Archive for the ‘Crispr’ Category

CRISPR, a new genome editing tool, could transform the field of biologyand a recent study on genetically-engineered human embryos has converted this promise into media hype. But scientists have been tinkering with genomes for decades. Why is CRISPR suddenly such a big deal?

The short answer is that CRISPR allows scientists to edit genomes with unprecedented precision, efficiency, and flexibility. The past few years have seen a flurry of firsts with CRISPR, from creating monkeys with targeted mutations to preventing HIV infection in human cells. Earlier this month, Chinese scientists announced they applied the technique to nonviable human embryos, hinting at CRISPRs potential to cure any genetic disease. And yes, it might even lead to designer babies. (Though, as the results of that study show, its still far from ready for the doctors office.)

In short, CRISPR is far better than older techniques for gene splicing and editing. And you know what? Scientists didnt invent it.

CRISPR is actually a naturally-occurring, ancient defense mechanism found in a wide range of bacteria. As far as back the 1980s, scientists observed a strange pattern in some bacterial genomes. One DNA sequence would be repeated over and over again, with unique sequences in between the repeats. They called this odd configuration clustered regularly interspaced short palindromic repeats, or CRISPR.

This was all puzzling until scientists realized the unique sequences in between the repeats matched the DNA of virusesspecifically viruses that prey on bacteria. It turns out CRISPR is one part of the bacterias immune system, which keeps bits of dangerous viruses around so it can recognize and defend against those viruses next time they attack. The second part of the defense mechanism is a set of enzymes called Cas (CRISPR-associated proteins), which can precisely snip DNA and slice the hell out of invading viruses. Conveniently, the genes that encode for Cas are always sitting somewhere near the CRISPR sequences.

Here is how they work together to disable viruses, as Carl Zimmer elegantly explains in Quanta:

As the CRISPR region fills with virus DNA, it becomes a molecular most-wanted gallery, representing the enemies the microbe has encountered. The microbe can then use this viral DNA to turn Cas enzymes into precision-guided weapons. The microbe copies the genetic material in each spacer into an RNA molecule. Cas enzymes then take up one of the RNA molecules and cradle it. Together, the viral RNA and the Cas enzymes drift through the cell. If they encounter genetic material from a virus that matches the CRISPR RNA, the RNA latches on tightly. The Cas enzymes then chop the DNA in two, preventing the virus from replicating.

There are a number Cas enzymes, but the best known is called Cas9. It comes from Streptococcus pyogenes, better known as the bacteria that causes strep throat. Together, they form the CRISPR/Cas9 system, though its often shortened to just CRISPR.

Top image: Screenshot from this MIT video explaining CRISPR

As this point, you can start connecting the dots: Cas9 is an enzyme that snips DNA, and CRISPR is a collection of DNA sequences that tells Cas9 exactly where to snip. All biologists have to do is feed Cas9 the right sequence, called a guide RNA, and boom, you can cut and paste bits of DNA sequence into the genome wherever you want.

DNA is a very long string of four different bases: A, T, C, and G. Other enzymes used in molecular biology might make a cut every time they see, say, a TCGA sequence, going wild and dicing up the entire genome. The CRISPR/Cas9 system doesnt do that.

Cas9 can recognize a sequence about 20 bases long, so it can be better tailored to a specific gene. All you have to do is design a target sequence using an online tool and order the guide RNA to match. It takes no longer than few days for the guide sequence to arrive by mail. You can even repair a faulty gene by cutting out it with CRISPR/Cas9 and injecting a normal copy of it into a cell. Occasionally, though, the enzyme still cuts in the wrong place, which is one of the stumbling blocks for wider use, especially in the clinic.

Mice whose genes have been altered or knocked out (disabled) are the workhorses for biomedical research. It can take over a year to establish new lines of genetically-altered mice with traditional techniques. But it takes just few months with CRISPR/Cas9, sparing the lives of many mice and saving time.

Traditionally, a knockout mouse is made using embryonic stem (ES) cells. Researchers inject the altered DNA sequence into mouse embryos, and hope they are incorporated through a rare process called homologous recombination. Some of first generation mice will be chimeras, their bodies a mixture of cells with and without the mutated sequence. Only some of the chimeras will have reproductive organs that make sperm with mutated sequence. Researchers breed those chimeras with normal mice to get a second generation, and hope that some of them are heterozygous, aka carrying one normal copy of the gene and one mutated copy of the gene in every cell. If you breed two of those heterozygous mice together, youll be lucky to get a third generation mouse with two copies of the mutant gene. So it takes at least three generations of mice to get your experimental mutant for research. Here it is summarized in a timeline:

But heres how a knockout mouse is made with CRISPR. Researchers inject the CRISPR/Cas9 sequences into mouse embryos. The system edits both copies of a gene at the same time, and you get the mouse in one generation. With CRISPR/Cas9, you can also alter, say, fives genes at once, whereas you would have to had to go that same laborious, multi-generational process five times before.

CRISPR is also more efficient than two other genome engineering techniques called zinc finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs). ZFN and TALENs can recognize longer DNA sequences and they theoretically have better specificity than CRISPR/Cas9, but they also have a major downside. Scientists have to create a custom-designed ZFN or TALEN protein each time, and they often have to create several variations before finding one that works. Its far easier to create a RNA guide sequence for CRISPR/Cas9, and its far more likely to work.

Most science experiments are done on a limited set of model organisms: mice, rats, zebrafish, fruit flies, and a nematode called C. elegans. Thats mostly because these are the organisms scientists have studied most closely and know how to manipulate genetically.

But with CRISPR/Cas9, its theoretically possible to modify the genomes of any animal under the sun. That includes humans. CRISPR could one day hold the cure to any number of genetic diseases, but of course human genetic manipulation is ethically fraught and still far from becoming routine.

Closer to reality are other genetically modified creaturesand not just the ones in labs. CRISPR could become a major force in ecology and conservation, especially when paired with other molecular biology tools. It could, for example, be used to introduce genes that slowly kill off the mosquitos spreading malaria. Or genes that put the brakes on invasive species like weeds. It could be the next great leap in conserving or enhancing our environmentopening up a whole new box of risks and rewards.

With the recent human embryo editing news, CRISPR has been getting a lot of coverage as a future medical treatment. But focusing on medicine alone is narrow-minded. Precise genome engineering has the potential to alter not just us, but the entire world and all its ecosystems.

More Reading:

Breakthrough DNA Editor Borne of Bacteria Quanta, Carl Zimmer

A CRISPR For-CAS-t The Scientist, Carina Storrs

Genetically Engineering Almost Anything NOVA NEXT, Tim De Chant and Eleanor Nelsen

This post has been updated to clarify that the the number of basepairs in guide RNA for CRISPR/Cas9 is different from the number of basepairs it recognizes in a target sequence.

Contact the author at sarah@gizmodo.com.

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Everything You Need to Know About CRISPR, the New Tool that ...

Agriculture has come a long way in the past century. We produce more food than ever before but our current model is unsustainable, and as the worlds population rapidly approaches the 8 billion mark, modern food production methods will need a radical transformation if theyre going to keep up. But luckily, theres a range of new technologies that might make it possible. In this series, well explore some of the innovative new solutions that farmers, scientists, and entrepreneurs are working on to make sure that nobody goes hungry in our increasingly crowded world.

Corn isnt the sexiest crop but its one of the most important. Its the most abundant grain on Earth, used as food and biofuel around the globe. In ancient times, Mesoamericans thrived on it, waged wars over it. Their myths claimed corn was the matter from which gods created mankind itself.

But, just as corn helped create these civilizations, these civilizations helped create corn through meticulous selective breeding. Todays grain hardly resembles its ancestors. Compared to the wild plant first cultivated by ancient Mexicans some ten thousand years ago, modern corn is a super mutant.

And yet, after all those thousands of years of cultivation, just two main genes are thought to be responsible for the evolution of the corn we eat today. Selective breeding is painstakingly slow and imprecise.

But thats all about to change.

Selective breeding is painstakingly slow and imprecise. But thats all about to change.

New gene editing tools like CRISPR/Cas9 now let scientists hack into genomes, make precise incisions, and insert desired traits into plants and animals. Well soon have corn with higher crop yields, mushrooms that dont brown, pigs with more meat on the bone, and disease resistant cattle. Changes that took years, decades, or even centuries, can now be made in a matter of months. In the next five years you might eat tortilla chips made from edited corn. By 2020 you might drink milk from an edited cow.

Dubbed the CRISPR Revolution these scientific advances in gene editing have huge potential that many experts think could help fortify our food system and feed an increasing population of farmers who are threatened by food scarcity caused, in part, by climate change.

But not everyone is so certain. Beyond the contentious legal battles that have thus far complicated CRISPR science, calling into question who can and cant use the technology, some consumer rights advocates think these tools will be used to maintain the status quo of an industry based primarily on corporate profit. Meanwhile, residual worry about genetically modified organisms (GMOs) may influence the public perception of gene-edited organisms, steering consumers towards the organic aisle despite scientific evidence.

Gene editing is, simply put, the act of making intentional changes to DNA in order to create an organism with a specific trait or traits. Its like using a word processor to edit the words in a sentence. Geneticists insist we dont confuse this with genetic modification (otherwise called genetic engineering), which introduces new genes from different species in order to achieve desired traits. The difference may sound trivial but experts say it could help calm the concerns associated with GMOs.

Consider this simplification. We have the sentence, The cat has a hat, but want to be more descriptive about the hats color. With modification, we would borrow the German word for black and write, The cat has a schwarz hat. The sentence makes sense (sort of) but its obvious that to some people it would be problematic and maybe even an improper use of language. With editing, we dont have to borrow a word from another language. We instead just insert the English word and write, The cat has a black hat.

In the older, more traditional system, scientists were taking a gene from one species and putting it into a plant to confer a particular trait on that plant, Rachel Haurwitz, co-founder of Caribou Biosciences, told Digital Trends. Thats not what were looking to do. Were looking to use CRISPR gene editing to achieve the same outcome as we can get from traditional breeding, just faster.

This ability to edit with such speed and precision is still relatively new, and due largely to CRISPR, which emerged straight from nature to become the most popular and powerful gene editing tool used today. Discovered in bacteria in the late eighties, it wasnt until 2005 that researchers began to unravel its role. Scientists found that when certain bacteria come under attack from viruses, they use special enzymes to cut, copy, and save a bit of the viral DNA. Later, if the intruder returns, the bacteria can quickly recognize it and react to defend itself.

A few years later, researchers realized this system could be used to cut and edit the DNA of any organism, not just viruses. In 2012, Jennifer Doudna and Emmanuelle Charpentier published the first paper demonstrating how CRISPR can be used to edit an organisms genome.

Were looking to use CRISPR gene editing to achieve the same outcome as we can get from traditional breeding, just faster.

Not only is this technique far cheaper, faster, and more precise than conventional genetic modification, it avoids many (if not all) of the issues raised by skeptics, whose main concerns point toward the creation of transgenic organisms.

But, whereas genetic modification entails combining DNA from multiple species, gene editing entails altering the DNA of one species with a trait that already exists naturally.

Gene editing is not at all about taking DNA from a foreign species and integrating it into a plant, Haurwitz said. Its really about working within the constraints of the plants own genome.

Just over four years ago, Haurwitz founded Caribou as a spin off from Doudnas lab at the University of California, Berkeley. Since then, her team has partnered with companies around the world, providing licensing rights to use the startups version of the gene editing tool. One of those partnerships may see the first CRISPR-edited organism come to market via DuPont Pioneer, one of the worlds biggest chemical companies.

The day before Halloween 2015, Yinong Yang submitted an Am I Regulated letter to the United States Department of Agricultures (USDA) Animal and Plant Health Inspection Service (APHIS). He and his colleagues at Penn State had used CRISPR to knock out a gene in white button mushrooms that makes them go brown over time. Without the browning gene, white buttons look better and last longer, and Yang wanted to know whether his mushrooms could legally go to market.

The following spring, the departments response resonated throughout the scientific and agricultural community. APHIS does not consider CRISPR/Cas9-edited white button mushroomsto be regulated, it wrote in an open letter.

Last year, researchers at DuPont Pioneer, the agriculture branch of the multi-billion-dollar conglomerate DuPont, published a study about a strain of corn engineered with CRISPR to be more resistant to drought. Its one of several CRISPR-modified crops that may soon be coming to market.

It was a landmark decision. Yangs mushrooms were the first gene-edited crop cleared for commercial sale by the USDA, which made a clear distinction between genetic modification and gene editing, and set a precedent for those to come.

A few days later, DuPont the fourth largest chemical corporation in the world received a similar response from the USDA regarding its CRISPR-edited waxy corn thats disease resistant and drought tolerant. DuPont wasted no time announcing plans to take its crop to market within the next five to ten years.

The USDA has said these products do not fall into their remit, as their remit is really focused on, say, plant pathogens or noxious weeds, said Haurwitz, whose company provides DuPont with its CRISPR technology. At the same time were seeing the FDA put out a call for information as theyre looking at their own remit to oversee the entire food supply, not just products made with modern biotechnology. And I think theyre looking to members of the scientific and business communities to really weigh in over the next few months.

Unlike most Button mushrooms, these ones dont brown or develop blemishes from being handled. This trait doesnt occur naturally it happens because the gene that makes the mushrooms turn brown was selectively removed from them via the CRISPR/Cas9 method. (Photo: Yang Lab)

For Yangs part, he intends to improve his mushrooms before making them commercially available. Although not legally required, he plans to seek approval from the Food and Drug Administration (FDA) and Environmental Protection Agency (EPA).

