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15 Worrying Things About the CRISPR Babies Scandal – The Atlantic

11. There is no way to tell whether Hes work did any good.

Both Nana and Lulu will be monitored at least until they turn 18. But the children were already at virtually no risk of contracting HIV, said Alta Charo, a bioethicist from the University of Wisconsin at Madison, in a statement. This means that there is no way to evaluate if this indeed conferred any benefit. If they remain HIV-negative, there is no way to show it has anything to do with the editing.

At the Hong Kong summit, He was asked whether the two children would be treated differently by their parents, who will know that they have been edited. I dont know how to answer this question, He said.

12. He has doubled down.

If He shows any contrition about how these events have unfolded, it has not been obvious. Speaking at the Hong Kong summit, he apologized, but only because news about his work leaked unexpectedly before he could present it in a scientific venue. That, He said, took away from the community. Regarding the experiment itself, he said: I feel proud.

13. Scientific academies have prevaricated.

In the wake of Hes bombshell, several scientists, including the CRISPR pioneer Feng Zhang and the stem-cell biologist Paul Knoepfler, have called for a temporary moratorium on similar experiments. By contrast, after the news first broke, the organizing committee of the Hong Kong summit, which includes representatives from scientific academies in Hong Kong, the United Kingdom, and the United States, released a bland statement in which it simply restated the conclusions from its earlier report. A second statement, released after the summit, was stronger, calling Hes claims deeply disturbing and his work irresponsible.

Read: A reckless and needless use of gene editing on human embryos

But the second statement still discusses the creation of more gene-edited babies as a goal that should be worked toward. The risks are too great to permit clinical trials of germ-line editing at this time, it says, but it is time to define a rigorous, responsible translational pathway toward such trials. George Daley from Harvard Medical School, who was one of the meetings co-organizers, made similar points during the event itself. Given that the world is still grappling with the implications of what has happened, no, its not time yet and its tone-deaf to say so, says Hank Greely.

Although the chair opened the summit by invoking Huxleys Brave New World, few of the discussions at the meeting, and nothing in the concluding statement, suggest a meaningful engagement with social consequences, says the Center for Genetics in Society, a watchdog group.

14. A leading geneticist came to Hes defense.

In an interview with Science, George Church, a respected figure from Harvard and a CRISPR pioneer, said that he felt an obligation to be balanced about the He affair. Church suggested that the man was being bullied and that the most serious thing about his experiment was that he didnt do the paperwork right. [Churchs] comments are incredibly irresponsible, says Alexis Carere, who is president-elect of the Canadian Association of Genetic Counsellors. If someone contravenes the rules that we have laid down, we are very justified in speaking out about it. The unfortunate effect of this is that it makes it seem like there is some kind of balance, and George is just in the middle. There is not.

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15 Worrying Things About the CRISPR Babies Scandal – The Atlantic

The Infamous Scientist Behind the CRISPR Baby Gene Editing Is …

From Popular Mechanics

When He Jiankui shocked the world last week by declaring he had successfully altered the genetic code of two babies, he was met with overwhelming skepticism and condemnation from the scientific community. Now, his case has gotten weirder. The South China Morning Post reports that the infamous scientist has gone missing.

Officials at He’s now-former university, the Shenzhen-based Southern University of Science and Technology, denied claims that He had been detained by the Chinese government. Right now nobodys information is accurate, only the official channels are, the official tells the SCMP.

On November 26, He Jiankui released a series of YouTube videos announcing that he had made science fiction real-using the genetic editing tool CRISPR, he had successfully edited the genetic code of two twin baby girls to make them more resistant to the HIV virus. He had not allowed any independent scientific inspection of his work, choosing to announce his breakthrough through mainstream journalism and social media.

After the highly unconventional announcement, He’s work has come under intense criticism in the realms of both ethics and pure science. Speaking at the International Human Genome Editing Summit, He falsely claimed that his results had “leaked,” although their release had been part of a carefully coordinated media release.

During a 20-minute talk with a question and answer period, He attempted to justify his study to his peers. Presenting himself as a champion working against discrimination of those with HIV, He said that he feels “proud” of his work which targeted CCR5, a known pathway for the virus.

The scientific community disagreed on both purely scientific and as well as moral grounds. Several scientists who observed He’s speech began challenging his work with the two girls, known as Lulu and Nana. One of the most thorough breakdowns of He’s work comes from Gaetan Burgio of Australia National University.

“If you look into details,” Burgio tells PopMech over the phone, “what they meant to target, they havent targeted. They targeted CCR5, which is correct, but they havent targeted the region known to show resistance to HIV.” Burgio says that its “likely” that at least one of the children has no additional resistance to HIV at all.

A particular failure of He’s, according to Burgio, was not recognizing what’s known as the “allele mosaic.” In genetics, a mosaic refers to two or more cell populations with differing genotypes (pieces of genetic material) in one individual. Alleles are crucial parts of our genetic code, variations on DNA that allow for unique traits like eye color. Like eye color, CCR5 has a wide variety of potential variations. Ignoring this mosaic while working on genes could end up in any number of results, ranging from the neutral to the deeply harmful.

He’s lack of transparency means that “we dont know what has been done to the genes” of the two infants, Burgio says.

There also appear to have been significant problems with an important part of any study this risky-informed consent of the parents. The consent form that patients signed has come under stern criticism from other scientists, comparing it to a “business form, of the kind that a company might use when subcontracting” while downplaying any risks of the procedure.

“If this was a mouse,” Burgio says, “I would not be concerned. But were talking about kids.” When asked about He’s motivations, Burgio felt sure that He wanted “to be first” in making the discovery. When asked about the possibility that He was genuine in his concern for HIV patients, Burgio laughed, noting that there are far safer ways to treat the disease.

Since He’s appearance at the summit, he has not been seen. His university, where He has apparently been on leave since February, has disavowed knowledge of his work. A graduate of Rice University in Texas, He found a collaborator in a professor from the school, Michael Deem. Rice has released a statement declaring that the work “violates scientific conduct guidelines and is inconsistent with ethical norms of the scientific community and Rice University.

Source: SCMP

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The Infamous Scientist Behind the CRISPR Baby Gene Editing Is …

What Happens to the CRISPR Twins? Their Lives Will … – time.com

For now, theyre known as Lulu and Nana, pseudonyms that are meant to give them some amount of anonymity amid the international uproar over their birth. As the first babies born after their genomes were edited (while they were embryos, by the genetics tool CRISPR) the twin girls, born in Shenzhen, China, are the subject of scientific and public scrutiny that will only escalate as they get older.

He Jiankui, a professor at the Southern University of Science and Technology, stunned the world when he claimed, both in a video posted by his lab and in an interview with a journalist, that he used CRISPR to disable a gene involved in helping HIV to enter healthy cells. By doing so, he gave the resulting edited embryos, including the twin girls, resistance to the virus. Doing so means He violated current guidelines prohibiting using CRISPR on human embryos for pregnancy. For now, Hes claims are only claims, since he has not published his work in a scientific journal for others to review and validate. While he did present his findings at a conference a few days after his YouTube announcement, researchers can only take the data at face value. He says he plans to publish the data, but now that the report has been released to the public, its difficult to predict which journals would accept the manuscript.

The Chinese researchers university denied knowledge of his experiment and said that He has been on leave since last February. Chinese authorities have now suspended Hes work, and Xu Nanping, vice minister of Chinas Ministry of Science and Technology, said Hes study was abominable in nature and violated Chinese laws and regulations, according to the governments Xinhua news.

The reason for the scientific censure boils down to the fact that He preempted a continuing debate over how and when CRISPR should be used in people. The technology, discovered in 2012, provides unprecedented precision and power to edit any genome, including the DNA of people, by snipping out portions of mutated genes and either allowing the genome to repair itself or by providing healthy versions of the gene. But because the approach is relatively new, scientists are still learning about exactly how precise their edits can be, and what some of the potential negative and long term consequences of altering human DNA could be.

Chinese geneticist He Jiankui of the Southern University of Science and Technology in Shenzhen, China, speaking during the Second International Summit on Human Genome Editing at the University of Hong Kong.

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Nearly all international genetics groups have guidelines prohibiting using CRISPR to edit human embryos and implanting them for pregnancy, as the Chinese researcher did. Experts fully support using CRISPR in cells that cant be passed down from generation to generation, like skin cells or blood cells.

