genome to fix mutations as they happen in real time, no snipping required. The first would let scientists treat human diseases where theres too much or too little of a certain substancesay insulinwithout permanently altering a patients DNA. The second could one day prevent diseases like cancer from occurring altogether. The specificity of Crispr, perhaps more than its actual cutting mechanism, will inspire applications we cant yet imagine.

Good at cutting DNA, great for knockouts. Already being replaced by newer base pair editors with more fine-tuned control.

Like Cas9 but not as sloppy. It leaves sticky DNA ends, which are easier to work with when making edits.

Cuts RNA not DNA. Could knock down protein levels without permanently changing your genome. Pair it with a reporter signal and youve got a diagnostic.

Cas3 gives zero f***. It offers no repair mechanismonce it finds that target DNA sequence it just starts cutting till there aint no DNA left.

Just discovered in an abandoned silver mine, we dont know yet what these tiny enzymes superpowers will be.

Meanwhile, consumers can expect to see their first Crisprd products lining grocery store shelves very soon. Because Crispr doesnt use plant pathogens to manipulate DNA (the old GMO-generating method), the USDA has given a free regulatory pass to gene-edited crops, allowing drought-tolerant soybeans and extra-starchy corn to ease into your favorite processed foods. Specialty fruits and vegetables will likely follow the commodity crops; the reduced regulatory burden and the cheapness of Crispr will allow companies appealing to consumers senses rather than farmers bottom lines to enter the market. Already a dozen or so startups have popped up to challenge the Bayer/Monsanto, DowDupont/Pioneers of the world.

This democratizing aspect of Crispr-based tech, combined with its nearly limitless commercial possibilities, make today a great time to be a molecular biologist. Want to make antibiotics that only target bad bugs without wiping out the entire microbiome? There are companies doing that. Want to make paper-based diagnostics that doctors can take into the field to test for diseases like dengue and Zika? There are research labs and startups doing that too. And as more tools come online, the backend Crispr ecosystem will continually expand to support, supply, and optimize them.

Crispr applications are only going to become more powerful, and when they do they will rightly invite more scrutiny, and probably more regulation. Were going to have to figure out if its OK to wipe out an entire species in the name of conservation and bring other ones back from extinction. Well have to wrestle with the possibility that gene editing tools might be used to produce biological weapons of unfathomable destruction. And yes, well eventually have to talk about designer babies; when is it acceptable to fix a genetic mutation? Would we ever start adding features? Where do we draw the line? Crispr, and all the tools that will one day make up the Crispr universe will undoubtedly force societiesnot just scientiststo confront these questions and ponder the oldest one of all; what does it mean to be human?

Everything You Need To Know About Crispr Gene EditingOkay, you get it, Crisprs a big deal. But now, arent you curious to know exactly how it works? You dont have to be a microbiologist to understand this step-by-step look inside the molecular multitool of the century.

What Good Is Crispr If It Cant Get Where It Needs To Go?It doesnt matter how good Crispr gets, in order to actually snip away humanitys worst diseases, it first has to get to the right cells. And thats way harder said than done. Its time to talk about Crisprs delivery problem.

First Human-Pig Chimera Is a Step Toward Custom OrgansScientists have long been dreaming of xenotransplantationputting animal organs into peopleas a possible solution to the current human organ shortage. But almost all attempts to do so have failed. Heres how Crispr is bringing new hope to the dream of animal organ farms.

Read This Before You Freak Out Over Gene-Edited SuperbabiesIn the last few years, scientists in the US and China have used Crispr to fix genetic mutations in human embryos, prompting concerns over the imminent takeover of genetically superior designer children. Breathe, people: You dont need to worry about that for a long, long, long time.

America Needs To Figure Out the Ethics of Gene Editing NowStill. All that successful human embryo modification has scientists around the world calling for varying levels of caution against it. And while pretty much everyone agrees on avoiding a Gattaca-type situation, thats where the consensus ends.

Process of EliminationFor decades conservationists have used medieval methods for eradicating invasive island predators like rats. And all those traps and guns and poisons still havent gotten the job done. Local species are still under threat of extinction. Now some scientists are turning to Crispr gene drives, a particularly potent genetic tool that could forever transform our power over nature. Emma Marris went to the Galapagos to see how they might work in the wild.

The FDA Wants to Regulate Gene-Edited Animals as DrugsWe get it. Its hard to contort the USs 1938 patchwork of laws around 21st century technology. But companies making hornless cows and tailless pigs and all-male beef cattle are pissed at the FDAs new interpretation of the rules, and talking about taking their tech elsewhere.

Easy DNA Editing Will Remake the World. Buckle Up.Still havent had enough Crispr? Amy Maxmens 2015 cover story is the definitive survey of this gene-editing technology; from its humble bacterial beginnings, to the trenches of its ferocious patent battle, to inside the companies already churning toward our Crispr-created future.

Plus! Crispr uploads a galloping horse GIF into a living bacteria and more WIRED gene editing coverage.