Edited waxy corn may find its way into the food system much sooner than white button mushrooms, if not as human food than as fodder for the growing number of livestock around the world. Meanwhile, these livestock are also undergoing genetic edits as researchers use the same tools to make animals healthier, meatier, and more productive.

Pigs harbor a lot of diseases and there are few as bad as porcine reproductive and respiratory syndrome (PRRS). It causes pregnant mothers to miscarry and makes it difficult for piglets to breathe. Its a problem for the pig farmers as well. Every year, the PRRS virus costs the industry nearly $1.6 billion dollars in Europe and another $664 million in the US.

The impacts of the disease for producers are often devastating, said Jonathan Lightner, Chief Scientific Officer at biotech company Genus. And the impacts on the animals themselves are terrible.

If we could integrate the polled phenotype into the dairy system, that would eliminate dehorning for at least seven or eight million animals a year.

But Lightner and his team are working on a solution. In December 2015, scientists at Genus and the University of Edinburghs Roslin Institute demonstrated how CRISPR could remove the CD163 molecule, a pathway through which the PRRS virus infects pig. Just last month, the researchers refined their work to remove just the portion of the gene that directly interacts with the virus. Lab tests, as published in a paper in the journal PLOS Pathogens, have shown that DNA in cells removed from these pigs successfully resist the virus. Next steps in the study will test whether the pigs themselves are resistant to the virus.

Swine are also the subject of research at Seoul National University in South Korea, where scientists led by Jin-Soo Kin are using a different gene-editing tool called TALEN to create meatier, double muscle pigs by removing a gene that inhibits muscle growth. We could do this through breeding, Kin told Nature back in 2015, but then it would take decades.

In fact, farmers have developed similar traits through breeding Belgian Blues, a type super-sculpted beef cattle prized for its lean meat and beefy build. It took over a hundred years to establish those traits in the breed.

Researchers at University of California, Davis and a startup called Recombinetics are using the same TALEN gene editing technique to cut decades down to days, removing the horned gene from common dairy cows and inserting the one that makes Angus beef cattle naturally dehorned or polled. Polled cattle are desirable because they pose less threat to their handlers and to each other. But, as Tad Sonstegard, Chief Science Officer of Acceligen (a Recombinetics subsidiary) explained, polled cattle in certain breeds are simply less productive.

Gene editing ala CRISPR/Cas9 has allowed scientists to not only produce polled (hornless) cows, but also cows that are immune to common diseases, such as tuberculosis. (Photo: Gregory Urquiaga/UC Davis)

The issue is that the top [dairy] bulls that everyone wants are horned, Sonstegard said. The animals that are polled that already exist have a difference of about $250 over their lifetime. If youre running a thousand head dairy [operation], thats a lot of money.

What many ranchers do instead is dehorn their cattle, a stressful practice when anesthesia is used, a painful practice when it isnt, and a significant expense for the ranchers either way.

If we could integrate the polled phenotype into the dairy system, that would eliminate dehorning for at least seven or eight million animals a year, Sonstegard said. If you include beef, thats up to fifteen million.

Recombinetics has already bred a couple gene-edited calves, which are undergoing care and monitoring at UC Davis. But, before any gene-edited cows produce the milk in our grocery stores, Sonstegard said scientists would need to prove that milk from these cows is similar to horned and polled cows that havent been gene edited. That would be simple though, he said, it would turn out the same.

As the global population grows, so does the demand for food. Meanwhile, farmers around the world face food scarcity generated in part by a changing climate that makes caring for plants and livestock an increasingly difficult task.

But CRISPR-like tools may be able to help.

On the plant side were looking at ways to breed plants that are more drought tolerant or in other ways can better survive the stresses of climate change, Haurwitz said. I think thats incredibly valuable and important as we look at the exploding global population. Caribou has also partnered with Genus in its project to breed PRRS virus resistant pigs.

Beyond his work at Recombinetics, Sonstegard sits on the scientific advisory board of the Centre for Tropical Livestock Genetics and Health, a Gates Foundation-backed initiative to improve the genetics of native livestock in tropical regions. Most productive livestock breeds cant survive the heat or diseases present in tropical environments, and breeds native to tropical environments havent had the same selective breeding programs that generate highly productive livestock.

Will CRISPR be used primarily for patenting foods in ways that fit in existing corporate profit models?

Most of the indigenous animals have not been under strict artificial selection, Sonstegard said. Its all been done anecdotally, since most farmers dont have that many cows and their systems arent that big. Meanwhile, most of the new DNA introduced to these herds is left over semen from bulls in developed countries, according to Sonstegard. Its cheap, he said, and no one in the developed country wants it anymore, so they ship it overseas.

There are a couple possible approaches to strengthening these indigenous breeds. One way would be to edit the DNA of bulls from productive breeds so that theyre more temperature tolerant and disease resistant within tropical climates. Those bulls could then be introduced to the native herds to reproduce and spread their productive genes. Alternatively, the DNA of indigenous bulls could be edited with genes likely to improve productivity of the herd, including milk production and carcass yield.

Right now the trend in those countries is that theres a linear growth in livestock numbers, Sonstegard said, because theyre not improving production but demand is increasing, so they just make more animals.Thats not sustainable.

Researchers are also using CRISPR to save dying and endangered species. This month some of Sonstegards colleagues published a paper showing they could develop surrogate hens that could help raise endangered species of birds. And in Florida, where an invasive disease known as citrus greening is decimating the states iconic orange industry, University of Florida scientists are using CRISPR to develop varieties of orange trees immune to the disease, according to the Tampa Bay Times.

But not everybody is so gung-ho.

UC Davis geneticist Alison Van Eenennaam, who collaborates with Recombinetics on gene-editing polled cows, is absolutely optimistic about the tool I think it can be used for very useful things, she said. Rather than ask why we should use, lets ask how. but shes also careful not to overstate the potential of gene editing. When asked whether the technology could be used to address world hunger, she said, I kind of think that idea is polyamorous. Show me anything that can magically solve world hunger. Lets not oversell this technology. Its useful but its useful for a fairly discreet purpose at this stage, which is making edits to a [gene] sequence that we know has a particular effect.

And CRISPR, of course, has its skeptics. Stacy Malkan, Co-Director of U.S. Right to Know, a nonprofit that calls for transparency and accountability in the food system, is both concerned about the inherent risk involved in gene editing and suspects it could ultimately perpetuate an already imbalanced food system.

Theres really no big difference between [gene editing] and conventional breeding.

Will CRISPR be used primarily for the purpose of patenting foods in ways that fit in existing corporate profit models, she asked, for example, to engineer commodity crops to withstand herbicides, or to engineer livestock to fit better in unhealthy confined feeding operations? Or will it be used to engineer foods that have consumer benefits? Will there be labeling, and safety assessments? There are many questions. Right now we hear a lot of marketing hype about possible benefits of CRISPR, but we heard the same promises about first-generation GMOs for decades and most of those benefits have not panned out.

For scientists like Van Eenennaam, the GMO discussion is over. Frankly, she said, Im over the debate. If someone isnt convinced by the evidence that every single major scientific society in the world says its safe, than nothing Im going to say is going to convince them any differently. When it comes to gene-edited organisms, most scientists are even more insistent about its safety. Theres really no big difference between [gene editing] and conventional breeding, Van Eenennaam added.

But there isnt complete consensus. Malkan points to an interview she recently had with Michael Hansen, senior scientist from Consumers Union, in which Hansen said of CRISPR-like gene editing tools, These methods are more precise than the old methods, but there can still be off-target and unintended effects. When you alter the genetics of living things they dont always behave as you expect. This is why its crucial to thoroughly study health and environmental impacts, but these studies arent required.

From Sonstegards perspective, mutations and off-target effects occur naturally anyway, and gene editing simply offers a more precise approach than selective breeding.

Still, Malkan and others have their reservations, grounded in the idea that its too early to determine the side effects. CRISPR is a powerful research tool for helping scientists understand genetics, how cells react, how entire plants and systems react, she said. In my view these experimental technologies should be kept in the lab, not unleashed in our food system, until those systems are better understood.

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CRISPR Gene Editing and the DNA of Future Food | Digital Trends

CRISPR/Cas9 is a gene editing technology thats revolutionizing science at a breathtaking pace.

One of its most exciting, taboo, and controversial applications is tweaking the genes of eggs, sperm, or early embryos to alter a human life. This could one day mean the ability to create smarter or more athletic humans (yes, designer babies), but also the chance to knock out disease-causing genetic mutations that parents pass on to their children. Were talking about eliminating mutations linked to diseases like breast and ovarian cancers or cystic fibrosis.

On Wednesday, a team of scientists reported that they have made major progress toward proving the latter is possible.

In a paper published in the prestigious journal Nature, a team led by Shoukhrat Mitalipov of Oregon Health and Science University described how it used CRISPR/Cas9 to correct a genetic mutation thats linked to a heart disorder called hypertrophic cardiomyopathy in human embryos. And they did it without the errors that have plagued previous attempts to edit human embryos with CRISPR.

To be clear, the new work from OHSU was an experiment the point was to test a concept, and the embryos used were never implanted into a womans uterus.

But the researchers were ultimately able to show that CRISPR/Cas9 can do what they hoped it would do. It cut the mutant gene sequence, prompted the embryos to repair the DNA with healthy copies of the gene, and eliminated the disease-causing mutation altogether from many of the embryos.

Lets pause for a minute and make sure were clear on what CRISPR/Cas9 is. You can read our full explainer here, but in a nutshell, its essentially a clever system built into bacterial DNA that allows them to recognize and fend off attackers, usually viruses. The way it works, as Brad Plumer described it, is that special enzymes in the CRISPR sequences known as Cas9 carry around stored bits of viral genetic code like a mug shot. When they find a match to the code, they will chop up the DNA and neutralize the threat.

The real breakthrough, which appeared in a series of landmark papers published in 2012 and 2013, was figuring out that it was possible to program CRISPR/Cas9 to find any kind of DNA code, not just viruses, and get the enzymes to snip it.

Mitalipov and colleagues created embryos in the lab with sperm from a carrier of the disease-causing mutation in the MYBPC3 gene, and eggs from 12 healthy donors. And they sent CRISPR/Cas9 into the fertilized egg.

As the embryos developed, they found that after CRISPR/Cas9 cut the sequence in the embryo DNA with the problematic gene. In most cases the embryos repaired the breaks with a healthy copy of the gene from the maternal donor.

In all, 36 out of 54 embryos ended up with mutation-free copies of MYBPC3. (Another, slight different round of the experiment yielded 42 out of 58 embryos with mutation-free copies of the gene.) Which means that had those embryos become children, the children would have had practically no chance of developing hypertrophic cardiomyopathy. Thats pretty significant since this a disease that affects one in 500 people and can cause sudden cardiac death and heart failure. If one parent has a mutant copy of MYBPC3, their child has a 50 percent chance of inheriting the condition.

These results are also promising for people (mainly older women and couples) who have a limited number of viable embryos to use to get pregnant with in vitro fertilization. Currently, reproductive medicine doctors use something called preimplantation genetic diagnosis, or PGD, to identify embryos with harmful mutations. And when they find embryos with mutations linked to disease, they often discard them, which can leave patients with few healthy embryos to try to transfer into the womb. (Transfer success rates are overall pretty low.)

The researchers say that in the future, their technique could be used with PGD to help fix the mutations in embryos that otherwise would be discarded, giving women and couples more embryos to transfer and a better chance of getting pregnant.

Were not ready for gene editing in embryos that would be implanted for pregnancy anytime soon. But this is a big advance because the researchers got stronger results than anyone who has ever tried to target disease-causing genes with CRISPR-Cas9 before.

And while the experiment focused only on this particular gene and disease, the researchers say they feel confident the technique would work for many of the thousands of other inherited disorders out there linked to one mutation because their approach has so far proved to be efficient, accurate, and safe.

But in a press conference on Tuesday, one of the co-authors, Paula Amato, an OB-GYN doctor at OHSU, stressed that many more safety tests would be needed before proceeding with a clinical trial. We want to replicate the study with other mutations and other [sperm and egg] donors, she said. In particular, she said shes interested in seeing if it works on BRCA1 and 2, mutations that increase the risk of breast and ovarian cancers.

Other researchers, including Nerges Winblad and Fredrik Lanner at Karolinska Institutet in Sweden, who wrote an accompanying article in Nature, are encouraged by the results but still cautious about the safety of the technology. They zeroed in on issues that have shown up in previous studies: off-target effects, or undesirable mutations in genome regions close to the targeted sequence, and mosaicism, where not all embryo cells make the desired changes. According to Winblad and Lanner, researchers will have to keep showing that they can reliably avoid these and other abnormalities in edited embryos before [the technology] can be used as a therapy for inherited diseases.

Amato and her co-authors said theres also plenty of room for other improvement. Some of their embryos DNA ended up with unintended additions or deletions. So their goal would be to get 80 to 90 percent of a large group of embryos mutation-free to ensure that the technique is reliable before attempting a clinical trial.

Again, this wasnt the first time scientists had tried to use CRISPR to edit human embryos. Chinese researchers have done it twice: once in 2015 to modify a gene linked to the blood disorder called beta thalassaemia, and then in 2016 to make genes resistant to HIV. But both of these experiments were smaller, and one used abnormal embryos while the other used immature eggs. And the results from both were messy, suggesting that embryo editing had a long, hard road ahead.