But what He did will forever change the twins DNA. Because he altered their genomes when they were embryos, those changes were picked up by every new cell that the embryos made as they continued to divide and develop, eventually forming the twins. So when the girls are ready to have children, their eggs may contain the CRISPR edits that He gave them, and they could pass on their altered genes to their children and all future generations of children in their lineage.

Having the gene itself is not necessarily a bad thing the edit He made is meant to protect people from getting infected with HIV but the problem is that scientists arent convinced yet that the HIV protection will be the only thing the CRISPR edit did to the twins genomes.

Its not clear, for example, that CRISPR is as precise as researchers would like it to be. It makes mistakes. In some cases, CRISPR may make unintended changes in random parts of the genome, like an autocorrect feature that mistakenly corrects typos to produce an entirely different word. In other cases, it may not make the edits as consistently as needed, so some cells may be edited while others are not, and some cells may even be partially edited, leaving a patchwork result scientists call mosaicism.

According to experts who reviewed some of the data He presented at a conference days after his stunning announcement, they say there is evidence that both girls born with the CRISPR edits showed such signs of mosaicism when they were embryos, meaning they are now likely to have the same mishmash of CRISPRd and unCRISPRd cells in their bodies. That means that they may not even benefit from the resistance to HIV that Hes grand experiment was meant to provide.

Theres also evidence that compromising the HIV gene may have other consequences for example, making people more susceptible to West Nile Virus and possibly the flu.

Its because of these unanswered questions and potential risks that scientists have favored a moratorium on using CRISPR in human embryos meant for pregnancy, at least until they have a better grasp on how CRISPR works and what some of the long term effects of editing might be. While the U.S. National Academy of Sciences in 2017 allowed for the eventual possibility of human babies whose genomes have been edited by CRISPR, it provided strict criteria for how that should happen: under strict monitoring and only in cases where there is no other medical option.

Neither of those criteria were met in the controversial CRISPR study. The university and the hospital where the births took place denied knowledge of Hes work, and the scientific community was blindsided that he had been proceeding with transferring human embryos for pregnancy. The gene he altered also does not represent an unmet medical need among the couples he worked with, only the fathers were HIV positive, meaning they were unlikely to pass on their infection to their children. Whats more, the fathers were on anti-HIV medications, which controlled their infection and make it even less likely they would infect their partners or their children.

In the twins case, what happens when they want to have children? Will they be allowed to have children naturally, and pass on their edited genes and whatever potential side effects might arise from their altered DNA? Or will regulatory or scientific authorities step in and attempt to control whether their genes continue into future generations by requiring the twins to have IVF and only implanting the embryos that do not show signs of the edited gene? Would those regulatory and scientific bodies even have the right to make such a request?

The implications go beyond just these twins, says Dr. Kiran Musunuru, professor of cardiovascular medicine and genetics at University of Pennsylvania Perelman School of Medicine. If we talk about the sanctity of human life, and the inherent dignity of human life, not much has been gained here. These babies were treated as subjects in a grand medical experiment, and we have to believe that they will be studied for the rest of their lives; its sad actually.

In his presentation and in his video, He justified his unorthodox actions by focusing on the personal. He said the father of the twins now feels motivated to find work and care for his family, and that altering the gene will protect future generations from HIV. But HIV experts say that judicious use and distribution of currently available drugs can effectively stop transmission of the virus, without taking such drastic steps of trying an proven genetic procedure and exposing people to its unknown risks.

While their identities are still protected for now, its unlikely the twins will remain anonymous for long. In bypassing ethical guidelines prohibiting the experiment that he conducted, He not only violated basic tenets of responsible scientific inquiry, he also forever changed how the girls will be viewed by society, and ultimately the decisions they make as a result of their involuntary status as the worlds first CRISPR babies.

Contact us at editors@time.com.

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What Happens to the CRISPR Twins? Their Lives Will … – time.com

Rogue Scientist Says Another Crispr Pregnancy Is Underway

On the second day of the Second International Summit on Human Genome Editing, the last session before lunch was already running long. But the crowd crammed into the Lee Shau Kee Lecture Centre at the University of Hong Kong wasnt budging. Neither were the 5,500 people around the world glued to their live video feeds. Everyone was waiting to hear from the the final speaker, the man who says he helped make the worlds first gene-edited babies.

That man is He Jiankui, the Chinese-born, American-trained biophysicist who claims to have Crisprd a pair of twin baby girls.

Robin Lovell-Badge, a biologist at the Francis Crick Institute in the UK, took to the podium to introduce the controversial speaker. Lovell-Badge reminded everyone that the National Academy of the Sciences, the global non-governmental science panel that helped convene this summit, did not know in advance about Hes work. He sent me the slides he was going to show in this session and they did not include any of the work he was going to talk about, said Lovell-Badge. Nothing involving human embryos that were implanted.

But after MIT Technology Review broke the news of Hes covert trials two days ago, Hes session at this event became the object of intense fascination. Folks following along on Twitter wondered if He would show at all. And for one long, agonizing minute after Lovell-Badge welcomed He to the stage, it looked like he might not. When He at last appeared, he began to deliver a different talk, packed with details about what hed been up to.

For the last two years, He has been working in secret, skirting ethical and scientific codes of conduct, and possibly even some laws, to make biological history. On Wednesday morning, Hong Kong time, he revealed to the world just how he did it. It will take scientists days to parse the 59 data-dense slides that describe Hes methods and results. Only then will a fuller picture begin to emerge about just how safe and effective the experiment was. But in the meantime, He still gave the rest of us plenty to think about.

Like the fact that Lulu and Nana, the twin girls, arent the only children Hes group has Crisprd. When pressed on the number of implantations that have taken place so far, the scientist disclosed that there is another potential pregnancy involving a gene-edited embryo. He hesitated to answer the question because the pregnancy is in an early stage. His research team has so far injected Crispr systems into 31 embryos that have developed to the blastocyst stage. He said 70 percent of them were successfully edited and await further screening and implantation in five remaining couples. But now thats all on hold. The trial is paused due to the current situation, said He.

He is now under investigation by his own university, and other legal bodies in China.

After Hes presentation, he took questions from the audience and the moderators, including Lovell-Badge and Matthew Porteus, a Stanford researcher and the scientific founder of Crispr Therapeutics, a company developing Crispr-based drugs to treat genetic diseases. Throughout, He remained calm and thoughtful, if not always fully forthcoming.

At one point, Harvard biochemist David Liu questioned the unmet medical need that He said his experiments were addressing. He recruited couples where the mother is HIV-negative and the father HIV-positive, editing their embryos to bestow them with a rare but natural traitthe ability to resist HIV infections. Given that there are ways to make sure HIV-positive parents dont transmit their disease to their babies without altering their DNA, Liu asked He to describe the unmet medical need, not of HIV in general, but of these patients in particular.

He responded that his trial was not just for these few patients, but for the millions of children suffering from HIV all over the world. He described personal experience with a village in China where 30 percent of the residents are infected and children have to live with their relatives for fear of contracting the virus. I feel proud, actually, said He.

Not everyone agreed with Hes take. Between question and answer sessions, Nobel laureate and summit chair David Baltimore interjected to announce that the organizing committee would issue a formal statement regarding Hes work on Thursday. Baltimore then shared a few personal thoughts, including that the experiments as described do not meet the criteria of the National Academy of Sciences for a responsible application of human germline editing. Personally I dont think it was medically necessary, said Baltimore. I think there has been a failure of self-regulation by the scientific community because of a lack of transparency, he added.

Other members of the organizing committee were similarly skeptical. Having listened to Dr. He, I can only conclude that this was misguided, premature, unnecessary and largely useless, Alta Charo, a bioethicist at the University of Wisconsin-Madison wrote in an email to WIRED. Charo co-chaired the 2017 National Academies consensus study that laid out the criteria for an ethical path to human germline editing. Her greatest concern, she said, is that the consent forms that Hes patients signed created the impression that his project was an AIDS vaccine trial, and may have conflated research with therapy by claiming participants were likely to benefit.

As to the other embryos hes edited, which are on ice while the trial is itself frozen? What will happen to those embryos, or even who decides what happens, Charo says, is unknown.

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Rogue Scientist Says Another Crispr Pregnancy Is Underway

Before the Claims of Crispr Babies, There Was Chinas One …

In China, many people have already ventured into that terrain. Even before Crispr, it has been possible to create so-called designer babies using in vitro fertilization and selecting egg donors with desirable genetic enhancements, such as looks and intelligence. Thats what many wealthy Chinese have been doing for years. The practice is fairly standard among rich consumers of any nationality, but I was told by fertility clinics and doctors in California that Chinese customers were frequently the most upfront and demanding, driving up prices of East Asian donor eggs to twice and even triple market rates.