This guide was last updated on April 26, 2018.

Enjoyed this deep dive? Check out more WIRED Guides.

Continued here:
What is Crispr Gene Editing? The Complete WIRED Guide | WIRED

We are in the midst of a gene-editing revolution.

For four decades, scientists have tinkered with our genes. Since the 1970s, they've experimentally switched them on and off, uncovering their functions; mapped their location within our genome; and even inserted or deleted them in animals, plants and human beings.

And in November 2018, aChinese scientist claimedto have created the world's first genetically modified human beings.

Though scientists have made great inroads into understanding human genetics, editing our genes has remained a complex process requiring imprecise, expensive technology, years of expertise and just a little luck, too.

In 2012, a pair of scientists developed a new tool to modify genes, reshaping the entire field of gene-editing forever: CRISPR. Often described as "a pair of molecular scissors," CRISPR is widely considered the most precise, most cost-effective and quickest way to edit genes. Its potential applications are far-reaching, affecting conservation, agriculture, drug development and how we might fight genetic diseases. It could even alter the entire gene pool of a species.

The field of CRISPR research is still remarkably young, yet we've already seen how it might be used to fight HIV infection, combat invasive species and destroy antibiotic-resistant bacteria. Many unknowns remain, however, including how CRISPR might damage DNA, leading to pathologies such as cancer.

Such a monumental leap in genetic engineering is full of complexities that ask big, often philosophical questions about science, ethics, how we conduct research and the future of humanity itself. With the confirmation that two human embryos were modified using CRISPR and carried to term, those questions have come sharply into focus. The future of gene-editing seemingly arrived overnight.

But what exactly is CRISPR and what are the outstanding concerns about such a powerful tool?

Let's break it all down.

CRISPR has the potential to be used in editing human embryos to create "designer babies."

Few predicted how important CRISPR would become for gene editing upon its discovery 30 years ago.

As early as 1987, researchers at Osaka University studying the function of Escherichia coli genes first noticed a set of short, repeated DNA sequences, but they didn't understand the significance.

Six years later, another microbiologist, Francisco Mojica, noted the sequences in a different single-celled organism, Haloferax mediterranei. The sequences kept appearing in other microbes and in 2002, the unusual DNA structures were given a name: Clustered regularly interspaced short palindromic repeats.

CRISPR.

Studying the sequences more intensely revealed that CRISPR forms an integral part of the "immune system" in bacteria, allowing them to fight off invading viruses. When a virus enters the bacteria, it fights back by cutting up the virus' DNA. This kills the virus and the bacteria stores some of the leftover DNA.

The leftover DNA is like a fingerprint, stored in the CRISPR database. If invaded again, the bacteria produce an enzyme called Cas9 that acts like a fingerprint scanner. Cas9 uses the CRISPR database to match the stored fingerprints with those of the new invader. If it can find a match, Cas9 is able to chop up the invading DNA.

Nature often provides great templates for technological advances. For instance, the nose of a Japanese bullet train is modeled on the kingfisher's beak because the latter is expertly "designed" by evolution to minimize noise as the bird dives into a stream to catch fish.

In a similar way, CRISPR/Cas9's ability to efficiently locate specific genetic sequences, and cut them, inspired a team of scientists to ask whether that ability could be mimicked for other purposes.

The answer would change gene editing forever.

In 2012, pioneering scientists Jennifer Doudna, from UC Berkeley, and Emmanuelle Charpentier, at Umea University Sweden, showed CRISPR could be hijacked and modified. Essentially, they'd turned CRISPR from a bacterial defense mechanism into a DNA-seeking missile strapped to a pair of molecular scissors. Their modified CRISPR system worked marvelously well, finding and cutting any gene they chose.

An illustration of the CRISPR-Cas9 gene editing complex. The Cas9 nuclease protein (white and green) uses a guide RNA (red) sequence to cut DNA (blue) at a complementary site.

Several research groups followed up on the original work, showing that the process was possible in yeast and cultured mouse and human cells.

The floodgates opened, and CRISPR research, which had long been the domain of molecular microbiologists, skyrocketed. The number of articles referencing CRISPR in preeminent research journal Nature has increased by over 6,000 percent between 2012 and 2018.

While other gene-editing tools are still in use, CRISPR provides a gigantic leap because of its precision and reliability. It's really good at finding genes and making accurate cuts. That allows genes to be cut out with ease, but it also provides an opportunity to paste new genes into the gap. Previous gene-editing tools could do this, too, but not with the ease that CRISPR can.

Another huge advantage CRISPR has over alternative gene-editing techniques is its expense. While previous techniques might cost a laboratory upward of $500 to edit a single gene, a CRISPR kit can do the same thing for under $100.

The CRISPR/Cas9 system has been adapted to enable gene editing in organisms including yeast, fungi, rice, tobacco, zebrafish, mice, dogs, rabbits, frogs, monkeys, mosquitoes and, of course, humans -- so its potential applications are enormous.