It was precisely those messy results, along with a host of other concerns, that prompted the Organizing Committee for the International Summit on Human Gene Editing at the National Academies of Sciences, Engineering, and Medicine to advise researchers in December 2015 to be extremely cautious about editing sperm, eggs, and embryos (known collectively as the human germline). Then in a report in February, it said clinical trials on human genome editing might one day be allowed, but in the meantime, researchers could attempt to correct mutations that cause a serious disease or condition and only when no reasonable alternatives exist. And definitely no research on enhancement of human traits like intelligence or strength for now.

At present, the US government does not fund any genomic editing of human embryos. (Mitalipov and his colleagues got funding from their university this new study.) And the Food and Drug Administration is prohibited by Congress from considering any clinical trials related to genetic editing of eggs, sperm, or embryos.

The impressive new findings in Nature raise huge questions about how the US should proceed with this field of research. How soon to allow clinical trials, for instance?

I believe [the National Academies] can reconsider what mutations and what cases the gene corrections can be used and must be used to allow clinical trials in the future to go forward, said Mitalipov. We may not be in agreement with the committees. The work is back and forth, and the committees hopefully will consider new options.

He added that hed be willing to move this research to the UK, if necessary. He also is sensitive about how his results are portrayed, given the American publics reticence, and in some cases fear, about genetic modification.

I dont like the word editing, he said. We didnt edit or modify anything. ... We used CRISPR to correct, using existing maternal genes.

Other CRISPR researchers have weighed in about where the field should go from here.

In my opinion, we still need to respect the recommendations in the [National Academy of Sciences] report published in February that recommended refraining from clinical use of human germline editing until and unless theres broad societal consensus about the value, Jennifer Doudna, a UC Berkeley molecular biologist and a leading CRISPR researcher, told the Los Angeles Times.

It may be quite a while before a clinical trial is approved. In the meantime, any prospective parents who want to avoid passing on disease-causing genes to their kids will have to continue to use PGD during in vitro fertilization.

As weve reported, scientists from myriad fields are using CRISPR to try to grow better food, destroy viruses, and clean up the environment.

Hank Greely explains for Vox why in 20 to 40 years, most Americans wont have sex to reproduce.

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Scientists successfully used CRISPR to fix a mutation that ...

Sharifnia, T., et. al. (2017) Cell Chemical Biology. 24:1075-1091. https://www.ncbi.nlm.nih.gov/pubmed/28938087

Rare cancers have traditionally been difficult to study due to low incidence and limited sample availability. However, new technologies, such as sequencing, have allowed for a greater understanding of the underlying genetic causes. In tandem with sequencing technologies, CRISPR/Cas and small molecule screens have allowed researchers to rapidly screen rare cancers for possible mechanisms and treatments.

Sharon Begley, STAT, 25 September 2017, https://www.statnews.com/2017/09/25/nobel-prize-predictions/

The season of Nobel Prize awards has arrived, and with it comes a slew of predictions. This year, STAT has identified who they believe has the best chance of winning the Nobel Prize in Medicine; including the CRISPR crowd of Emmanuelle Charpentier, George Church, Jennifer Doudna, and Feng Zhang. The only problem being each Nobel Prize can only be awarded to three people.

Rachael Lallensack, Nature News, 18 September 2017, http://www.nature.com/news/crispr-reveals-genetic-master-switches-behind-butterfly-wing-patterns-1.22628

Two new studies in the Proceedings of the National Academy of Sciences (http://www.pnas.org/content/early/2017/08/29/1709058114, http://www.pnas.org/content/early/2017/08/29/1708149114) provide insight into butterfly wing color. The studies identified two genes, WntA is responsible for creation of the coloring pattern and borders, while optix fills the color within the borders. Understanding butterfly coloration could provide insights into adaptations such as mimicry.

Vella, M.R. et. al. (2017) Scientific Reports 7:11038. https://www.ncbi.nlm.nih.gov/pubmed/28887462

CRISPR/Cas gene drives could be used to eliminate vector-borne diseases such as malaria and Lyme disease. However, release of modified organisms is controversial in part due to unforeseen consequences. Developing strategies for gene drive reversal could prove useful if such problems arise. This paper develops models to evaluate the effectiveness of gene drive counter-measures in order to evaluate their potential use.

Bikard, D., Barrangou, R., (2017) Current Opinion in Microbiology, 37:155-160. https://www.ncbi.nlm.nih.gov/pubmed/28888103

Self-targeting bacteria with CRISPR usually proves fatal. This observation could lead to a new type of antimicrobial where the CRISPR/Cas system is introduced to fight infection. This review discusses how CRISPR/Cas could target bacterial infections, as well as how the system may be delivered to the infection site.

David Nield, Science Alert, 9 September 2017, https://www.sciencealert.com/now-scientists-are-using-crispr-to-change-the-colour-of-flowers

The Japanese morning glory plant has traditionally had violet flowers, however using CRISPR to disrupt a single gene, scientists have altered the flower color to white. White morning glories can be found; however, it took 850 years for the white version to appear. CRISPR accomplished the task in less than 12 months. This is the first time CRISPR has been used to alter flower color in higher plants.

Liu, X., et. al. (2017) Cell 170:1028-1043. https://www.ncbi.nlm.nih.gov/pubmed/28841410

Many genes are regulated by cis-regulatory elements, though the molecular composition of these elements remains unknown. In a new study published in Cell, Liu et. al. describe a new technique called CAPTURE (CRISPR affinity purification in situ of regulatory elements) that uses a biotin labeled dCas9 to isolate cis regulatory elements in an unbiased fashion, allowing for insights into genome structure and function.

Stanford Medicine News Center, 29 August 2017, http://med.stanford.edu/news/all-news/2017/08/online-game-challenges-players-to-design-on-off-switch-for-crispr.html

Researchers at Stanford University School of Medicine have created a new online computer game called Eterna where players design RNA molecules that could act as an on/off switch for Cas9. Molecular biologists at Stanford will then create the most promising molecules and test them in living cells. Researchers aim to have 100,000 players contribute 10 solutions each. As the research team tests the molecules in the lab, they will provide information to the players for further refinement.

Julia Franz and Katie Hiler, WUNC Science Friday, 27 August 2017, http://wunc.org/post/new-developments-human-gene-editing-face-ethical-and-regulatory-quagmire-us#stream/0

Despite the results of Augusts CRISPR edited embryo paper being called into question, its publication has resulted in an increase in the ethics debate. Scientists agree that CRISPR gene editing will continue to improve and society must grapple with the ethical problems. Ira Flatow sits down with the author of the August Nature article and with Kelly Ormond, genetics professor at Stanford University and member of the Stanford Center for Biomedical Ethics, to discuss the results and how to proceed.

Dieter et. al. (2017) BioRxiv, 181255. http://www.biorxiv.org/content/early/2017/08/28/181255

On 02 August 2017, a Nature article claimed a major breakthrough in CRISPR genome editing. Researchers from around the world, including the United States, announced that they had successfully corrected viable human embryos heterozygous for the MYBPC3 mutation that results in heart disease, without mosaicism. Recently, the results of this article have been called into question with the publishing of a BioRxiv article. The authors of the new paper identify other possible mechanisms that could have caused the observed results and suggest additional experiments to effectively prove CRISPR gene editing.

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CRISPR Update CRISPR Updates, News and Articles

Gene-editing technology CRISPR has revealed a clue in how human embryos begin to develop, possibly reducing the risk of miscarriage in those crucial first few weeks of pregnancy.

CRISPR Cas9 can modify or snip out genetic defects thought to contribute to miscarriage, but until now it wasnt clear why some embryos continued to form into a fetus and others did not. However,findings, published Wednesday in the journal Nature, hold genetic clues.

British scientists conducting the study found that a certain human genetic marker called OTC4 played an important role in the formation and development in the early stages of embryonic development. The scientists used CRISPR Cas9 to knock out this important gene in days-old human embryos and found that without it, these embryos ceased to attach or grow properly.

The findings could not only help us better understand why some women suffer more miscarriages than others, but it could also potentially greatly increase the rate of successful in vitro fertilization (IVF) procedures.

IVF is sometimes the only way a couple can make a baby using their own genes, but even with technological improvements over the years, the rates of success are still poor.Only about 36 percent of IVF cycles result in a viable pregnancy, and a mere 24 percent produce a baby, according to the Centers for Disease Control.

Of course, this is not the first time scientists have tested on human embryos. The practice has sparked a fierce international debate, but earlier this year, U.S. scientists used CRISPR technology to cut out a gene known to cause heart defects in three-day old human embryos.

None of the embryos in that study or this latest one were meant to go on to become human beings and were discarded after the study was finished. However, both studies hint at the potential CRISPR could have in the formation of human life in the future.

It will likely take years before putting this breakthrough into practice on viable embryos meant to develop beyond a few days, and theres likely still much more research needed, but it does give hope for those whove suffered a miscarriage and wanting to ensure they can one day carry a healthy baby to full term.

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CRISPR breakthrough could drop miscarriage rates | TechCrunch

Usually, this type of study is conducted on mice, which are easier to come by and carry less ethical considerations. But, in this case, scientists knocked out the gene in 41 human embryos donated by couples who had undergone in-vitro fertilization (IVF). The researchers claim the switch allowed them to highlight key differences between the role of OCT4 in human and mouse models. The team are hoping their findings can help scientists better grasp why some women suffer more miscarriages than others. Additionally, the study could also increase the rate of successful IVF procedures.

This isn't the first time scientists have used human embryos. Earlier this year, a team of researchers from Oregon became the first to use CRISPR tech to cut out genes that cause inherited diseases in humans. Before that, scientists in China utilized the technique to repair a gene that can bring about a fatal blood disorder.

The new study is being hailed as a compelling first step. "We were surprised to see just how crucial this gene is for human embryo development, but we need to continue our work to confirm its role," Norah Fogarty of the Francis Crick Institute told CNN. "Other research methods, including studies in mice, suggested a later and more focused role for OCT4, so our results highlight the need for human embryo research."

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CRISPR gene-editing could result in more successful birth rates

Have you heard? A revolution has seized the scientific community. Within only a few years, research labs worldwide have adopted a new technology that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier. This technology is referred to as CRISPR, and it has changed not only the way basic research is conducted, but also the way we can now think about treating diseases [1,2].

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. While seemingly innocuous, CRISPR sequences are a crucial component of the immune systems [3] of these simple life forms. The immune system is responsible for protecting an organisms health and well-being. Just like us, bacterial cells can be invaded by viruses, which are small, infectious agents. If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by destroying the genome of the invading virus [4]. The genome of the virus includes genetic material that is necessary for the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR immune system protects bacteria from ongoing viral infection.

Figure 1 ~ The steps of CRISPR-mediated immunity. CRISPRs are regions in the bacterial genome that help defend against invading viruses. These regions are composed of short DNA repeats (black diamonds) and spacers (colored boxes). When a previously unseen virus infects a bacterium, a new spacer derived from the virus is incorporated amongst existing spacers. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules. The CRISPR RNA associates with and guides bacterial molecular machinery to a matching target sequence in the invading virus. The molecular machinery cuts up and destroys the invading viral genome. Figure adapted from Molecular Cell 54, April 24, 2014 [5].

Interspersed between the short DNA repeats of bacterial CRISPRs are similarly short variable sequences called spacers (FIGURE 1). These spacers are derived from DNA of viruses that have previously attacked the host bacterium [3]. Hence, spacers serve as a genetic memory of previous infections. If another infection by the same virus should occur, the CRISPR defense system will cut up any viral DNA sequence matching the spacer sequence and thus protect the bacterium from viral attack. If a previously unseen virus attacks, a new spacer is made and added to the chain of spacers and repeats.

The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps [5]:

Step 1) Adaptation DNA from an invading virus is processed into short segments that are inserted into the CRISPR sequence as new spacers.

Step 2) Production of CRISPR RNA CRISPR repeats and spacers in the bacterial DNA undergo transcription, the process of copying DNA into RNA (ribonucleic acid). Unlike the double-chain helix structure of DNA, the resulting RNA is a single-chain molecule. This RNA chain is cut into short pieces called CRISPR RNAs.

Step 3) Targeting CRISPR RNAs guide bacterial molecular machinery to destroy the viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences acquired during adaptation, they are exact matches to the viral genome and thus serve as excellent guides.

The specificity of CRISPR-based immunity in recognizing and destroying invading viruses is not just useful for bacteria. Creative applications of this primitive yet elegant defense system have emerged in disciplines as diverse as industry, basic research, and medicine.

In Industry

The inherent functions of the CRISPR system are advantageous for industrial processes that utilize bacterial cultures. CRISPR-based immunity can be employed to make these cultures more resistant to viral attack, which would otherwise impede productivity. In fact, the original discovery of CRISPR immunity came from researchers at Danisco, a company in the food production industry [2,3]. Danisco scientists were studying a bacterium called Streptococcus thermophilus, which is used to make yogurts and cheeses. Certain viruses can infect this bacterium and damage the quality or quantity of the food. It was discovered that CRISPR sequences equipped S. thermophilus with immunity against such viral attack. Expanding beyond S. thermophilus to other useful bacteria, manufacturers can apply the same principles to improve culture sustainability and lifespan.

In the Lab

Beyond applications encompassing bacterial immune defenses, scientists have learned how to harness CRISPR technology in the lab [6] to make precise changes in the genes of organisms as diverse as fruit flies, fish, mice, plants and even human cells. Genes are defined by their specific sequences, which provide instructions on how to build and maintain an organisms cells. A change in the sequence of even one gene can significantly affect the biology of the cell and in turn may affect the health of an organism. CRISPR techniques allow scientists to modify specific genes while sparing all others, thus clarifying the association between a given gene and its consequence to the organism.