Wendie Wilson-Miller, who runs an egg donor agency in Southern California, told me that her Chinese clients almost always want taller, at least 5 foot 5. And they have questions about eyelids; they want to see baby pictures to see if the donors had eyelid surgery.

For years, B.G.I. Shenzhen, one of the worlds largest gene-sequencing facilities, has been running a project to explore the genetic basis for human intelligence, with the goal of eventually enabling parents to boost their offsprings I.Q. before birth. While it may not be possible to isolate human intelligence to a purely genetic component, the company clearly believes theres huge potential demand for such a service. One of its co-founders, Wang Jiang, recently caused a furor when he said in a speech that employees would not be allowed to have children with birth defects because they would be a disgrace.

No society is uniform, and news of the Crispr babies has generated much condemnation and outrage within China, particularly by Dr. Hes peers, who consider him an irresponsible rogue scientist. A top Chinese bioethicist, Qiu Renzong, compared his actions to using a cannon to shoot a bird.

But at the same time, a recent poll indicated wide support in China for gene editing to treat disease, with 24 percent in favor of legalizing gene editing for enhancing intelligence. By contrast, 68 percent of Americans say they are worried about gene editing and its effects, according to Pew Research.

Much is still unknown about the so-called Crispr babies. But it is almost certain that more will follow; Dr. He has already said his experiments have generated another pregnancy. It is also almost certain someone will attempt gene editing to make stronger, smarter, more attractive babies. Pandoras box is wide open in China.

Mei Fong, a Pulitzer Prize-winning journalist, is the author of One Child: The Story of Chinas Most Radical Experiment.

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Scientist Who Crisprd Babies Bucked His Own Ethics Policy

We said dont freak out, when scientists first used Crispr to edit DNA in non-viable human embryos. When they tried it in embryos that could theoretically produce babies, we said dont panic. Many years and years of boring bench science remain before anyone could even think about putting it near a womans uterus. Well, we might have been wrong. Permission to push the panic button granted.

Late Sunday night, a Chinese researcher stunned the world by claiming to have created the first human babies, a set of twins, with Crispr-edited DNA. Two beautiful little Chinese girls, Lulu and Nana, came crying into the world as healthy as any other babies a few weeks ago, the scientist, He Jiankui, said in the first of five promotional videos posted to YouTube hours after MIT Technology Review broke the news.

Lulu and Nana are reported to have a genetic mutation, courtesy of Crispr, that makes it harder for HIV to invade and infect their white blood cells. The claim, which has yet to be independently verified or backed up by published data, has ignited furious criticism, international outrage, and multiple investigations. The scientific outcry has been so swift because Hes purported work, conducted in secret, bulldozes past existing ethical guidance on so-called germline editing, in which alterations to an embryos DNA will be passed down to subsequent generations.

Whats perhaps most strange is not that He ignored global recommendations on conducting responsible Crispr research in humans. He also ignored his own advice to the worldguidelines that were published within hours of his transgression becoming public.

On Monday, He and his colleagues at Southern University of Science and Technology, in Shenzhen, published a set of draft ethical principles to frame, guide, and restrict clinical applications that communities around the world can share and localize based on religious beliefs, culture, and public-health challenges. Those principles included transparency and only performing the procedure when the risks are outweighed by serious medical need.

The piece appeared in the The Crispr Journal, a young publication dedicated to Crispr research, commentary, and debate. Rodolphe Barrangou, the journals editor in chief, where the peer-reviewed perspective appeared, says that the article was one of two that it had published recently addressing the ethical concerns of human germline editing, the other by a bioethicist at the University of North Carolina. Both papers authors had requested that their writing come out ahead of a major gene editing summit taking place this week in Hong Kong. When half-rumors of Hes covert work reached Barrangou over the weekend, his team discussed pulling the paper, but ultimately decided that there was nothing too solid to discredit it, based on the information available at the time.

Now Barrangou and his team are rethinking that decision. For one thing, He did not disclose any conflicts of interest, which is standard practice among respectable journals. Its since become clear that not only is He at the helm of several genetics companies in China, He was actively pursuing controversial human research long before writing up a scientific and moral code to guide it.Were currently assessing whether the omission was a matter of ill-management or ill-intent, says Barrangou, who added that the journal is now conducting an audit to see if a retraction might be warranted. Its perplexing to see authors submit an ethical framework under which work should be done on the one hand, and then concurrently do something that directly contravenes at least two of five of their stated principles.

One is transparency. Reporting by Tech Review and The Associated Press has raised questions about whether He misled trial participants and Chinese regulators in his ambitions to make the first Crisprd baby. Two is medical necessity.

Take the gene Hes group chose to edit: CCR5. It codes for a receptor that HIV uses to infiltrate white blood cells, like a key to a locked door. No key, no access. Other controversial Crispr firsts have attempted to correct faulty versions of genes responsible for inherited, often incurable disorders, reverting them back to the healthy version. In contrast, Hes group crippled normal copies of CCR5 to lower the risk of future possible infection with HIVa disease that is easily prevented, treated, and controlled by means that dont involve forever changing someones DNA. Drugs, condoms, needle-exchange programs are all reasonable alternatives.

There are all sorts of questions these issues raise, but the most fundamental is the risk-benefit ratio for the babies who are going to be born, says Hank Greely, an ethicist at Stanford University. And the risk-benefit ratio on this stinks. Any institutional review board that approved it should be disbanded if not jailed.

Reporting by Stat indicates that He may have just gotten in over his head and tried to cram a self-guided ethics education into a few short months. The young scientistrecords indicate He is just 34has a background in biophysics, with stints studying in the US at Rice University and in bioengineer Stephen Quakes lab at Stanford. His resume doesnt read like someone steeped deeply in the nuances and ethics of human research. Barrangou says that came across in the many rounds of edits Hes framework went through. The editorial team did spend a significant amount of time improving both the language and the content, he says.

Its too soon to say whether Hes stunt will bring him fame or just infamy. Hes still scheduled to speak at the human genome editing summit on Wednesday and Thursday. And Chinas central government in Beijing has yet to come down one way or another. Condemnation would make He a rogue and a scientific outcast. Anything else opens the door for a Crispr IVF cottage industry to emerge in China and potentially elsewhere. Its hard to imagine this was the only group in the world doing this, says Paul Knoepfler, a stem cell researcher at UC Davis who wrote a book on the future of designer babies called GMO Sapiens. Some might say this broke the ice. Will others forge ahead and go public with their results or stop what theyre doing and see how this plays out?

What happens next makes all the difference. The fact that two babies now exist with one gene changed by Crispr to a less common form doesnt change the world overnight. What changes the world is how society reacts, and whether it decides to let such DNA-altering procedures become common.

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Scientist Who Crisprd Babies Bucked His Own Ethics Policy

Chinese Scientist Claims to Use Crispr to Make First …

Ever since scientists created the powerful gene editing technique Crispr, they have braced apprehensively for the day when it would be used to create a genetically altered human being. Many nations banned such work, fearing it could be misused to alter everything from eye color to I.Q.

Now, the moment they feared may have come. On Monday, a scientist in China announced that he had created the worlds first genetically edited babies, twin girls who were born this month.

The researcher, He Jiankui, said that he had altered a gene in the embryos, before having them implanted in the mothers womb, with the goal of making the babies resistant to infection with H.I.V. He has not published the research in any journal and did not share any evidence or data that definitively proved he had done it.

But his previous work is known to many experts in the field, who said many with alarm that it was entirely possible he had.

Its scary, said Dr. Alexander Marson, a gene editing expert at the University of California in San Francisco.

While the United States and many other countries have made it illegal to deliberately alter the genes of human embryos, it is not against the law to do so in China, but the practice is opposed by many researchers there. A group of 122 Chinese scientists issued a statement calling Dr. Hes actions crazy and his claims a huge blow to the global reputation and development of Chinese science.

If human embryos can be routinely edited, many scientists, ethicists and policymakers fear a slippery slope to a future in which babies are genetically engineered for traits like athletic or intellectual prowess that have nothing to do with preventing devastating medical conditions.