For research scientists, CRISPR is a tool that provides better, faster tinkering with genes, allowing them to create models of disease in human cell lines and mouse models with much higher proficiency. With better models of say, cancer, researchers are able to fully understand the pathology and how it develops, and that could lead to improved treatment options.

One particular leap in cancer therapy options is the genetic modification of T cells, a type of white blood cell that's critical for the human immune system. A Chinese clinical trial extracted T cells from patients, used CRISPR to delete a gene that usually acts as an immune system brake, and then reintroduced them into the patients in an effort to combat lung cancer. And that's just one of the many trials underway using CRISPR edited cells to fight particular types of cancer.

Beyond cancer, CRISPR has the potential to treat diseases caused by a mutation in a single gene, such as sickle cell anemia or Duchenne muscular dystrophy. Correcting a defective gene is known as gene therapy, and CRISPR is potentially the most powerful way to perform it. Using mouse models, researchers have demonstrated the efficacy of such treatments but human gene therapies using CRISPR remain untested.

Mosquitoes will be targeted using CRISPR gene drives, which could potentially drive malaria-carrying species to extinction.

Then there are CRISPR gene drives, which use CRISPR to guarantee a genetic trait will be passed from parent to offspring -- essentially rewriting the rules of inheritance. Guaranteeing certain genes will spread through a population provides an unprecedented opportunity to tackle mosquito-borne diseases such as malaria, enabling scientists to create infertile mosquitoes in the lab and release them in the wild to crash the population -- or even render a species extinct.

And CRISPR's potential benefits don't end there. The tool opens up new ways of creating antimicrobials to combat rising levels of antibiotic resistance, targeted manipulation of agricultural crops such as wheat to make them hardier or more nutritious, and, potentially, the ability to design human beings, gene by gene.

CRISPR may be the most precise way to cut DNA we've yet discovered, but it's not always perfect.

One of the chief barriers to getting CRISPR effectively working in humans is the risk of "off-target effects." When CRISPR is tasked with hunting down a gene, it sometimes finds genes that look very similar to its target and cuts them, too.

An unintended cut may cause mutations in other genes, leading to pathologies such as cancer, or it may have no effect at all -- but with safety a major concern, scientists will need to ensure CRISPR acts only on the gene it's intended to impact. This work has already begun, and several teams of researchers have tinkered with CRISPR/Cas9 to increase its specificity.

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To date, CRISPR work in humans has been confined to cells that don't pass on their genome to the next generation. But gene editing can also be used to edit embryos and thus, change the human gene pool. In 2015, an expert panel of CRISPR scientists suggested that such editing -- known as germline editing -- would be irresponsible until consensus can be reached on safety, efficacy, regulation and social concerns.

Still, research into germline editing has been occurring for several years. In 2017, scientists in the UK edited human embryos for the first time, and researchers in the US used CRISPR to correct a defective gene that causes heart disease. The ability to edit embryos begins to raise ethical concerns about so-called designer babies, wherein scientists may select beneficial genes to increase physical fitness, intelligence or muscle strength, creeping into the controversial waters of eugenics.

That particular future is likely a long way off -- but the era of editing the human genome has already begun.

On Nov. 25, 2018, Chinese scientist Jiankui He said he had created the world's first CRISPR babies. By using CRISPR, He was able to delete a gene known as CCR5. The modified embryos resulted in the birth of twin girls, known by the pseudonyms Lulu and Nana.

The scientific community widely condemned the research, criticizing He's lack of transparency and asking whether there was an unmet medical need for the two girls to receive such a modification. In the wake of the research, several high-profile researchers involved with CRISPR's creation even suggested a global moratorium on using the tool for germline editing.

Few would argue that He's work highlights a need for stricter regulatory controls and effective oversight of clinical trials in which embryos are edited. While He maintains his own experiment was concerned with improving the health of the twin girls by making them HIV-resistant, the experiment was deemed reckless and ethically wrong and the potential consequences overlooked. In January 2019, the Chinese government said that He acted both unlawfully and unethicallyand would face charges. He was later dismissed by his university.

Jiankui He claimed to have created the world's first gene-edited babies.

The most recent International Summit for Human Genome Editing, in November 2018, concluded, as it did in 2015, "the scientific understanding and technical requirements for clinical practice remain too uncertain and the risks too great to permit clinical trials of germline editing at the time."

He's work, which remains unpublished, heralds the first clinical trial and birth of genetically modified human beings -- which means, whether it was the intention or not, a new era for CRISPR has begun.

We've already seen CRISPR transform the entire field of molecular biology -- and that effect has rippled across the biological and medical fields at lightning speed. In only six years, CRISPR went from an evolutionary adaptation in bacteria to a gene-editing tool that, potentially, created the very first genetically modified human beings.

As the revolution surges forward, the greatest challenges will lie in oversight and regulation of the technology, the technical hurdles that science must overcome to ensure it is precise and safe, and the larger societal concerns of tinkering with the stuff that makes us us.

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