Rather than relying on bacteria to generate CRISPR RNAs, scientists first design and synthesize short RNA molecules that match a specific DNA sequencefor example, in a human cell. Then, like in the targeting step of the bacterial system, this guide RNA shuttles molecular machinery to the intended DNA target. Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence of a gene (Figure 2)! This type of gene editing can be likened to editing a sentence with a word processor to delete words or correct spelling mistakes. One important application of such technology is to facilitate making animal models with precise genetic changes to study the progress and treatment of human diseases.

Figure 2 ~ Gene silencing and editing with CRISPR. Guide RNA designed to match the DNA region of interest directs molecular machinery to cut both strands of the targeted DNA. During gene silencing, the cell attempts to repair the broken DNA, but often does so with errors that disrupt the geneeffectively silencing it. For gene editing, a repair template with a specified change in sequence is added to the cell and incorporated into the DNA during the repair process. The targeted DNA is now altered to carry this new sequence.

In Medicine

With early successes in the lab, many are looking toward medical applications of CRISPR technology. One application is for the treatment of genetic diseases. The first evidence that CRISPR can be used to correct a mutant gene and reverse disease symptoms in a living animal was published earlier this year [7]. By replacing the mutant form of a gene with its correct sequence in adult mice, researchers demonstrated a cure for a rare liver disorder that could be achieved with a single treatment. In addition to treating heritable diseases, CRISPR can be used in the realm of infectious diseases, possibly providing a way to make more specific antibiotics that target only disease-causing bacterial strains while sparing beneficial bacteria [8]. A recent SITN Waves article discusses how this technique was also used to make white blood cells resistant to HIV infection [9].

Of course, any new technology takes some time to understand and perfect. It will be important to verify that a particular guide RNA is specific for its target gene, so that the CRISPR system does not mistakenly attack other genes. It will also be important to find a way to deliver CRISPR therapies into the body before they can become widely used in medicine. Although a lot remains to be discovered, there is no doubt that CRISPR has become a valuable tool in research. In fact, there is enough excitement in the field to warrant the launch of several Biotech start-ups that hope to use CRISPR-inspired technology to treat human diseases [8].

Ekaterina Pak is a Ph.D. student in the Biological and Biomedical Sciences program at Harvard Medical School.

1. Palca, J. A CRISPR way to fix faulty genes. (26 June 2014) NPR < http://www.npr.org/blogs/health/2014/06/26/325213397/a-crispr-way-to-fix-faulty-genes> [29 June 2014]

2. Pennisi, E. The CRISPR Craze. (2013) Science, 341 (6148): 833-836.

3. 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.

4. 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., and van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960964.

5. Barrangou, R. and Marraffini, L. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity (2014). Molecular Cell 54, 234-244.

6. Jinkek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. (2012) 337(6096):816-21.

7. CRISPR reverses disease symptoms in living animals for first time. (31 March 2014). Genetic Engineering and Biotechnology News. <http://www.genengnews.com/gen-news-highlights/crispr-reverses-disease-symptoms-in-living-animals-for-first-time/81249682/> [27 July 2014]

8. Pollack, A. A powerful new way to edit DNA. (3 March 2014). NYTimes < http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html?_r=0> [16 July 2014]

9. Gene editing technique allows for HIV resistance? <http://sitn.hms.harvard.edu/flash/waves/2014/gene-editing-technique-allows-for-hiv-resistance/> [13 June 2014]

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CRISPR: A game-changing genetic engineering technique ...

Why it matters to you

Pigs could be a solution to the shortage of transplant organs. CRISPR gene editing makes them safer candidates.

There is a massive shortage of transplant organs worldwide, and scientists are desperate to come up with a solution whether that be boosting patients immune systems to let them accept otherwise incompatible organs, or creating technology for preserving organs after they are harvested. A new international research initiative has another approach: Using CRISPR gene editing on pigs to make them into safe organ donor candidates for humans.

The reason pigs are desirable as possible sources of organs is that their organs are similar to humans in both size and anatomy. Unfortunately, they also carry viruses known as porcine endogenous retroviruses (PERVs) embedded in their DNA. As this research demonstrated, this can be passed on to humans, although gene editing can be used to eradicate it.

Currently, the major problem of human transplants is the great shortage of transplantable human organs, Lin Lin, a researcher in the department of biomedicine at Denmarks Aarhus University, told Digital Trends. While using pig organs, we can in principle use as many as we need. Eradicating PERVs makes porcine organs safer for human transplants. However, there are still several other barriers that we have to cross in order to make pig organs better for human transplants. This is now achievable with the great development in CRISPR gene editing.

Using an optimized CRISPR-Cas9 gene editing technology and porcine somatic cell nuclear transfer, this work successfully generated viable pigs that are 100 percent PERV-inactivated.Thirty-seven PERV-inactive piglets have so far been born, with 15 remaining alive. The oldest of these is four months old, which means it will need to be monitored for a longer period of time to make sure it suffers no ill-effects.

The next major step is to solve the problem of vigorous immune responses, such as complement activation, coagulation and thrombosis, triggered by xenotransplantation, Lin said. Many previous works have demonstrated that the immunological incapability can be alleviated through tailoring the pig genome. Thus, a serial of very sophisticated gene editing and modifications will be further introduced into the PERV-inactivated pigs and tested in higher primates.

A paper describing the research was recently published in the journal Science.

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Thanks to CRISPR, gene-edited pigs could be organ donors for ...

CRISPR or CRISPR Cas9 is commonly used to refer to a revolutionary genome editing technology that enables efficient and precise genomic modifications in a wide variety of organisms and tissues.

Definition: Clustered Regularly Interspaced Short Palindromic Repeat or CRISPR (pronounced 'crisper') was identified in a prokaryotic defence system. CRISPR are sections of genetic code containing short repetitions of base sequences followed by spacer DNA segments

Identified in archaea and bacteria, short nucleic acid sequences are captured from invading pathogens and integrated in the CRISPR loci amidst the repeats. Small RNAs, produced by transcription of these loci, can then guide a set of endonucleases to cleave the genomes of future invading pathogens, thereby disabling their attacks.

Definition: CRISPR ASsociated protein 9 (Cas 9) is an endonuclease used in an RNA-guided gene editing platform. It uses a synthetic guide RNA to introduce a double strand break at a specific location within a strand of DNA

Cas9 was the first of several restriction nucleases (or molecular scissors) discovered that enable CRISPR genome editing. The CRISPR Cas9 mechanism has since been adapted into a powerful tool that puts genome editing into the mainstream.

In the laboratory, CRISPR Cas9 genome editing is achieved by transfecting a cell with the Cas9 protein along with a specially designed guide RNA (gRNA) that directs the cut through hybridization with its matching genomic sequence. When the cell repairs that break, errors can occur to generate a gene knockout or additional genetic modifications can be introduced. Our CRISPR gene editing technology is particularly good for the efficient generation of complete knockout of genes on multiple alleles.

Use of wild-type Cas 9 has been shown to lead to off-target cleavage, but a modified version introduces only single strand nicks to the DNA, which in pairs still stimulate the repair mechanisms while significantly decreasing the risk of off-target cutting.

Horizon has licensed gene editing IP from Harvard University, the Broad Institute and ERS Genomics with the goal of being able to ensure that we will be able to offer uninterrupted use of CRISPR tools to our customers. Our scientists have extensive knowledge of CRISPR technology including the benefits of using each Cas9 structure.

Other Gene Editing Systems

Genome editing can be achieved using the widely used S. Pyogenes (spCas9), and also utilising CRISPR Cas 9 protocol for S. Aureus (scCas9), Cpf1, HiFi Cas9, Nickase Cas9, Nuclease Cas9, NgAgo gDNA and even synthetic spCas9 with alternative PAM sites.

Our genome editing knowledge also includes rAAV and ZFNs.

Continue your CRIPSR/Cas9 research with ourpopular education and training webinars:

Find out more about our exciting upcoming eventwhere the future of CRISPR will be discussed:

The CRISPR Forum 2017

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007.CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819): 1709-1712.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.

Continued here:
CRISPR - CRISPR-Cas9 | Gene Editing

B

ioengineer Luhan Yang swiped through the photos on her phone until she got to one that made her beam: It showed her crouching down by a pudgy, wide-eyed newborn she calls my baby.

This newborn is a pig, and its the first to be born with dozens of genetic changes that could enable scientists to turn swine into a source of organs for human transplants, Yang and her colleaguesreported on Thursday in Science.

Theynamed the piglet Laika, after the first dog to orbit Earth in 1957. The new Laika, born this year in China after numerous miscarriages and other setbacks, could be a pioneer in her own right. Using the genome-editing technology CRISPR-Cas9, Yang and her team at the biotech startup eGenesis knocked out pig DNA that has long been considered a deal-breaker for efforts to use pigs as organ donors. Laika and 36 other designer piglets are completely free of it.

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There are additional Olympic-level hurdles to overcome before people facing death from organ failure get replacement kidneys, hearts, livers, or lungs from the species that provides their bacon and pork chops.Other genetic changes will be necessary. And regulators require stringent tests in lab primates before a single patient could get a CRISPRd pig organ; that will take years.

But after decades of dashed hopes, experts say, xenotransplantation might actually be in the offing.

Its an elegant tour de force of genetic engineering, so my hat is off to them, said Dr. A. Joseph Tector, of the University of Alabama, Birmingham, who has also made genetically modified pigs aimed at producing transplantable organs. But if you want to move xenotransplantation to the hospital, there are many more things youll have to do.

Doctors wont have to do much persuasion, however, to get patients to accept organs from another species. There is so much desperation among people on transplant lists, and 20 a day are dying as they wait, said Dr. Adam Griesemer, a xenotransplantation researcher and transplant surgeon at Columbia University Medical Center. This could be a path to a transplant for them. Colleagues keep asking me when were going to do it.

Pigs are scientists first choice because their organs and physiology are pretty close matches to humans, and they come with less ethical baggage than, say, chimps or baboons. But for years, the path to xenotransplantation has been paved with disappointment. Pig organs with genetic changes, transplanted into baboons and other lab animals,kept failing within weeks, even though the recipients received immune-suppressing drugs to prevent organ rejection.

Yang believes that CRISPR can accomplish what previous approaches have not: make multiple, simultaneous changes in pig DNA so that the animals organs work, and work safely, in people.

The team at Massachusetts-based eGenesis, working with scientists in China, used the Dolly recipe to clone pigs. They started with cells from adult pigs, and used an electrical jolt to fuse them with pig ova whose DNA had been removed. They grew the resulting embryos in lab dishes and then transferred healthy ones to sows, hoping for pregnancies.

The adult cells were not as nature made them, however. In a key step, the scientists used the genome-editor CRISPR to cripple all 25 copies of PERV genes DNA in the pig genome that makes potentially dangerous viruses that could infect anyone who receives a pig organ. (PERV stands for porcine endogenous retroviruses.) Initially, in about one-third of the CRISPRd pig cells, the PERV genes were almost all gone. In most of the rest, CRISPR missed its mark. That wasnt unexpected; for all the hype around CRISPR, it isnt perfect.

The unwelcome surprise was that cells that were effectively CRISPRd the ones the scientists needed to clone designer pigs were dying like orchids in the tundra. Apparently, in its zeal to attack so many PERV genes, CRISPR had shredded the cells genomes fatally.

Its quite a problem, when you move to so many targets, said Yang, the chief scientific officer at eGenesis. If there are multiple cuts in the genome at the same time, chromosomes rearrange themselves. That can happen when you make two or three [CRISPR edits], and were dealing with 25.

The eGenesis scientists, many of them alums of George Churchs lab at Harvard Medical School, scrambled for a solution. They eventually stumbled on a cocktail of molecules that both increased the number of PERV targets that CRISPR hit and, even better, kept the well-CRISPRd cells alive. We were able to get cells to grow even with very aggressive gene editing, Yang said: 100 percent of the cells doused with the chemical cocktail were 100 percent PERV-free.

As is typical with cloning, very few of the cloned embryos were healthy enough to implant into sows, and few implanted embryos resulted in births. Crucially, however, of the 37 piglets born from 17 sows, all were PERV-free. And CRISPR did not change any DNA it wasnt supposed to; there were no off-target effects.

The oldest pigs are nearly 5 months old, or adolescents; 15 remain alive. The rest were killed so the scientists could see whether their organs were developing normally.

So far, so good, Yang said, showing that pigs dont need PERVs to live: Weve shown you can produce PERV-free pigs which could serve as a source for future xenotransplants.

Among eGenesiss next experiments: see if the pigs are fertile and, if so, whether their CRISPRd genetic changes, including inactivating PERVs, are inherited. That could provide an easier source of transplantable organs than cloning.

Other scientists have also used CRISPR to produce pigs with altered genomes, including pigs in which a genethat triggers organ rejection was eliminated. Last year, scientists announcedthat hearts from genetically-modified pigs survived in baboons for up to 945 days, a record.

UABs Tector and his colleagues, with financial backing from United Therapeutics Corp., are using CRISPR not on PERVs but on other pig genes. Knocking out threein particular could protect pig organs from being attacked by the human immune system, he said; lab macaques that received kidneys from the pigs have survived as long as 499 days. We have a pig we are very confident we can make work for kidney transplants, Tector said.

There is disagreement about whether pig organs would have to be PERV-free to be successfully transplanted into people. Tector said transplant patients could take anti-retroviral drugs, just as they take immune-suppressing drugs, to kill the viruses.