While those possibilities might seem far in the future, a different concern is urgent and immediate: safety. The methods used for gene editing can inadvertently alter other genes in unpredictable ways. Dr. He said that did not happen in this case, but it is a worry that looms over the field.

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Dr. He made his announcement on the eve of the Second International Summit on Human Genome Editing in Hong Kong, saying that he had recruited several couples in which the man had H.I.V. and then used in vitro fertilization to create human embryos that were resistant to the virus that causes AIDS. He said he did it by directing Crispr-Cas9 to deliberately disable a gene, known as CCR, that is used to make a protein H.I.V. needs to enter cells.

Dr. He said the experiment worked for a couple whose twin girls were born in November. He said there were no adverse effects on other genes.

In a video that he posted, Dr. He said the father of the twins has a reason to live now that he has children, and that people with H.I.V. face severe discrimination in China.

Dr. Hes announcement was reported earlier by the MIT Technology Review and The Associated Press.

In an interview with the A.P. he indicated that he hoped to set an example to use genetic editing for valid reasons. I feel a strong responsibility that its not just to make a first, but also make it an example, he told the A.P. He added: Society will decide what to do next.

It is highly unusual for a scientist to announce a groundbreaking development without at least providing data that academic peers can review. Dr. He said he had gotten permission to do the work from the ethics board of the hospital Shenzhen Harmonicare, but the hospital, in interviews with Chinese media, denied being involved. Cheng Zhen, the general manager of Shenzhen Harmonicare, has asked the police to investigate what they suspect are fraudulent ethical review materials, according to the Beijing News.

The university that Dr. He is attached to, the Southern University of Science and Technology, said Dr. He has been on no-pay leave since February and that the school of biology believed that his project is a serious violation of academic ethics and academic norms, according to the state-run Beijing News.

In a statement late on Monday, Chinas national health commission said it has asked the health commission in southern Guangdong province to investigate Mr. Hes claims.

Many scientists in the United States were appalled by the developments.

I think thats completely insane, said Shoukhrat Mitalipov, director of the Center for Embryonic Cell and Gene Therapy at Oregon Health and Science University. Dr. Mitalipov broke new ground last year by using gene editing to successfully remove a dangerous mutation from human embryos in a laboratory dish.

Dr. Mitalipov said that unlike his own work, which focuses on editing out mutations that cause serious diseases that cannot be prevented any other way, Dr. He did not do anything medically necessary. There are other ways to prevent H.I.V. infection in newborns.

Just three months ago, at a conference in late August on genome engineering at Cold Spring Harbor Laboratory in New York, Dr. He presented work on editing the CCR gene in the embryos of nine couples.

At the conference, whose organizers included Jennifer Doudna, one of the inventors of Crispr technology, Dr. He gave a careful talk about something that fellow attendees considered squarely within the realm of ethically approved research. But he did not mention that some of those embryos had been implanted in a woman and could result in genetically engineered babies.

What we now know is that as he was talking, there was a woman in China carrying twins, said Fyodor Urnov, deputy director of the Altius Institute for Biomedical Sciences and a visiting researcher at the Innovative Genomics Institute at the University of California. He had the opportunity to say Oh and by the way, Im just going to come out and say it, people, theres a woman carrying twins.

I would never play poker against Dr. He, Dr. Urnov quipped.

Richard Hynes, a cancer researcher at the Massachusetts Institute of Technology, who co-led an advisory group on human gene editing for the National Academy of Sciences and the National Academy of Medicine, said that group and a similar organization in Britain had determined that if human genes were to be edited, the procedure should only be done to address serious unmet needs in medical treatment, it had to be well monitored, it had to be well followed up, full consent has to be in place.

It is not clear why altering genes to make people resistant to H.I.V. is a serious unmet need. Men with H.I.V. do not infect embryos. Their semen contains the virus that causes AIDS, which can infect women, but the virus can be washed off their sperm before insemination. Or a doctor can inject a single sperm into an egg. In either case, the woman will not be infected and neither will the babies.

Dr. He got his Ph.D., from Rice University, in physics and his postdoctoral training, at Stanford, was with Stephen Quake, a professor of bioengineering and applied physics who works on sequencing DNA, not editing it.

Experts said that using Crispr would actually be quite easy for someone like Dr. He.

After coming to Shenzhen in 2012, Dr. He, at age 28, established a DNA sequencing company, Direct Genomics, and listed Dr. Quake on its advisory board. But, in a telephone interview on Monday, Dr. Quake said he was never associated with the company.

Austin Ramzy contributed reporting from Hong Kong and Elsie Chen contributed research from Beijing.

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Chinese Scientist Claims to Use Crispr to Make First …

CRISPR babies: new details on the experiment that shocked …

He Jiankui at the Human Genome Editing Conference in Hong Kong

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By Michael Le Page

On Monday, the world was stunned by an Associated Press story claiming that the first gene-edited babies had been born in China. On Wednesday, the scientist responsible revealed far more details during a talk at a gene-editing summit in Hong Kong, including that there is another pregnancy.

There hasnt yet been any independent verification that two gene-edited girls really have been born. But the technical details revealed by He Jiankui today may have been enough to convince many of the scientists in attendance. However, questions still remain over the ethicsand safety of the experiment.

The stated aim of theproject was to make individuals immune to HIV by disabling the gene for a protein called CCR5, which is exploited by the virus. However, disabling this gene does not provide complete protection against HIV and the broader consequences of knocking out this gene which is involved in immune function are unclear.

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The team began by using the CRISPR gene editing method to disable CCR5 in mice and monkeys, He said, and found no health or behavioural issues. But one of the organisers of the summit, Robin Lovell-Badge of the Francis Crick Institute in London, pointed out that immune genes affect the entire body, and that a different mouse study found that deleting CCR5improved their cognitive abilities.

Have you inadvertently caused an enhancement? Lovell-Badge asked He after the talk. The mouse study needed verification, He replied. I am against using genome editing for enhancement.

Another big safety issue is off-target effects the risk that CRISPRcausesunintended, harmful mutations elsewhere in the genome. To try to prevent this, Hes team sequenced the entire genomes of both parents. They then removed 3 to 5 cells from each of the edited embryos before implantation in the mother and fully sequenced them, too, to check for unwanted mutations.

Comparing the genomes revealed several new mutations in the two edited embryos for which resultshave been released. Only one of these mutations found in the embryo of the girl nicknamed Lulu might be due to CRISPR, He concluded. Its possible that this may be the case, because every individual has up to 100 new mutations by chance anyway.

The possible off-target mutation was judged by the team to be harmless because itisin a region of DNA that is far from any genes. According to the slides He presented, the parents were told about it and decided to proceed.

But CRISPR expert Gaetan Burgio of the Australian National University tweeted thatthe checks for off-target mutations were not good enough. For instance, they would not have detected any very large deletions of DNA, he said.

The final big safety issue with using CRISPR on embryos is something called mosaicism. If the eggs started dividing before the gene editing took place, the twin girls might have a mixture of cells with and without the edit. Whether they do or not was unclear from Hes talk.

Tests on the placenta and umbilical cord blood and tissue found exactly the same mutations in each sample for both twins, the slides reveal. But the potential off-target mutation was found only in the cells taken from the embryo and not in later samples, which does imply mosaicism. And Burgio told New Scientist that the results suggest both twins are mosaics. I cant believe they went ahead and implanted the embryos, he says.

Mosaicism is an issue for two reasons. Firstly, if an embryo is a mosaic then removing a few cells for testing is not enough to check the health and status of an embryo. Secondly, if Lulus immune cells developed from non-edited cells, they would still be completely vulnerable to HIV.

We know the other twin, Nana, is definitely still completely vulnerable to HIV. She has a 15-DNA-letter long deletion in one of the two copies of the CCR5 gene that probably will not be enough to disable the protein. And the other copy was not edited at all.

Questions had been raised over why Nanas embryo was implanted at all, but He said the parents were informed and decided to implant it.

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CRISPR babies: new details on the experiment that shocked …

EXCLUSIVE: Chinese scientists are creating CRISPR babies …

When Chinese researchers first edited the genes of a human embryo in a lab dish in 2015, it sparked global outcry and pleas from scientists not to make a baby using the technology, at least for the present.

It was the invention of a powerful gene-editing tool, CRISPR, which is cheap and easy to deploy,that made the birth of humans genetically modified in an in vitro fertilization (IVF) center a theoretical possibility.

Now, it appears it may already be happening.