Nevertheless, eGenesis scientists achievement with their 25 DNA edits, the eGenesis pigs set the record for genome modifications suggests that however many edits are needed to make pigs into organ donors might be feasible. The challenge is to identify which pig genes are necessary and sufficient to change so that the animals organs have a shot at working in people.

Senior Writer, Science and Discovery

Sharon covers science and discovery.

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Birth of CRISPR'd pigs advances hopes for turning pigs into ...

Excision BioTherapeutics has raised money to move what it sees as a cure for HIV into the clinic. Stemcentrx backer Artis Ventures led the $10 million seed round to equip Excision to start human testing of its CRISPR-enabled attack on latent HIV virus.

Philadelphia-based Excision is built on research conducted at Temple Universitys Lewis Katz School of Medicine. The work led to a paper published last year, in which Excision co-founder Kamel Khalili, Ph.D., and his partners administered a multiplex of guide RNAs (gRNAs) and Staphylococcus aureus Cas9 to HIV-infected mice. The team designed the treatment to remove a large, essential DNA fragment from HIV.

Results from the study furthered Excisions belief its candidate can wipe out HIV provirus from all tissues in the body without causing genotoxic effects and off-target editing.

That belief prompted the founding of Excision in 2015. Having generated animal data to back up the belief, Excision has high hopes for the approach.

We're in this to cure patients of HIV, Excision CEO Thomas Malcolm, Ph.D., said.

Excision sees an HIV CRISPR Cas9/gRNA multiplex biologic based on Khalilis workEBT101as its best shot of meeting this lofty goal. The plan is to wrap up IND-enabling studies of the candidate in the months to come and get it into the clinic around the end of next year. That small trial will act as an early test of the safety and, to a lesser extent, the efficacy of EBT101 and its delivery system.

Some of Khalilis projects used adeno-associated virus (AAV) vectors to deliver sgRNAs and Cas9. But Excision is now looking at a lentiviral approach.

It's really more specific for the types of cells that have that latent virus. HIV itself is a lentivirus so it makes sense to use a lentiviral shell to deliver the therapeutic, Malcolm said. Were showing we can easily access all of these reservoirs with this approach.

The plan for later trials is to use EBT101 to target these reservoirs in patients taking cocktails of HIV inhibitors to control the virus. These cocktails, such as Gileads Genvoya, lower HIV viral loads to undetectable levels in most patients. But, while that has improved outcomes significantly, Excision is confident a product that eradicates the virus would still find a market.

This confidence is based on what Malcolm calls the baggage that comes with cocktails. That term covers the risk of noncompliance to the daily treatment regimen and the comorbidities common in people who live with HIV, although there is evidence suggesting treatment with modern antiretroviral therapy cuts the risk of these complications.

The other shortcoming, which is linked to the risk of noncompliance, stems from the potential for HIV to develop resistance to drugs. That is happening today. A CDC study found 16% of patients diagnosed with HIV in 10 metropolitan areas from 2007 to 2010 carried antiretroviral-resistant virus. A WHO study found more than 10% of patients starting treatment in six of 11 surveyed countries in Africa, Asia and Latin America had a resistant strain.

Malcolm sees this causing big problems down the line.

It's a ticking time bomb, he said. It's just a matter of time before you're going to get another patient zero who is going to be completely unsusceptible to these inhibitor cocktails and we're going to be right back to where we were in the '80s.

Excision plans to head off that scenario by developing EBT101. In parallel, the biotech is working on a clutch of earlier-stage programs, two of which it will move into animal studies using the seed money. Success in those studies would tee Excision up to move candidates against JC virusthe cause of progressive multifocal leukoencephalopathyand herpes simplex virus into the clinic in the next couple of years.

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Artis funds Excision to test whether CRISPR can cure HIV ... - FierceBiotech

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New gene editing tech promises to be even better than CRISPR Digital Trends Just when we were getting used to the CRISPR/Cas9 gene editing revolution, a new fourth-generation DNA base editor has come along. University of South Carolina to provide Transomic Technologies ...Markets Insider all 4 news articles »

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New gene editing tech promises to be even better than CRISPR - Digital Trends

I've written a number of times about the tremendous rise in citizen science game platforms that allow players to contribute to scientific research. Arguably the most well known of these games is Stanford based Eterna.

Last year a paperhighlighted how players of the game were playing an ever increasing role in scientific research.

We see that in particular researchers in the natural sciences have collected and classified data with the help of interested volunteers. In the social sciences, there has been a focus on inviting select parts of the public to find out the effects of science on peoples everyday lives. This may for example concern environment problems and risks,the authors say.

The game began life by asking players to help scientists understand RNA, but it has recently branched out into new fields. For instance, last year they developed a version that aimed to further understanding of TB.

The latest version sees players tasked with designing a CRISPR-controlling molecule. The design of the challenge is to develop an RNA molecule that's capable of acting as an on/off switch for CRISPR. The resulting molecules will then be tested by molecular biologists.

The ability to turn off CRISPR is crucial as the editor is incredibly powerful and may have unexpected effects on the cells, so being able to turn it off is key to its safe usage. The researchers also believe the functionality could allow CRISPR to be deployed on a kind of timer so that it can be activated and deactivated according to a schedule.

"Great ideas can come from anywhere, so this is also an experiment in the democratization of science," the team say. "A lot of people have hidden talents that they don't even know about. This could be their calling. Maybe there's somebody out there who is a security guard and a fantastic RNA biochemist, and they don't even know it."

The aim is to get up to 100,000 players to contribute, with each player contributing around 10 solutions each. Should that number of players participate, it gives the team a good amount of data to work with, and their initial tests will then go into refining the game further to guide future players in their designs.

In addition to producing some invaluable inputs into scientific research, the team also hope to enhance interest in science among the wider population.

"The Eterna game is a powerful way to engage lots and lots of people," they say. "They're not just passive users of information but actually involved in the process."

As with other computer games, Eterna aims to incentivize players by allowing them to earn points, build expertise and advance to higher levels. The best players then gain the chance to have their designs implemented in a lab environment.

Citizen science games like Eterna have proven incredibly popular. For instance, I wrote recently about the Sea Hero Quest game developed by Deutsche Telekom to promote research into dementia. The original mobile game has been downloaded over 3 million times, providing data equivalent to 12,000 years of lab research.

As such, the Eterna team believe that the game is as much about the sociology of science as it is about the hard science itself.

"There is a misconception of science as something that happens in an ivory tower by someone in a white coat with a long beard. And they are saying things and drawing things that nobody understands. But it's not like that! It's really like a puzzle that anybody can get engaged with," they say.

You can play the game for free by clicking here, or alternatively watch the video below to learn more about it.

See the original post here:
Eterna Citizen Science Game Turns Its Attention To CRISPR - HuffPost

Morning Glory. Credit: University of Tsukuba

In a world-first, Japanese scientists have used the revolutionary CRISPR, or CRISPR/Cas9, genome- editing tool to change flower colour in an ornamental plant. Researchers from the University of Tsukuba, the National Agriculture and Food Research Organization (NARO) and Yokohama City University, Japan, altered the flower colour of the traditional Japanese garden plant, Japanese morning glory (Ipomoea nil or Pharbitis nil), from violet to white, by disrupting a single gene. This research highlights the huge potential of the CRISPR/Cas9 system to the study and manipulation of genes in horticultural plants.

Japanese morning glory, or Asagao, was chosen for this study as it is one of two traditional horticultural model plants in the National BioResource Project in Japan (NBRP). Extensive genetic studies of this plant have already been performed, its genome sequenced and DNA transfer methods established. In addition, as public concern with genetic technologies such as CRISPR/Cas9 is currently a social issue in Japan, studies using this popular and widely-grown plant may help to educate the public on this topic.

The research team targeted a single gene, dihydroflavonol-4-reductase-B (DFR-B), encoding an anthocyanin biosynthesis enzyme, that is responsible for the colour of the plant's stems, leaves and flowers. Two other, very closely related genes (DFR-A and DRF-C) sit side-by-side, next to DFR-B. Therefore, the challenge was to specifically and accurately target the DFR-B gene without altering the other genes. The CRISPR/Cas9 system was used as it is currently the most precise method of gene editing.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system is based on a bacterial defense mechanism. It is composed of two molecules that alter the DNA sequence. Cas9, an enzyme, cuts the two strands of DNA in a precise location so that DNA can be added or removed. Cas9 is guided to the correct location by gRNA, or guide RNA, a small piece of RNA that has been designed to be complementary to the target DNA sequence. Cas9 cuts the two strands of DNA at the target location, allowing DNA to be removed and/or added.

As reported on 30 August 2017 in Scientific Reports, a short DNA sequence in the Japanese morning glory DFR-B gene was selected as the target for the CRISPR/Cas9 system. This sequence contains the active site of the enzyme produced by the DFR-B gene. Disruption of this sequence should therefore de-activate the enzyme, resulting in an absence of the colour pigment, anthocyanin. The CRISPR/Cas9 system was inserted into tissue-cultured embryos of Japanese morning glory plants using the DNA-transferring capabilities of the plant bacterium Rhizobium. As expected, the DFR-B enzyme was successfully inactivated, resulting in approximately 75%of the transgenic plants with green stems and white flowers. Non-transformed plants with an active enzyme had violet stems and flowers. These changes in stem colour were observed very early in the tissue culture process. A series of genetic analyses confirmed that the DNA target sequence had been altered in the transgenic plants, with either DNA insertions or deletions in both copies of the DFR-B gene (so-called bi-allellic mutants). The other related genes, DFR-A and DFR-C, were examined and no mutations were found, confirming the high specificity of the CRISPR/Cas9 system.

Next, the researchers examined the inheritance of the CRISPR/Cas9-induced mutations by analyzing plants from the next generation. These plants looked exactly like their parents. Among these plants were some without any sign of the introduced DNA. This raises some interesting questions in terms

of the regulation of genetically modified organisms (GMOs), as these next-generation plants are considered transgenic, based on process-based definitions (how they were made), and non- transgenic, based on product-based definitions (the presence of foreign DNA in the final product).

This technology is also extremely useful in confirming the function of genes. Experiments in the 1930s and 1990s used 'forward' genetic screening techniques to find the genes responsible for flower colour production in the Japanese morning glory. The CRISPR/Cas9 system described here is the 'reverse' genetic approach, used to find out what an organism looks like after a known gene is disrupted, and confirms that the DFR-B gene is the main gene responsible for colour in Japanese morning glory plants.

Currently, CRISPR/Cas9 technology is not 100% efficient, that is, not all targeted plants will be transgenic. The mutation rate in this study, 75%, however, was relatively high. This is one of the reasons this research will greatly facilitate those interested in the modification of flower colours and shapes using the CRISPR/Cas9 system in ornamental flowers or vegetables.

The story of the Japanese morning glory started in the 8th century AD, with the introduction of wild blue-flowered plants into Japan from China. In 1631, the first white-flowered Japanese morning glory was painted in Japan. What took nature nearly 850years to achieve has taken less than one using the CRISPR/Cas9 system, indicating both its power and its potential.

Explore further: Modifying fat content in soybean oil with the molecular scissors Cpf1

More information: Kenta Watanabe et al. CRISPR/Cas9-mediated mutagenesis of the dihydroflavonol-4-reductase-B (DFR-B) locus in the Japanese morning glory Ipomoea (Pharbitis) nil, Scientific Reports (2017). DOI: 10.1038/s41598-017-10715-1

Read the original:
Scientists use CRISPR technology to change flower colour - Phys.org - Phys.Org

Experts at a one-day workshop in Washington DC next week will discuss public interest aspects of patents and two breakthrough new medical technologies related to gene editing (CRISPR) and cancer treatment (CAR T).

The 15 September event, entitled, Patents, the Public Interest and Two New Medical Technologies: CRISPR and CAR T, will feature a unique mix of key health advocates, academics, licensing and standards experts, congressional staff and others.

A live webcast on YouTube will be available here.

Event moderators include William New of Intellectual Property Watch and Sarah Karlin-Smith of Politico.

The full KEI event announcement is reprinted below:

Workshop: Patents, the Public Interest and Two New Medical Technologies: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), Chimeric Antigen Receptors (CAR) technologiesOn September 15th, 2017, Knowledge Ecology International will be hosting a workshop on: Patents, the Public Interest and Two New Medical Technologies: CRISPR and CAR T.

CRISPR related inventions include breakthrough technologies to modify genes, which have broad applications for innovations in medicine, agriculture and other fields.

CAR T therapies are an exciting new approach to treating cancer and other diseases, including previously incurable cancers.

Both technologies were developed with significant funding from the U.S. federal government.

There are controversies over the licensing of several CRISPR related inventions, and over the pricing of new CAR Treatments, including most recently the decision by Novartis to charge $475,000 for Kymriah, a CAR T treatment for leukemia.

The workshop will bring feature a diverse group of experts and stakeholders to discuss the public policy challenges appropriate governance of CRISPR and CAR Ts intellectual property.

Date: Friday, September 15, 2017Location: Kaiser Permanente Center for Total Health, 700 Second St. NE (near Union Station), Washington, DC 20002

To Register: use this form.

A PDF version of the program is available here.

Related

See original here:
Workshop Next Week On Public Interest And CRISPR Gene Editing, CAR T Cancer Treatment - Intellectual Property Watch

CRISPR: The Next Generation

The latest news in genetic science has been dominated by the CRISPR/Cas9 technique over the past fiveyears. But a new fourth-generation DNA base editor could see CRISPR dethroned, according to a recent study published in Science Advances.

The fourth-generation base editor a tool used to modify the building blocks of genetic code, only now with an inhibitor added to protect DNA from accidental changes heightens editing accuracy and reduces unintentional DNA changes, which occur with current base-editing technologies.

DNA is a series of base pairs, or nucleotides: adenine, thymine, guanine and cytosine, called A-T-G-C for short. When CRISPR converts a C:G base pair to a T:A pair, sometimes it inadvertently changes the C:G base pair to a G:C or A:T base pair. Thismight seem like an insignificant mess of letters, but on the genetic level, even a single mistaken nucleotide can have devastating consequences for an organism.

Approximately two-thirds of known human genetic variants associated with disease are point mutations, said study co-author David R. Liu, Harvard University Professor of Chemistry and Chemical Biology and Howard Hughes Medical Institute Investigator, in an email interview with Futurism.The fourth-generation base editors to my knowledge are the most effective forms of these molecular machines that can directly correct certain types of point mutations.

Liu, co-author Alexis Komor, and their colleagues found that the number of undesired editing products depends on the level of a cutting enzyme called uracil N-glycosylase (UNG). Uracil is one of the four base pairs found in RNA, which is involved in the process of transcribing DNA so that its code is physically expressed by the organism.

Mechanistically, it made sense that [this]was leading to the undesired products we would occasionally observe, Liu explained. The teamhad a hunch that UNG, which initiates base excision repair at uracils, might be the culprit. Indeed, when we performed base editing in cells lacking UNG, essentially all undesired product formation went away.

Komoret al. then designed a fourth-generation base editor combined with an inhibitor, called BE4, which blocks UNG from cutting and altering DNA inadvertently. BE4 shuts down the cellular troublemaker (UNG) more effectively than our previous base editors, which results in higher efficiency of C to T base editing, and also fewer undesired products, said Liu.

Using BE4 formed with the bacteriaStreptococcus pyogenes, this base editing procedure increased the efficiency of swapping C:G to T:A by 50 percent, while halving the frequency of undesired byproducts. Click to View Full Infographic

This comes at a time when the number of incredible things CRISPR is doing extends beyond the medical field: into ecology, with the possibility of artificially designing algae to create a more efficient biofuel, and national security, with the U.S. Advanced Research Projects Agency (DARPA) investing $65 million in a project called Safe Genes.The DARPA Safe Genes program is very forward-thinking and focused on important issues including safest practices for genome editing, as well as helping to advance these technologies to realize their full potential, commented Liu.

Ultimately, the optimum balance lies in protecting the public with restrictions on gene editing technology, but not implementing so many that it decreases the number and diversity of efforts to use these technologies for the public good, Liu said.

For a nominal charge, Lius team has made the base editor available ona nonprofit genetic repository called Addgene: We definitely want the scientific community to use these tools.

Read the original post:
A Fourth-Generation DNA Base Editor Could Replace CRISPR - Futurism

Newly fertilized (left) and later-stage (right) human embryos that have had a disease mutation corrected by the CRISPR editing system.

OHSU

By Kelly ServickAug. 31, 2017 , 12:28 PM

When the first U.S. team to edit human embryos with CRISPR revealed their success earlier this month, the field reeled with the possibility that the gene-editing technique might soon produce children free of their parents genetic defects. But the way CRISPR repaired the paternal mutation targeted in the embryos was also a surprise. Instead of replacing the gene defect with strands of DNA that the researchers inserted, theembryos appeared to use the mothers healthy gene as a template for repairing the cut made by CRISPRs enzyme.

But such a feat has not been observed in previous CRISPR experiments, and some scientists are now questioning whether the repairs really happened that way. In a paper published online this week on the preprint server bioRxiv, a group of six geneticists, developmental biologists, and stem cell researchers offers alternative explanations for the results. And uncertainty about exactly how the embryos DNA changed after editing leaves many questions about the techniques safety, they argue. (The authors declined to discuss the paper while its being reviewed for publication.)

Embryologist Shoukhrat Mitalipov of Oregon Health and Science University in Portland, who led the now-disputed experiments, released a statement saying that his team stands by its explanation. We based our finding and conclusions on careful experimental design involving hundreds of human embryos, it says.

In the 2 August Nature paper, Mitalipov and his collaborators showed they could bump up the efficiency of human embryo editing by inserting the CRISPR machinery earlier in development than previous experiments. When they combined healthy eggs with sperm bearing a disease-causing mutation and immediately added CRISPR, they found that 72% of the resulting embryos were free of the mutationrather than the expected 50% that would have avoided inheriting the harmful gene anyway.

Although the researchers inserted short strands of DNA as templates for repair, the cells didnt seem to take them up; those specific sequences were absent from the embryos. The cells must have relied instead on the nonmutated sequence in the egg donors DNA when making the repairs, the team concluded.

The bioRxiv response, led by developmental biologist Maria Jasin of Memorial Sloan Kettering Cancer Center in New York City and Columbia University stem cell biologist Dieter Egli, challenges that interpretation. The authors, which also include well-known CRISPR researcher and Harvard University geneticist George Church, say that the Nature paper goes against conventional wisdom about how embryos are organized early in development. Right after an egg is fertilized, the DNA from the sperm and the egg arent believed to be in close enough proximity to interact or share genes, they explain.

Stem cell researcher Junjiu Huang of Sun Yat-Sen University in Guangzhou, China, who led the first published study of CRISPR editing of a human embryo, isnt on the bioRxiv paper, but shares that concern. Its not unexpected for a cell to use its own sequences to guide repair, he notes. In his groups study, which used nonviable embryos, a gene related to the CRISPR-targeted gene seemed to function as a template. But that gene was on the same chromosome as CRISPRs edits. Here, the sperm and egg nuclei are seemingly too far apart to cooperate in the repairs, he says.

The preprint authors lay out two other scenarios for what Mitalipovs team saw. Its possible that some of the embryos didnt take up paternal DNA at all, and thus never inherited the mutation to begin with. In some in vitro fertilization procedures, embryos can occasionally start to develop from maternal DNA alone, and the study didnt rule out this phenomenon for every embryo, they say.

They also suggest that mutated paternal gene could have been snipped out of young embryos but never actually replaced with a healthy version. CRISPRs cuts can sometimes cause chunks of DNA to be removed from the strand before the two cut ends are rejoined, they note. That would mean no detectable mutationbut it could also mean missing sections of DNA that could have unknown consequences for the embryo.

This possibility of allele dropout has been the subject of discussion in the field ever since the Nature paper was published, says developmental biologist Robin Lovell-Badge of the Francis Crick Institute in London. Many scientists are now waiting for a response from Mitalipov, he says.

In his statement, Mitalipov promised to respond to [the] critiques point by point in the form of a formal peer-reviewed response in a matter of weeks. He also urged follow-up to resolve the matter. We encourage other scientists to reproduce our findings by conducting their own experiments on human embryos and publishing their results.

*Update, 1 September, 1:30 p.m.: The new version of this story has additional comments from several researchers and clarifies the authorship of the preprint.

The rest is here:
Skepticism surfaces over CRISPR human embryo editing claims ... - Science Magazine

E. coli might best be known for giving street food connoisseurs occasional bouts of gastric regret. But the humble microbial workhorse, with its easy-to-edit genome, has given humankind so much moreinsulin, antibiotics, cancer drugs, biofuels, synthetic rubber, and now: a place to keep your selfies safe for the next millennium.

Scientists have already used plain old DNA to encode and store all 587,287 words of War and Peace, a list of all the plant material archived in the Svalbard Seed Vault, and an OK Go music video. But now, researchers have created for the first time a living library, embedded within, you guessed it: E. coli. In a paper published today in Nature, Harvard researchers1 describe using a Crispr system to insert bits of DNA encoded with photos and a GIF of a galloping horse into live bacteria. When the scientists retrieved and reconstructed the images by sequencing the bacterial genomes, they got back the same images they put in with about 90 percent accuracy.

The study is an interestingif slightly gimmickyway to show off Crispr's power to turn living cells into digital data warehouses. (As if E. coli didnt already have enough on its plate, what with securing global insulin supplies and weaning the world off fossil fuels.) But the real question: Why would anyone want to do this?

To the left are a series of frames from Eadweard Muybridges Human and Animal Locomotion. To the right are the frames after multiple generations of bacterial growth, recovered by sequencing bacterial genomes.

Seth Shipman

If youre Jeff Nivala, its not to preserve visual messages for people in the far-off future. Its so he can turn human cells like neurons into biological recording devices. The E. coli is just a proof of concept to show what cool things you can do with this Crispr system, says Nivala, a coauthor on the paper and geneticist at Harvard. Our real goal is to enable cells to gather information about themselves and to store it in their genome for us to look at later. That concept is called the molecular ticker tape. Its something George Church thought up before Nivala, a postdoc, arrived in his lab. But its a challenge Nivala thinks is uniquely suited to Crispr.

In case youve been living in a bunker, Crispr-Cas9 is a revolutionary molecular tool that combines special proteins and RNA molecules to precisely cut and edit DNA. It was discovered in bacteria, which use it as a sort of ancient immune system to fend off viral attackers. Cas9 is the protein that does all the cutting, i.e. gene editings heavy lifting. Lesser known are Cas1 and Cas2. Theyre the ones that tell Cas9 where to do the cutting.

Church's lab plans to leverage that system to get human brain cells to show how exactly they develop into neurons. Nivala thinks theyll be able to do that because of how Cas1 and Cas2 work. During a viral invasion, the proteins go out and grab a piece of the attackers DNA, which they slip into the bacterial genome for another enzyme to turn into a matching guide RNA. Thats what helps Cas9 find (and then chop up) copies of the virus in the cell. The really cool bit is that Cas1 and Cas2 dont just insert viral DNA into the genome at random. As they encounter new threats, they add DNA in the order in which it arrives. That turns a cells genome into a temporal recordthink ice cores for molecular historyof whatever the cell encounters.

To the left is an image of a human hand, which was encoded into nucleotides and captured by the Crispr-Cas adaptation system in living bacteria. To the right is the image after multiple generations of bacterial growth, recovered by sequencing bacterial genomes.

Seth Shipman

One day, Nivala thinks scientists will be able to use that system to record synaptic activity. Like a guest book at a wedding, embedded signals in the genome could tell researchers exactly which neurons were talking to each other at different times, in response to different stimuli.

If you think of a cell as a processor, this adds a thumb drive, which stores information for later processing, says Karin Strauss, lead researcher on Microsoft's own DNA storage project. Last year the company set a new record200 megabytesand has plans to get a DNA storage system up and running by the end of this decade. As for DNA data storage in the IT industry, it is more well served by standard DNA synthesis and sequencing at the moment because they are easier to control and a lot denser than whole cells, says Strauss, who is unconnected to the Harvard research.

Companies that make custom DNA, such as Twist Biosciences, are already selling to customers using it for storage purposes. But its still only a small piece of their businessabout 5 percent. Costs have to come down by a factor of about 10,000 before DNA becomes competitive with traditional storage methods. But the long-term benefits will be huge: Properly stored in a cold, dry place, DNA can keep data intact for at least 100,000 years.

Thats why scientists such as Ewan Birney, director of the European Bioinformatics Institute, are working on better tools and methods to make DNA storage truly scalable. In that endeavor he doesnt see a place for live cells, which start out at less than 100 percent accuracy and are susceptible to mutations over time that could further degrade data integrity. Its cute, and I wish Id done it, Birney says of the Nature paper. But it doesnt add much on the DNA storage side of things. What did impress me was the amount of edits they achieved with high fidelity. Its a real tour de force of Crispr.

So, at least for now, theres no reason to think your family photo albums will one day be backed up on an E. coli drive. More likely, the memories cells store will be their own.

1Disclosure: One of these researchers is married to a WIRED editor.

Read more:
Scientists Upload a Galloping Horse GIF Into Bacteria With ...

CRISPR is typically used to edit disease-causing gene mutations, but is increasingly being tapped for broader applications. The latest? Identifying sequences that activate genes, which could help unravel the causes of autoimmune disease.

While CRISPR shows great promise in the treatment of genetic disease, the genes it cuts outthose that code directly for proteinsonly make up 2% of the human genome. The other 98% consists of regulatory gene sequences, including promoters, which switch on genes next to them, and enhancers, which activate genes that may sit far away from them in the genome.

When the balance of promoters and enhancers is out of whack, it can lead to disease. But its difficult to pinpoint just which regulators have a hand in causing disease, as specific regulators play a role in specific cells, under specific conditions.

Jacob Corn and Alexander Marson at the University of California, San Francisco, focused on T cells and the IL2RA protein, which tells T cells if they should step up or dampen an inflammatory response. If the enhancers that switch on IL2RA are faulty, the T cells dont suppress inflammation, which could cause autoimmune disorders, such as Crohns and inflammatory bowel disease (IBD).

Corn and Marson usedCRISPR activation, or CRISPRa, to homein on the IL2RA gene. This methoduses a guide RNA to target sections of the genome, much like regular CRISPR, but instead of cutting them, it activates those sequences to see how they affect gene expression.

They created 20,000 guide RNAs for use with CRISPRa: "We essentially performed 20,000 experiments in parallel to find all the sequences that turn on this gene," said Marson, an assistant professor of microbiology and immunology at UC San Francisco, in a statement.

The teamturned up several sequences that might be important for ILR2A expression, including a common genetic variant that was already linked to increased IBD riskbut was not well understood. The findings are published in Nature.

"This starts to unlock the fundamental circuitry of immune cell regulation, which will dramatically increase our understanding of disease," Marson said.