According to Chinese medical documents posted online this month (hereand here), a team at the Southern University of Science and Technology, in Shenzhen, has been recruiting couples in an effort to create the first gene-edited babies. They planned to eliminate a gene called CCR5 inhopes of rendering the offspring resistant to HIV, smallpox, and cholera.

Southern University of Science and Technology

The clinical trial documents describe a studyin which CRISPR is employed to modify human embryosbefore they are transferred into womens uteruses.

The scientist behind the effort, He Jiankui, did not reply to a list of questions about whether the undertaking had produced a live birth. Reached by telephone, he declined to comment.

However, data submitted as part of the trial listing shows that genetic tests have been carried out on fetuses as late as 24 weeks, or six months. Its not known if those pregnancies were terminated, carried to term, or are ongoing.

[After this story was published, the Associated Press reported that according to He, onecouple in the trialgave birth to twingirls this month,though the agency wasn’t able to confirm his claim independently. He also released a promotional video about his project.]

The birth of the first genetically tailored humans would be a stunning medical achievement, for both He and China. But it will prove controversial, too. Where some see anew form of medicinethat eliminates genetic disease, others see a slippery slope to enhancements, designer babies, and a new form of eugenics.

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The step toward genetically tailored humans was undertaken in secrecy and with the clear ambition of a stunning medical first.

In this ever more competitive global pursuit of applications for gene editing, we hope to be a stand-out, He and his team wrote in an ethics statement they submitted last year. They predicted their innovation will surpass the invention of in vitro fertilization, whose developer was awarded a Nobel Prize in 2010.

Gene-editing summit

Theclaim that China has already made genetically altered humans comes just as the worlds leading experts are jetting into Hong Kong for the Second International Summit on Human Genome Editing.

The purpose of the international meetingis to help determine whether humans should begin to genetically modify themselves, and if so, how. That purpose now appears to have been preempted by the actions of He, an elite biologist recruited back to China from the US as part of its Thousand Talents Plan.

The technology is ethically charged because changes to an embryo would be inherited by future generations and could eventually affect the entire gene pool.We have never done anything that will change the genes of the human race, and we have never done anything that will have effects that will go on through the generations, David Baltimore, a biologist and former president of the California Institute of Technology, who chairs the international summit proceedings, said in a pre-recorded message ahead of the event, which begins Tuesday, November 27.

It appears the organizers of the summit were also kept in the dark about Hes plans.

Regret and concern

The genetic editing of a speck-size human embryo carries significant risks, including the risks of introducing unwanted mutationsor yielding a baby whose body is composed of some edited and some unedited cells. Data on the Chinese trial site indicate that one of the fetuses is a mosaic of cells that had been edited in different ways.

A gene-editing scientist, Fyodor Urnov, associate director of the Altius Institute for Biomedical Sciences, a nonprofit in Seattle, reviewed the Chinese documents and said that, while incomplete, they do show that this effort aims to produce a human with altered genes.

Urnov called the undertaking cause for regret and concern over the fact that gene editinga powerful and useful techniquewas put to use in a setting where it was unnecessary. Indeed, studies are already under way to edit the same gene in the bodies of adults with HIV. It is a hard-to-explain foray into human germ-line genetic engineering that may overshadow in the mind of the public a decade of progress in gene editing of adults and children to treat existing disease, he says.

Big project

In a scientific presentation in 2017 at Cold Spring Harbor Laboratory, which is posted to YouTube, He described a very large series of preliminary experiments on mice, monkeys, and more than 300 human embryos. One risk of CRISPR is that it can introduce accidental or off target mutations. But He claimed he found few or no unwanted changes in the test embryos.

He is also the chairman and founder of a DNA sequencing company called Direct Genomics. A new breed of biotech companies could ultimately reap a windfall should the new methodsof conferring health benefits on children be widely employed.

The National Academies of Science, Engineering and Medicine

According to the clinical trial plan, genetic measurements would be carried out on embryos and would continue during pregnancy to check on the status of the fetuses. During his 2017 presentation, He acknowledged that if the first CRISPR baby were unhealthy, it could prove a disaster.

We should do this slow and cautious, since a single case of failure could kill the whole field, he said.

A listing describing the study was posted in November, but other trial documents are dated as early as March of 2017. That was only a month after the National Academy of Sciences in the US gave guarded support for gene-edited babies, although only if they could be created safely and under strict oversight.

Currently, using a genetically engineered embryo to establish a pregnancy would be illegal in much of Europe and prohibited in the United States. It is also prohibited in China under a 2003 ministerial guidance to IVF clinics. It is not clear if He got special permission or disregarded the guidance, which may not have the force of law.

Public opinion

In recent weeks, He has begun an active outreach campaign, speaking to ethics advisors, commissioning an opinion poll in China, and hiring an American public-relations professional, Ryan Ferrell.

My sense is that the groundwork for future self-justification is getting laid, says Benjamin Hurlbut, a bioethicist from Arizona State University who will attend the Hong Kong summit.

The new opinion poll, which was carried out by Sun Yat-Sen University, found wide support for gene editing among the sampled 4,700 Chinese, including a group of respondentswho were HIV positive. More than 60% favored legalizing edited children if the objective was to treat or prevent disease. (Polls by the Pew Research Center have found similar levels support in the US for gene editing.)

Hes choice to edit the gene called CCR5 could prove controversial as well. People without working copies of the gene are believed to be immune or highly resistant to infection by HIV. In order to mimic the same result in embryos, however, Hes team has been using CRISPR to mutate otherwise normal embryos to damage the CCR5 gene.

The attempt to create children protected from HIV also falls into an ethical gray zone between treatment and enhancement. That is because the procedure does not appear to cure any disease or disorder in the embryo, but instead attempts to create a health advantage, much as a vaccine protects against chicken pox.

For the HIV study, doctors and AIDS groups recruited Chinese couples in which the man was HIV positive. The infection has been a growing problem in China.

So far, experts have mostly agreed that gene editing shouldnt be used to make designer babies whose physical looks or personality has been changed.

He appeared to anticipate the concerns his study could provoke. I support gene editing for the treatment and prevention of disease, He posted in November to the social media site WeChat, but not for enhancement or improving I.Q., which is not beneficial to society.

Still, removing the CCR5 gene to create HIV resistance may not present a particularly strong reason to alter a babys heredity. There are easier, less expensive ways to prevent HIV infection. Also,editing embryos during an IVF procedure would be costly, high-tech, and likely to remain inaccessible in many poor regions of the world where HIV is rampant.

A person who knows He said his scientific ambitions appear to be in line with prevailing social attitudes in China, including the idea that the larger communal good transcends individual ethics and even international guidelines.

Behind the Chinese trial also lies some bold thinking about how evolution can be shaped by science. While the natural mutation that disables CCR5 is relatively common in parts of Northern Europe, it is not found in China. The distribution of the genetic trait around the worldin some populations but not in othershighlights how genetic engineering might be used to pick the most useful inventions discovered by evolution over the eons in different locations and bring them together in tomorrows children.

Such thinking could, in the future, yield people who have only the luckiest genes and never suffer Alzheimers, heart disease, or certain infections.

The text of an academic website that He maintains shows that he sees the technology in the same historic, and transformative, terms. For billions of years, life progressed according to Darwins theory of evolution, it states. More recently, industrialization has changed the environment in radical ways posing a great challenge that humanity can meet with powerful tools to control evolution.

It concludes: By correcting the disease genes we human[s] can better live in the fast-changing environment.

Note: This story was updated after publication to include claims by He Jiankui thatthe trial had produced live births.

Read more:
EXCLUSIVE: Chinese scientists are creating CRISPR babies …

Chinese University ‘Shocked’ By News of First CRISPR …

Its not the usual way that reputable scientists announce their breakthroughs to the world, but on Monday, Jiankui He released a video proclaiming that he had produced the worlds first human babies whose genomes were edited using the powerful technique called CRISPR. He had also previously spoken with the Associated Press about his study, which he says resulted in twin girls born with the first genomes edited by man.

The report was met with instant concern and skepticism by the scientific community. Hes experiment altered the genomes of embryos produced through IVF; their genetic changes will therefore be passed on to any future generations. Whats more, most experts in CRISPR are not convinced that the technology is ready or safe for treating humans.

Given the current early state of genome editing technology, Im in favor of a moratorium on implantation of edited embryos until we have come up with a thoughtful set of safety requirements first, Feng Zhang, one of the co-discoverers of CRISPR and from the Broad Institute of MIT and Harvard, said in a statement responding to the report. Not only do I see this as risky, but I am also deeply concerned about the lack of transparency surrounding this trial.