The utility of CRISPR is growing by the day. Recently, UC San Diego researchers created a new version of CRISPR that targets RNA rather than DNA, which could be used to treat diseases caused by errant repeats in RNA sequences, including Huntington disease and a type of amyotrophic lateral sclerosis.

And scientists at eGenesis have deployed the gene-editing tool in the organ transplant field. By snipping out a family of viruses in the pig genome, they have overcome one obstacle in xenotransplantation, or using animal organs for human transplant.

The next step forthe UCSF researchers isto modify their method so that it can screen for enhancers of many different genes at once.

"Not only can we now find these regulatory regions, but we can do it so quickly and easily that it's mind-blowing," said Corn, assistant adjunct professor of molecular and cell biology at Berkeley. "It would have taken years to find just one before, but now it takes a single person just a few months to find several."

Read this article:
'Gentler' CRISPR sheds light on autoimmune disease | FierceBiotech - FierceBiotech

A few years ago, a team of researchers at Stanford University launched a video game called Eterna. The game was designed to harness the brain power of thousands of gamers, challenging them to design new chemical sequences of RNA. A new follow-up game has just been launched, and this time players are challenged to create a new RNA molecule that can essentially function as an on/off switch for the CRISPR/Cas9 gene editing process.

When Eterna was first launched in 2011 it was a bit of an experiment. Half-intended to simply educate people in an entertaining way, researchers hoped once the game scaled up to enough players the results would start to become clinically significant. And significant they became.

As players refined their RNA molecule-making skills, the game grew in complexity. Up to 100,000 registered players were engaging with the game at its peak, and in early 2016 a paper, co-authored by the game players, was published in the peer-reviewed Journal of Molecular Biology. The paper established a set of rules developed by the game players determining the difficulty of designing appropriate RNA molecular structures.

Earlier in 2016 Eterna players were tasked with designing a novel molecule to assist with the creation of a simple and accurate blood test for tuberculosis. Now researchers are turning their sights on CRISPR, hoping this gaggle of game players can inspire a new molecular structure that will help focus the gene-editing technology.

"Great ideas can come from anywhere, so this is also an experiment in the democratization of science," says Stanford's Professor Howard Chang. "A lot of people have hidden talents that they don't even know about. This could be their calling. Maybe there's somebody out there who is a security guard and a fantastic RNA biochemist, and they don't even know it."

The new Eterna challenge asks players to design a unique RNA molecule that can do several things, from being recognized by the CRISPR-associated enzyme to guiding it to a targeted gene. The researchers suspect that this new challenge may be slightly easier than other, more mathematically orientated Eterna challenges, but they are looking for thousands of diverse solutions that could be applied into laboratory outcomes.

"We're not sure yet if there will be unforeseen problems with the Cas9 protein experimentally," says Rhiju Das, the principle investigator for Eterna. "That's partially why we want as many diverse solutions as possible for the Greenleaf and Chang labs to test, even in this pilot round. We're hoping for 10,000 to 100,000 players to contribute 10 solutions each. If we get that many, we'll indeed work to get that many synthesized and tested."

This real-life laboratory outcome makes the Eterna game unique as it gives game players the possibility of having their designs actually created and tested in the lab. And anyone can play the game as long as they have access to the internet and an interest in learning how to play.

"There is a misconception of science as something that happens in an ivory tower by someone in a white coat with a long beard," says Das. "And they are saying things and drawing things that nobody understands. But it's not like that! It's really like a puzzle that anybody can get engaged with."

Learn more about the Eterna CRISPR challenge in the video below and join the Eterna community and play the game here.

Source: Stanford University

Excerpt from:
Video gamers tasked with helping develop new molecule for controlling CRISPR - New Atlas

Early embryos two days after co-injection with a gene-correcting enzyme. (OHSU)

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A few weeks ago, two stories crossed paths. In MIT Technology Review, we learned that, for the first time in the United States, researchers had used the gene-editing technique known as CRISPR to modify a human embryo. Severaldays later, CBS Newsreleased a report that through nearly universal prenatal testing followed by selective abortion, Iceland has virtually eliminated Down syndrome. Ad Policy

The CRISPR story shows that we are on the cusp of an enormous leap of capability when it comes to shaping the genetic potential of our offspring. Meanwhile, Ive contended that the past decades of testing, genetic consultation, and decision-making about abortion related to prenatal diagnoses of Down syndrome have served as a kind of test run for the future of human procreation. Can we make informed choices? Can we understand that probability doesnt equate to outcome when were talking genetic makeup? Can we use science to build a more just, happier humanity?

If whats happening in Iceland is, indeed, a test run, its a test were failing. Prospective parents are making decisions based on fear and stigma, helped along by the medical profession. As our tools to make such decisions get even more powerful, we have to shift how we talk about genetic diversity.

Cards on the table: Im the father of a boy with Down syndrome. I am pro-choice, anti-eugenics, and pro-information. In preparation for the age of CRISPR, well need to develop new ways to talk about whats normal and whats good, because we face decisions that are nearly unprecedented in human history. I say nearly, because with Down syndrome prenatal testing, we have a body of evidence for what happens when we expand our power to determine who gets born without building systems to ensure that we make informed decisions.Related Article

CRISPR (short for Clustered Regularly Interspaced Short Palindromic Repeats) is wickedly powerful. It makes reasonably precise changes to a targeted cells DNA by means of a technique adapted from naturally occurring DNA-editing defense mechanisms in bacteria. Chinese scientists first modified human embryos two years ago. The researchers in Oregon used it to change the DNA of a large number of one-celled embryos with the goal of demonstrating both that the technique could be used at scale and that the genes causing disease could be effectively identified and eliminated.

Each new development, as previously covered in The Nation, sparks rounds of debates between those optimistic about fighting diseases and those concerned about implications. For example, sickle-cell patients hope for a cure, while the intelligence community worries that terror groups could weaponize CRISPR. Earlier this year, the National Academy of Sciences, Engineering, and Medicineagreedthat genome editing could be used to modify embryos, but should be allowed only for treating or preventing diseases or disabilities at this time. Ethicists demand more robust engagement of the questions we are about to face, as techniques move from the research to the practical stage. Still, most of the debates remain locked in abstract thought experiments.Most Popular

Prenatal testing followed by selective abortion is not genetic engineering. It is, however, a space in which we have real-world data about how people make choices about procreation when granted additional information about the genetic makeup of their potential offspring. It turns out, perhaps unsurprisingly, that fear, misinformation, and bias shape our decision-making.Current Issue

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Take Iceland. In the small island country, all pregnant women are informed of the availability of genetic screening. Between 80 and 85 percent take the test, and nearly 100 percent of all positive tests for Down syndrome result in termination. What are we to make of such an outcome? Each person hearing the words Down syndrome applied to their fetus does so as a consumer of a culture that, broadly speaking, denigrates life with developmental disabilities. Geneticist Kari Stefansson characterized the counseling as heavy-handed in favor of termination, and so thats where the momentum is. If Icelandic doctors, nurses, and genetic counselors dont find ways to mitigate that, the disability largely disappears.

This is typically the moment in essays about prenatal testing in which I assure you that my son is happy. He is. Hes 10. He likes Hamilton and Harry Potter, and is a wonderfully inventive communicator. We are privileged to live in a good community with good schools, and when we encountered obstacles to his long-term supports in one state, we could move. Weve never denied that there are challenges, but the greatest ones are constructed by an ableist society, not inherent to his disability. Society can be changed. His genes dont need to be.

But the decision whether or not to terminate is not about my sons outcomes, but accepting two general principles. First, with good social supports, theres no reason that people with Down syndrome cant lead good lives included within communities. For a doctor to assert the probability that Down syndrome leads to despair is simply not true. Second, in general, probabilities never guarantee outcomes. Our genes encode an array of probabilities into our bodies.

In recent years, rather than focusing on the abortion itself (or decision to carry to term), North American activists in Down-syndrome advocacy communities have tried to look at the communication in the period between the positive test and the decision about whether or not to terminate. The goal is to provide materials to better inform the tens of thousands of doctors, nurses, and counselors who encounter women in the context of prenatal testing. These efforts have coalesced around the nonpartisan rubric: pro-information.

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I e-mailed Stephanie Meredith,Lettercase program director at the University of Kentuckys Human Development Institute, where she has helped develop resources for distribution to people who talk to women about prenatal testing. She told me that many genetic counselors and obstetricians offer compassionate, sensitive, and balanced support following prenatal testing, but some clinicians may provide insufficient, outdated, or unintentionally biased counseling. Outcomes for people with Down syndrome and related conditions have changed remarkably over the past 50 years. But too many people involved in prenatal care lack up-to-date information, and theres no easy way for institutes like Merediths to reach every clinician in the country. Meanwhile, companies selling prenatal tests want to increase their market share. Meredith said that clinicians are constantly inundated by marketing from testing labs with very little educational support regarding the conditions included in the test.

Ive spent years talking to parents who received prenatal tests (we did not). Some were told flatly untrue statements about Down syndrome, breaking up marriages and leaving families overwhelmed by stress. In fact, theres evidence that families like mine divorce at lower ratesthan other families. Others were presented with outdated statistics about early death as a likely outcome. Its not (although the premature death rates of African Americans with the condition remainfar too high). Its true that people with Down syndrome once tended to die young and learn little, but thats a fact linked to the era of mass institutionalization. Inclusion has radically changed the probabilities. My son has as good a chance to live as long, happy, productive life as anyone of our socioeconomic status. But expectant parents hearing the words Down syndrome for the first time will only know this if theyre told.

Unfortunately, politics is making it hard to hold the pro-information coalition together, thanks to Americananti-choice efforts. Around the country, the GOP is proposing and passing lawsbanning abortion if a woman tells her doctor shes doing it because of a prenatal diagnosis. We cant be pro-information if we criminalize such conversations. Such a bill ispending right now in Ohio.

What does all this have to do with CRISPR? Right now, were still in a liminal state when it comes to predicting genetic outcomes for fetuses. Our tools, from amniocentesis (developed in the 1950s and 60s) to contemporary screenings that locate fetal blood cells in the mothers bloodstream, are reactive and postconception. Soon, theyll shift to preconception and proactive. What will the tens of thousands of clinicians tell would-be parents as they get flooded with messaging from companies eager to sell their high-tech CRISPR product lines?

Preventing this potentially dystopian future where altered genes separates the haves from the have-nots starts by shifting discourse. A pro-information approach demands that everyone involved in genetic counseling have access to the best data and presents it in a value-neutral way. We must build systems now that grow as our tools evolve. If we do not, genetic diversity will gradually become code for poverty, and new stigmas will run all the way to the DNA.

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We're Failing Our Test Run for the Age of CRISPR | The Nation - The Nation.

In the new Eterna challenge, called OpenCRISPR, players will design a guide RNA molecule that leads CRISPR to the right sequence of DNA for editing or binding. The RNA is the part that confers gene specificity. Its the thing that says, Go after gene A, not gene B, said Chang.

The difficulty for Eterna players is to come up with an RNA molecule that does several things, said Greenleaf. The guide RNA has to be recognized by the CRISPR-associated enzyme. The CRISPR-enzyme system has to be able to recruit biochemical activity to the targeted gene. And lastly, the activity of the CRISPR-enzyme system has to be controlled by a small-molecule drug, so there needs to be a binding pocket for that small molecule. The RNA molecule has to function so that the CRISPR system is active when the small-molecule drug is present and inactive when its not. So far, experts have not been able to create such a drug-activated CRISPR, which is why Chang and Greenleaf are calling on the community of Eterna gamers for help.

The new puzzle will be quite different from the recent challenge in which Eterna players had to design a molecule that could do a mathematical calculation for a tuberculosis diagnostic test. The CRISPR puzzle actually should be pretty easy to solve in silico, even for new players who get to the switch design levels, said Rhiju Das, PhD, associate professor of biochemistry and principal investigator for Eterna.

How those Eterna-designed switches will behave in living cells is a big question. Das said the team will be asking players for different possible solutions to the same problem. Were not sure yet if there will be unforeseen problems with the Cas9 protein experimentally. Thats partially why we want as many diverse solutions as possible for the Greenleaf and Chang labs to test, even in this pilot round, Das said.

It will be an iterative process, said Greenleaf. His Stanford lab will test the first round of solutions and then return these data to the players with refinements that will guide their design work.

Were hoping for 10,000 to 100,000 players to contribute 10 solutions each. If we get that many, well indeed work to get that many synthesized and tested, Das said.

One of the goals of Stanfords Center for Personal Dynamic Regulomes is to get people interested in science, said Chang. The Eterna game is a powerful way to engage lots and lots of people, he said. Theyre not just passive users of information but actually involved in the process.

Like other computer games, Eterna allows players to accumulate points, build expertise and advance to higher levels. The best players have a chance of having their designs implemented in the lab.

One thing that makes the project exciting, said Chang, is that it is an experiment in the sociology of science. There is a misconception of science as something that happens in an ivory tower by someone in a white coat with a long beard. And they are saying things and drawing things that nobody understands. But its not like that! Its really like a puzzle that anybody can get engaged with, he said.

Anyone interested in playing Eterna can sign up here.

In addition to the funding from NIGMS and Stanfords Center for Personal Dynamic Regulomes, the new Eterna challenge is being launched with collaborative support from the Innovative Genomics Institute at the University of California-Berkeley. Stanfords departments of Biochemistry and of Genetics also supported the work.

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Online game challenges players to design on/off switch for CRISPR ... - Stanford Medical Center Report

Have researchers taken a step closer to developing an eventual cure for HIV? A Temple University-led team hopes so, by using a gene editing technique to successfully remove HIV infection from lab mice. The gene-editing tool calledCRISPR which allows scientists to basically cut out and insert specific portions of DNA was used to excise HIV DNA from the mice.