In 2015, prominent members of the scientific community familiar with the technology, including Zhang and another co-discoverer, Jennifer Doudna from University of California, Berkeley, agreed to voluntarily stop research on using CRISPR in human embryos because the safety and long term consequences of the technology were too uncertain. The researchers support studies in which CRISPR is used to develop treatments that would affect cells that arent passed on to the next generation i.e. anything except egg and sperm but say that more research is needed before CRISPR is used to make changes in genomes that can be carried by generation after generation.

While editing the DNA of a human embryo is not currently allowed in the U.S., in 2017, an international committee of the National Academy of Sciences called for loosening the moratorium and allowing trials of CRISPR in human embryos, under strict oversight, to treat rare genetic diseases that cant be addressed in any other way. In the U.K., officials approved studies of CRISPR in human embryos in 2016, but those embryos will not be transplanted to create a pregnancy. Those trials call for destroying the embryos after a week, since the technologys safety remains unclear.

He, on the other hand, has apparently jumped ahead to producing the first human babies born with CRISPR editing. He is on the faculty of Southern University of Science and Technology in Shenzhen China, but in a statement released in response to Hes videos, the university said he is on unpaid leave from February 2018 to January 2021; officials did not provide a reason for the leave.

The University was deeply shocked by this event and has taken immediate action to reach Dr. Jiankui He for clarification, the officials said in the statement. The research was conducted outside of the campus and was not reported to the University nor the Department [to which He belongs]. The statement went on to note that the university believes that Dr. Jiankui Hes conduct in utilizing CRISPR/Cas9 to edit human embryos has seriously violated academic ethics and codes of conduct The University will call for international experts to form an independent committee to investigate this incident, and to release the results to the public.

CRISPR, first described in 2012, gives scientists the most precise and effective way to edit the human genome by snipping out offending mutations or genes and either allowing the genome to repair itself or providing researchers with the ability to insert new genetic material to correct disease genes. But studies suggest that controlling CRISPR in human cells remains a challenge; in some cases CRISPR may cut unintended parts of the genome.

In his promotional video, He describes targeting the CCR5 gene, which helps the HIV virus enter healthy human cells. He worked with seven heterosexual couples in which the male partner was HIV positive and the women were HIV negative. After the couples produced embryos through IVF, he used CRISPR to cut the CCR5 gene, disabling it in the hopes of making the embryos less vulnerable to HIV infection. He claims that of 22 embryos, 16 showed signs of successful CRISPR editing, and 11 were implanted, resulting in a single pregnancy with twin girls who were born in November. One twin, according to Hes tests, showed signs that both copies of the CCR5 gene it inherited (one from its mother and one from its father) were successfully altered, while the other twin showed that one version of the gene it inherited was altered.

That so-called mosaicism, in which some but not all of the embryos cells are altered, is troubling since in this case, it would mean that girl may not be entirely protected from HIV infection like her sister. Thats one of the reasons why researchers are concerned about the report. Normally such scientific milestones are reported in scientific journals complete with detailed descriptions of how the researcher accomplished the feat along with data supporting their claims. Without such documentation, its impossible to verify whether the girls indeed showed successful CRISPR editing or not.

He, who created two companies based on his studies, is scheduled to present his findings at the Second International Summit on Human Genome Editing, and will certainly be the target of numerous questions from the leading gene-editing scientists in attendance.

Contact us at editors@time.com.

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Chinese University ‘Shocked’ By News of First CRISPR …

CRISPR – Simple English Wikipedia, the free encyclopedia

CRISPR is a term used in microbiology. It stands for Clustered Regularly-Interspaced Short Palindromic Repeats. These are a natural segment of the genetic code found in prokaryotes: most bacteria and archaea have it.[1]

CRISPR has a lot of short repeated sequences. These sequences are part of an adaptive immune system for prokaryotes. It allows them to remember and counter other organisms that prey on them, such as bacteriophages.

They have the potential to modify the genes of almost any organism. They are part of a tool that allows precisely targeted cutting and insertion of genes in genetic modification (GM). Work is under way to find how they can be used to attack virus diseases in humans.[2]

Each repetition is followed by short segments of “spacer DNA” from previous exposures to a bacterial virus or plasmid.[2] CRISPR spacers recognize and cut up the foreign genetic elements in a manner like RNA interference in eukaryotic organisms.

In effect, the spacers are fragments of DNA from viruses that have previously tried to attack the cell line. The foreign source of the spacers was a sign to researchers that the CRISPR/cas system could have a role in adaptive immunity in bacteria.[3]

The actual cutting is done by a nuclease called Cas9. Cas9 has two active cutting sites, one for each strand of the DNA’s double helix. Cas9 does this by unwinding foreign DNA and checking whether it is complementary to the 20 basepair spacer region of the guide RNA (the spacer region RNA). If it is, the foreign DNA gets chopped up.

The technology has been used to switch off genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuel and to genetically modify crop strains.[4]

See original here:
CRISPR – Simple English Wikipedia, the free encyclopedia

CRISPR NIH Director’s Blog

Posted on September 11th, 2018 by Dr. Francis Collins

Caption: A CRISPR/cas9 gene editing-based treatment restored production of dystrophin proteins (green) in the diaphragm muscles of dogs with Duchenne muscular dystrophy.Credit: UT Southwestern

CRISPR and other gene editing tools hold great promise for curing a wide range of devastating conditions caused by misspellings in DNA. Among the many looking to gene editing with hope are kids with Duchenne muscular dystrophy (DMD), an uncommon and tragically fatal genetic disease in which their musclesincluding skeletal muscles, the heart, and the main muscle used for breathinggradually become too weak to function. Such hopes were recently buoyed by a new study that showed infusion of the CRISPR/Cas9 gene editing system could halt disease progression in a dog model of DMD.

As seen in the micrographs above, NIH-funded researchers were able to use the CRISPR/Cas9 editing system to restore production of a critical protein, called dystrophin, by up to 92 percent in the muscle tissue of affected dogs. While more study is needed before clinical trials could begin in humans, this is very exciting news, especially when one considers that boosting dystrophin levels by as little as 15 percent may be enough to provide significant benefit for kids with DMD.

Posted In: News

Tags: animal models, beagles, Cavalier King Charles Spaniel, CRISPR, CRISPR/Cas9, diaphragm muscle, DMD, dogs, Duchenne muscular dystrophy, dystrophin, gene editing, genetic diseases, heart, muscle, muscular dystrophy, rare diseases, Somatic Cell Genome Editing

Posted on October 10th, 2017 by Dr. Francis Collins

About a month ago, I had the pleasure of welcoming the Juip (pronounced Yipe) family from Michigan to NIH. Although youd never guess it from this photo, two of the Juips five children9-year-old Claire and 11-year-old Jake (both to my left)have a rare genetic disease called Friedreichs ataxia (FA). This inherited condition causes progressive damage to their nervous systems and their hearts. No treatment currently exists for kids like Claire and Jake, yet this remarkable family has turned this serious health challenge into an opportunity to raise awareness about the need for biomedical research.

One thing that helps keep the Juips optimistic is the therapeutic potential of CRISPR/Cas9, an innovative gene editing systemthat may someday make it possible to correct the genetic mutations responsible for FA and many other conditions. So, Im sure the Juips were among those encouraged by the recent news that NIH-funded researchers have developed a highly versatile approach to CRISPR/Cas9-based therapies. Instead of relying on viruses to carry the gene-editing system into cells, the new approach uses tiny particles of gold as the delivery system!

Posted In: Health, Science, technology, Uncategorized

Tags: CRISPR, CRISPR-Gold, CRISPR/Cas9, DMD, Duchenne muscular dystrophy, dystrophin, FA, Friedreichs ataxia, gene editing, Juip, rare diseases, stem cells

Posted on July 18th, 2017 by Dr. Francis Collins

Credit: Seth Shipman, Harvard Medical School, Boston

Theres a reason why our cells store all of their genetic information as DNA. This remarkable molecule is unsurpassed for storing lots of data in an exceedingly small space. In fact, some have speculated that, if encoded in DNA, all of the data ever generated by humans could fit in a room about the size of a two-car garage and, if that room happens to be climate controlled, the data would remain intact for hundreds of thousands of years! [1]

Scientists have already explored whether synthetic DNA molecules on a chip might prove useful for archiving vast amounts of digital information. Now, an NIH-funded team of researchers is taking DNAs information storage capabilities in another intriguing direction. Theyve devised their own code to record information not on a DNA chip, but in the DNA of living cells. Already, the team has used bacterial cells to store the data needed to outline the shape of a human hand, as well the data necessary to reproduce five frames from a famous vintage film of a horse galloping (see above).

But the researchers ultimate goal isnt to make drawings or movies. They envision one day using DNA as a type of molecular recorder that will continuously monitor events taking place within a cell, providing potentially unprecedented looks at how cells function in both health and disease.

Posted In: Health, Science, Video

Tags: biosensor, biotechnology, Cas1, Cas2, CRISPR, CRISPR-Cas, DNA, DNA movie, DNA storage, E. coli, film, gene editing, genomics, Human and Animal Locomotion, imaging, information storage, molecular recorder, movie, spacers

Posted on May 4th, 2017 by Dr. Francis Collins

Jesse Dixon

As a kid, Jesse Dixon often listened to his parents at the dinner table discussing how to run experiments and their own research laboratories. His father Jack is an internationally renowned biochemist and the former vice president and chief scientific officer of the Howard Hughes Medical Institute. His mother Claudia Kent Dixon, now retired, did groundbreaking work in the study of lipid molecules that serve as the building blocks of cell membranes.

So, when Jesse Dixon set out to pursue a career, he followed in his parents footsteps and chose science. But Dixon, a researcher at the Salk Institute, La Jolla, CA, has charted a different research path by studying genomics, with a focus on understanding chromosomal structure. Dixon has now received a 2016 NIH Directors Early Independence Award to study the three-dimensional organization of the genome, and how changes in its structure might contribute to diseases such as cancer or even to physical differences among people.

Posted In: Health, Science

Tags: 2016 NIH Directors Early Independence Award, 3D genome structure, chromatin, chromatin structure, CRISPR, CRISPR/Cas9, DNA, DNA packaging, ENCODE, Encyclopedia of DNA Elements, enhancer, gene editing, genome, genomics, histones, TAD, topologically associated domains

Posted on January 24th, 2017 by Dr. Francis Collins

Caption: This image represents an infection-fighting cell called a neutrophil. In this artists rendering, the cells DNA is being edited to help restore its ability to fight bacterial invaders.Credit: NIAID, NIH

For gene therapy research, the perennial challenge has been devising a reliable way to insert safely a working copy of a gene into relevant cells that can take over for a faulty one. But with the recent discovery of powerful gene editing tools, the landscape of opportunity is starting to change. Instead of threading the needle through the cell membrane with a bulky gene, researchers are starting to design ways to apply these tools in the nucleusto edit out the disease-causing error in a gene and allow it to work correctly.

While the research is just getting under way, progress is already being made for a rare inherited immunodeficiency called chronic granulomatous disease (CGD). As published recently in Science Translational Medicine, a team of NIH researchers has shown with the help of the latest CRISPR/Cas9 gene-editing tools, they can correct a mutation in human blood-forming adult stem cells that triggers a common form of CGD. Whats more, they can do it without introducing any new and potentially disease-causing errors to the surrounding DNA sequence [1].

When those edited human cells were transplanted into mice, the cells correctly took up residence in the bone marrow and began producing fully functional white blood cells. The corrected cells persisted in the animals bone marrow and bloodstream for up to five months, providing proof of principle that this lifelong genetic condition and others like it could one day be cured without the risks and limitations of our current treatments.

Posted In: Health, Science

Tags: adult stem cells, bacteria, CGD, chronic granulomatous disease, clinical trials, CRISPR, CRISPR-Cas, CRISPR/Cas9, DNA editing, fungi, gene therapy, genetics, hematopoietic stem cells, immunodeficiency, immunology, infectious disease, inherited immuodeficiency, neutrophil, rare disease, translational medicine, X chromosome, X-linked chronic granulomatous disease

Originally posted here:
CRISPR NIH Director’s Blog

What is CRISPR?

In this video Paul Andersen explains how the CRISPR/Cas immune system was identified in bacteria and how the CRISPR/Cas9 system was developed to edit genomes.

Do you speak another language? Help me translate my videos:http://www.bozemanscience.com/transla…

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Intro Title: I4dsong_loop_main.wavArtist: CosmicDLink to sound: http://www.freesound.org/people/Cosmi…Creative Commons Atribution License

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All of the images are licensed under creative commons and public domain licensing:Adenosine. (2009). English: Artistic rendering of a T4 bacteriophage. The colours grey and orange do not signify anything, they are just used to illustrate structure. Created for Wikipedia. Retrieved from https://commons.wikimedia.org/wiki/Fi…E. coli Bacteria. (n.d.). Retrieved February 17, 2016, from https://www.flickr.com/photos/niaid/1…Fioretti, B. F. Hallbauer &. (2015). English: Director, Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology. Visiting professor The Laboratory for Molecular Infection Medicine Sweden MIMS; http://www.mpiib-berlin.mpg.de/resear…. Retrieved from https://commons.wikimedia.org/wiki/Fi…Foresman, P. S. ([object HTMLTableCellElement]). English: Line art drawing of a chimera. Retrieved from https://commons.wikimedia.org/wiki/Fi…Magladem96. (2014). English: Picture of DNA Base Flipping. Retrieved from https://commons.wikimedia.org/wiki/Fi…project, C. wiki. (2014). English: Crystal Structure of Cas9 bound to DNA based on the Anders et al 2014 Nature paper. Rendition was performed using UCSFs chimera software. Retrieved from https://commons.wikimedia.org/wiki/Fi…Providers, P. C. (1979). English: Photomicrograph of Streptococcus pyogenes bacteria, 900x Mag. A pus specimen, viewed using Pappenheims stain. Last century, infections by S. pyogenes claimed many lives especially since the organism was the most important cause of puerperal fever and scarlet fever. Streptococci. Retrieved from https://commons.wikimedia.org/wiki/Fi…RRZEicons. (2010). English: zipper, open, close. Retrieved from https://commons.wikimedia.org/wiki/Fi…UC Berkeley. (n.d.). Gene editing with CRISPR-Cas9. Retrieved from https://www.youtube.com/watch?v=avM1Y…

Read more from the original source:
What is CRISPR?

Addgene: CRISPR Guide

Generating a Knockout Using CRISPR

You can use CRISPR to generate knockout cells or animals by co-expressing an endonuclease like Cas9 or Cpf1 and a gRNA specific to the gene to be targeted. The genomic target can be any 20 nucleotide DNA sequence, provided it meets two conditions:

The PAM sequence is essential for target binding, but the exact sequence depends on which Cas protein you use. We’ll use the popular S. pyogenes Cas9 (SpCas9) as an example, but check out our list of additional Cas proteins and PAM sequences. Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation into an active DNA-binding conformation. Importantly, the spacer region of the gRNA remains free to interact with target DNA.

Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8-10 bases at the 3 end of the gRNA targeting sequence) will begin to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3 to 5 direction. The zipper-like annealing mechanics of Cas9 may explain why mismatches between the target sequence in the 3 seed sequence completely abolish target cleavage, whereas mismatches toward the 5 end distal to the PAM often still permit target cleavage.

The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (3-4 nucleotides upstream of the PAM sequence).

The resulting DSB is then repaired by one of two general repair pathways:

The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations (for more information, jump to Plan Your Experiment.) In most cases, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene. However, the strength of the knockout phenotype for a given mutant cell must be validated experimentally. Learn more about non-homologous end joining (NHEJ).

Browse Plasmids: Double-Strand Break (Cut)

CRISPR specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in the genome. Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called off-targets and need to be considered when designing a gRNA for your experiment (see the Plan Your Experiment section below).

In addition to optimizing gRNA design, CRISPR specificity can also be increased through modifications to Cas9. As discussed previously, Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB.

Thus, two nickases targeting opposite DNA strands are required to generate a DSB within the target DNA (often referred to as a double nick or dual nickase CRISPR system). This requirement dramatically increases target specificity, since it is unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB. Therefore, if high specificity is crucial to your experiment, you might consider using the dual nickase approach to create a double nick-induced DSB. The nickase system can also be combined with HDR-mediated gene editing for specific gene edits.