This was the first time CRISPR has been used to shut down HIV replication and eliminate the virus from animal cells. Think of CRISPR as working somewhat like microscopic scissors that snip out an unwanted piece of DNA and then replace that with a new piece. The research, published in the journal Molecular Therapy, involved three animal models, including a "humanized" model where human immune cells infected with the virus were transplanted in lab mice.

"Over our years of research, all of this was frankly a big surprise. This research, so far, has yielded all pleasant surprises, frankly. I never thought that this CRISPR system was going to be working out so beautifully with such efficiency and precision when it first came onto the scene," Kamel Khalili, director of Temple's center for neurovirology, told CBS News.

Khalili led the study along with Wenhui Hu, associate professor in Temple University School of Medicine's Center for Metabolic Disease Research and the Department of Pathology, and Won-Bin Young, who was at that time an assistant professor in the Department of Radiology at the University of Pittsburgh School of Medicine.

This work builds off the team's previously published research last year in which they introduced the HIV-1 DNA into the tissue of rat and mice subjects, and then removed these fragments using CRISPR. This new study is the first time this has been done in three animal models.

While the work signals progress, the medical community still sees years of work ahead before there's a reliable cure for HIV. According to the World Health Organization, 36.7 million people were reported to be living with HIV globally by the end of 2015. Since the start of the HIV/AIDS epidemic, more than 70 million people have been infected with the virus that has resulted in 35 million deaths.

The stakes are high, and the Temple team is one of many trying to find a cure for the virus, which has proven exceptionally difficult to eliminate from the body. While current drug treatments can reduce the virus to virtually undetectable levels enabling many patients to live longer, healthier lives HIV continues to lurk in hidden reservoirs and comes roaring back if treatment stops. In late 2015, theamfAR Institute for HIV Cure Research set the ambitious goal of developing a basis for cure for HIV by the end of 2020.

"The basic science community in HIV research is now very focused on finding a cure," Paul Volberding, head of the institute, wrote in an email to CBS News. "It still feels a long way off but the tools we now have definitely including the gene editing used in this report is accelerating our work and raising optimism. The cure field is in very close contact and collaborations are active world wide. It's really quite exciting!"

Volberding is also the director of the UCSF AIDS Research Institute and has a place in history for founding the first inpatient ward for people with AIDS at San Francisco General Hospital in 1983. How promising does he view this new research out of Temple?

"Gene editing is a potent and still rather new tool in HIV research and many other areas as well," he wrote. "It faces a challenge in scalability getting the technology simplified and inexpensive but is certainly worth following."

Since first being developed a mere five years ago, CRISPR has generated excitement and controversy in equal measures. While it was named "Breakthrough of the Year" in 2015 by Science magazine, ethical debate has swirled around CRISPR over how it could be used for good or ill to make changes to our DNA down the line.

Ellen Jorgensen, a molecular biologist and science communicator whose latest project is the yet-to-launch Biotech Without Borders, said she thinks it's important to focus on the potential of CRISPR, rather than feed into the "hysteria" that can surround such life-altering scientific technologies.

"I think CRISPR is an example of why the general public should embrace the chance to learn more about this sort of technology that will be more and more relevant to everyone's daily life as time goes on," Jorgensen told CBS News. "We are in an age of biotechnology as opposed to the last century, which was the 'age of physics.' There is an equal potential here to disrupt technologies, but it also creates ethical questions that the general public has to weigh in on. My thing is, I want them to weigh in on them, but have the understanding that this technology is something that is powerful and that can spur a lot of change moving forward."

In the case of this latest HIV research advance, Jorgensen, who cofounded Genspace, a nonprofit devoted to fostering better science literacy, said she believes there is "great potential" in finding a cure for something like HIV through gene editing technology.

Moving forward, Khalili and his team plan to try their technique on primate subjects, whose DNA is obviously closer to humans. He said they are working on securing more funding to move on to primate clinical trials.

Volberding added that "primates are a very good model for human trials," and that research like this is promising in the continued fight against HIV.

"I think that CRISPR and tools like it are revolutionizing the medical field and will bring about new ways for the treatment and cure for a broad range of diseases," Khalili said. "When it comes to treating HIV or cancer or other genetic diseases, I think there are a lot of good things that will come out of this."

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CRISPR gene editing eliminates HIV infection in mice ...

1. The gene editing tool CRISPR-Cas9 was used to correct a mutant paternal MYBPC3 allele in human preimplantation embryos.

2. No off-target effects were detected.

Evidence Rating Level: 1 (Excellent)

Study Rundown: A dominant mutation in the gene MYBPC3 causes hypertrophic cardiomyopathy (HCM), the most common cause of sudden death in otherwise healthy young athletes. While most current therapies focus on relieving symptoms of HCM, researchers in this study aimed to prevent transmission of the causative gene mutation by correcting it in preimplantation embryos.

Healthy donor eggs were injected with sperm that were heterozygous for the MYBPC3 mutation. After fertilization, recombinant Cas9 protein and single guide RNA that targeted MYBPC3 were microinjected into the zygotes. A majority of treated embryos survived and lost the mutation in this gene, without other genes being impaired. CRISPR-Cas9 targeting of MYBPC3 was found to be highly specific in the treated embryos.

This study was the first to use CRISPR-Cas9 to correct a harmful mutation without causing significant off-target effects. Although this genome editing technique is still far from clinical use and requires full discussion from a bioethics perspective, this research suggests the potential clinical efficacy of this therapy for in vitro fertilization and the correction of fatal mutations.

Click to read the study in Nature

Relevant Reading: Genome engineering through CRISPR/Cas9 technology in the human germline and pluripotent stem cells

In-Depth [in vitro study]: Human zygotes were produced by fertilizing 70 oocytes without MYBPC3 mutations with sperm from an HCM patient with a heterozygous mutation in MYBPC3. Eighteen days after fertilization, recombinant Cas9 protein, short guide RNA, and single-stranded oligodeoxynucleotideswere microinjected into the cytoplasm of the zygotes. A majority of zygotes survived this procedure, with a survival rate of 97.1%. Three days after injection of the Cas9 protein, 54 injected embryos were sequenced and 66.7% were found to be homozygous for the wild-type (WT) allele of MYBPC3. Almost half of the blastomeres from mosaic embryos were also found to be homozygous for the WT allele of this gene, demonstrating that the heterozygous mutation was repaired through homology-directed repair. These analyses demonstrated the efficient targeting by CRISPR-Cas9 in human embryos.

To improve the efficacy of gene correction, CRISPR-Cas9 was mixed with sperm and injected into 75 oocytes in metaphase II. This method resulted in an increase in WT embryos, with 72.4% successfully removing the mutation. Additionally, a majority of these oocytes developed into the eight-cell stage and then blastocysts, demonstrating no significant effect on embryonic development due to this therapy.

Finally, off-target effects were assessed through whole genome sequencing, digested genome sequencing, and whole exome sequencing. No insertions or deletions were detected in the WT blastomeres at 23 off-target loci, demonstrating the high targeting efficacy and potential safety of this treatment.

Image: PD

2017 2 Minute Medicine, Inc. All rights reserved. No works may be reproduced without expressed written consent from 2 Minute Medicine, Inc. Inquire about licensing here. No article should be construed as medical advice and is not intended as such by the authors or by 2 Minute Medicine, Inc.

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CRISPR-Cas9 corrects hypertrophic cardiomyopathy gene mutation in human preimplantation embryos [PreClinical] - 2 Minute Medicine

Gene editing made great strides this month when scientists reported success using a technique called CRISPR Clustered Regularly Interspaced Short Palindromic Repeats to correct a serious, disease-causing mutation in human embryos. Researchers fixed a mutation that leads to hypertrophic cardiomyopathy, a relatively common inherited disease of the heart muscle that affects about 1 in 500 people. The public response was wildly enthusiastic. But any new technology can spur confusion and hyperbole, and this one is no exception. Here are five myths about what CRISPR can and cant do.

Myth No. 1

CRISPR can build customized babies.

In February 2016, one CRISPR critic predicted in Mother Jones, We are this close to designer babies. And this month, biologist Richard Dawkins mused that the genetically edited designer babies on the horizon shouldnt be any more worrisome than children who are pushed by their parents to hone their natural talents.

But CRISPR is not on the cusp of creating a super-race for one main reason: We dont know how to do that. We dont know how to build baby Einsteins or order up a finely chiseled and uber-flexible Simone Biles, because there is no single smart gene or spunky, lithe gymnast gene.

Much of what goes on inside our bodies and our brains is influenced by a combination of genes and environment, nature and nurture. Beauty, athleticism and musicality dont hinge on a single sequence of base-pairs. Instead, these characteristics are considered complex traits that are shaped by the input of multiple genes, along with lifestyle and environmental factors. This is especially true of intelligence. Studies, many of which have tracked adopted children and twins, have indicated that just 50 percent of the variation in intelligence among people can be chalked up to genetics.

Myth No. 2

CRISPR is the only hope for would-be parents with genetic conditions.

The Genetic Literacy Project, a group dedicated to increasing the publics understanding of gene research, wrote this year that parents worried about passing on genetic disorders to their children have hope: Gene editing. Likewise, an Australian newspaper greeted this months CRISPR news with an ebullient headline: Hope for parents as science deletes mutant killer gene.

While its undeniable that the ability to home in on and fix a genetic error would enable some would-be parents to sidestep the possibility of transmitting a disease to their offspring, gene editing is not the only option in such cases. Preimplantation genetic diagnosis has been used for decades to help couples who go through IVF ensure that they select healthy embryos from among those fertilized in a clinic. The technology has allowed carriers of genetic disease to conceive unaffected children, starting in 1991, when it was first used to avoid cystic fibrosis.

In the event that not enough healthy embryos are created during the IVF process, CRISPR could one day lend a helping hand and repair defective embryos, giving a couple more choices. Still, an essay that accompanied this months research report, published in Nature, concluded that embryo genetic testing during IVF remains the standard way to prevent the transmission of inherited diseases in human embryos.

Myth No. 3

CRISPR will be available for widespread use soon.

I think its really likely that in the not-too-distant future it will cure genetic disease, Jennifer Doudna, one of the scientists behind CRISPR, said at a recent conference . The Chicago Tribunes editorial board shared the sentiment in April 2016, claiming that for some people born with debilitating genetic diseases, scientists could give them relief from their symptoms and maybe even cure them in the not-too-distant future.

Not so fast. In the United States, a human-embryo research ban has been in place since 1996, prohibiting the use of federal money to support research in which embryos are created, destroyed or discarded. Recent embryo-editing studies were paid for by universities and foundations, but the lack of federal funding slows the science down.

Moreover, just because one experiment was successful doesnt mean the next one will be. In fact, even though most embryos were successfully repaired in the recently reported study, more than a quarter werent. Another concern is that CRISPR may solve one problem while unintentionally creating another. A challenge is to avoid off-target edits or mosaicism, a condition that occurred in previous attempts, in which CRISPR successfully edited the specific mutation in some but not all cells. The technique needs much more practice before its ready for widespread public use.

Myth No. 4

CRISPR means a future without genetic diseases.

There is widespread interest in using CRISPR, which allows the targeted editing of specific genes, to potentially end genetic disease in humans, Vice reported in December 2015 . A more recent headline from Wired cheered that CRISPR may cure all genetic disease one day.

While that would certainly be nice, its impossible to edit out all genetic diseases, because not all genetic diseases are simply inherited. There are about 10,000 single-gene disorders that weve discovered diseases caused by a specific, individual gene mutation. But there are thousands more that are caused by multiple genetic factors. Moreover, some genetic conditions are the result of new, spontaneous changes in DNA, called de novo mutations.

Cancer is a prime example. While some types of cancer can be inherited, many others dont appear to have a primary genetic component, and often respond to a variety of environmental factors and other outside causes. Ending genetic disease is a worthy goal, but an extremely complicated one that will require more than eliminating heritable disease.

Myth No. 5

CRISPR technology will one day be broadly available.

Recent advances in gene-editing technology have made the process cheaper , causing some commentators to predict a quick CRISPR proliferation on the horizon. Gene Editing Is Now Cheap and Easy, one 2015 headline claimed. A Wall Street Journal article concerned with amateurs imitating CRISPRs technology likewise fretted that DIY gene editing is fast, cheap and worrisome.

CRISPR may be cheaper than it once was, but its hard to foresee a future when all prospective parents who could benefit will be able to afford it. As a rule, genetic technologies are very expensive: Patients dont pay just for the supplies used, but for doctors time, labor and equipment, often over a number of appointments. You dont have to look any further than IVF to be reminded that using science to have babies costs a lot of money: The median cost of a single IVF cycle is $7,500. It is unclear whether insurance would cover CRISPR gene editing, but its highly unlikely considering that few pay for preimplantation genetic diagnosis or IVF in the first place.

If CRISPR were to become a safe, accepted embryo-editing technique, its likely that only the well-to-do would be able to afford it, essentially making genetic diseases into diseases of poverty. Its not too hard to imagine a wildly disparate economic playing field a dystopian vision, in the words of StatNews writer Jim Kozubek, in which these treatments will be available to only the wealthiest among us who can pay for them.

Twitter:@brochman

Five myths is a weekly feature challenging everything you think you know. You can check out previous myths, read more from Outlook or follow our updates on Facebook and Twitter.

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Five myths about gene editing - The Washington Post - Washington Post