In 2015, researchers used rational mutagenesis to develop two high fidelity Cas9s: eSpCas9(1.1) and SpCas9-HF1. eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing through alanine substitutions that disrupt Cas9’s interactions with the DNA phosphate backbone. HypaCas9, developed in 2017, contains mutations in the REC3 domain that increase Cas9 proofreading and target discrimination. All three high fidelity enzymes generate less off-target editing than wildtype Cas9.

Browse Plasmids: Single-Strand Break (Nick)

While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions like the addition of a fluorophore or tag.

In order to utilize HDR for gene editing, a DNA repair template containing the desired sequence must be delivered into the cell type of interest with the gRNA(s) and Cas9 or Cas9 nickase. The repair template must contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms.) The length of each homology arm is dependent on the size of the change being introduced, with larger insertions requiring longer homology arms.

Depending on the application, the repair template may be a single-stranded oligonucleotide, double-stranded oligonucleotide, or a double-stranded DNA plasmid. When designing the repair template, do not include the PAM sequence present in the genomic DNA. This step prevents the repair template from being a suitable target for Cas9 cleavage. For example, you could alter the DNA sequence of the PAM with a silent mutation that does not change the amino acid sequence.

The efficiency of HDR is generally low (

Since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a large portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, the resulting population of cells will contain some combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally and to isolate clones containing the desired edit (see the validation section in Plan Your Experiment). Learn more about homology directed repair (HDR).

Browse Plasmids: Endogenous Tagging

As discussed above, the efficiency of HDR is very low due to the number of DSBs repaired by NHEJ. To make point mutations without using HDR, researchers have developed CRISPR base editors that fuse Cas9 nickase or dCas9 to a cytidine deaminase like APOBEC1. Base editors are targeted to a specific locus by a gRNA, and they can convert cytidine to uridine within a small editing window near the PAM site. Uridine is subsequently converted to thymidine through base excision repair, creating a C->T change (or G->A on the opposite strand.) This class of base editors is available with multiple Cas9 variants and using high fidelity Cas9s. In addition, new base editors have been engineered to convert adenosine to inosine, which is treated like guanosine by the cell, creating an A->G (or T->C) change. Learn more about CRISPR DNA base editors.

Browse Plasmids: Base Edit

Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Fusing an ADAR2(E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA base editor that converts adenosine to inosine in RNA (termed REPAIR.) Since inosine is functionally equivalent to guanosine, the result is an A->G change in RNA. dPspCas13b does not appear to require a specific sequence adjacent to the RNA target, making this a very flexible editing system. Editors based on ADAR2(E488Q/T375G) display improved specificity, and editors carrying the delta-984-1090 ADAR truncation retain RNA editing capabilities and are small enough to be packaged in AAV particles.

Browse Plasmids: RNA Editing

CRISPR is a remarkably flexible tool for genome manipulation, as Cas enzymes bind target DNA independently of their ability to cleave target DNA. Specifically, both RuvC and HNH nuclease domains can be rendered inactive by point mutations (D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. The dCas9 molecule retains the ability to bind to target DNA based on the gRNA targeting sequence.

Early experiments demonstrated that targeting dCas9 to transcription start sites was sufficient to repress transcription by blocking initiation. dCas9 can also be tagged with transcriptional repressors or activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcriptional repression or activation of downstream target genes. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator, A (e.g. VP64) or transcriptional repressor, R (e.g. KRAB; see panel A to the right).

Additionally, more elaborate activation strategies have been developed for more potent activation of target genes in mammalian cells. These include: co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag system, panel B), dCas9 fused to several different activation domains in series (e.g. dCas9-VPR, panel C) or co-expression of dCas9-VP64 with a modified scaffold gRNA and additional RNA-binding helper activators (e.g. SAM activators, panel D). Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA.

Browse Plasmids: Activate, Repress/Interfere

Cas enzymes can be fused to epigenetic modifiers like p300 and TET1 to create programmable epigenome-engineering tools. Like CRISPR activators and repressors, these tools alter gene expression without inducing double-strand breaks. However, they are much more specific for particular chromatin and DNA modifications, allowing researchers to isolate the effects of a single epigenetic mark.

Another potential advantage of CRISPR epigenetic tools is their persistence and inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated/removed from the system. In contrast, epigenetic marks left by targeted epigenetic modifiers may be more frequently inherited by daughter cells. In certain cases, epigenetic modifiers may work better than activators/repressors in modulating transcription. However, since the effects of these tools are likely cell type- and context-dependent, it might be beneficial to try multiple CRISPR strategies when setting up your experimental system.

Browse Plasmids: Epigenetics

Expressing several gRNAs from the same plasmid ensures that each cell containing the plasmid will express all of the desired gRNAs, thus increasing the likelihood that all desired genomic edits will be carried out by Cas9. Such multiplex CRISPR applications include:

Current multiplex CRISPR systems enable researchers to target anywhere from 2 to 7 genetic loci by cloning multiple gRNAs into a single plasmid. These multiplex gRNA vectors can conceivably be combined with any of the aforementioned CRISPR derivatives to not only knock out target genes, but activate or repress target genes as well. Read more about Cas9 multiplexing and Cpf1 multiplexing.

Browse Plasmids: Multiplex gRNA vectors

The ease of gRNA design and synthesis, as well as the ability to target almost any genomic locus, make CRISPR the ideal genome engineering system for large-scale forward genetic screening. Forward genetic screens are particularly useful for studying diseases or phenotypes for which the underlying genetic cause is not known. In general, the goal of a genetic screen is to generate a large population of cells with mutations in a wide variety of genes and then use these mutant cells to identify the genetic perturbations that result in a desired phenotype.

Before CRISPR, genetic screens relied heavily on shRNA technology, which is prone to off-target effects and false negatives due to incomplete knockdown of target genes. In contrast, CRISPR is capable of making highly specific, permanent genetic modifications that are more likely to ablate target gene function. CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis. Currently, the most popular method for conducting genome-wide screens using CRISPR involves the use of pooled lentiviral CRISPR libraries.

Pooled lentiviral CRISPR libraries (often referred to as CRISPR libraries) are a heterogenous population of lentiviral transfer vectors, each containing an individual gRNA targeting a single gene in a given genome.

Guide RNAs are designed in silico and synthesized (see panel A below), then cloned in a pooled format into lentiviral transfer vectors (panel B). CRISPR libraries have been designed for common CRISPR applications including genetic knockout, activation, and repression for human and mouse genes.

Each CRISPR library is different, as libraries can target anywhere from a single class of genes up to every gene in the genome. However, there are several features that are common across most CRISPR libraries. First, each library typically contains 3-6 gRNAs per gene to ensure modification of every target gene, so CRISPR libraries contain thousands of unique gRNAs targeting a wide variety of genes. Guide RNA design for CRISPR libraries is usually optimized to select for guide RNAs with high on-target activity and low off-target activity.

Keep in mind that the exact region of the gene to be targeted varies depending on the specific application. For example, knockout libraries often target 5 constitutively expressed exons, but activation and repression libraries will target promoter or enhancer regions. Be sure to check the library page/original publication to see if a library is suitable for your experiment. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.

CRISPR libraries from Addgene are available in two formats: as DNA, or in select cases, as pre-made lentivirus.

In the case of DNA libraries, the CRISPR library will be shipped at a concentration that is too low to be used in experiments. Thus, the first step in using your library is to amplify the library (panel C) to increase the total amount of DNA. When amplifying the library, it is important to maintain good representation of gRNAs so that the composition of your amplified library matches that of the original library. You’ll use next-generation sequencing (NGS) to verify that this is the case. Learn more about library verification.

Once the library has been amplified/verified, the next step is to generate lentivirus containing the entire CRISPR library (panel D). Then, you will transduce cells with the lentiviral library (panel E). Remember – if you are using a 2-vector system, you will transduce cells that are already expressing Cas9. After applying your screening conditions, you will look for relevant genes (hits) using NGS technology. For more detail on using CRISPR for both positive and negative screens, see our pooled library guide.

As noted above, forward genetic screens are most useful for situations in which the physiology or cell biology behind a particular phenotype or disease is well understood, but the underlying genetic causes are unknown. Therefore, genome-wide screens using CRISPR libraries are a great way to gather unbiased information regarding which genes play a causal role in a given phenotype. With any experiment, it is important to verify that the hits you identify are actually important for your phenotype of interest. You can individually test the gRNAs identified in your screen to ensure that they reproduce the phenotype of interest.

Find more information on factors to consider before starting your pooled library experiment in Practical Considerations for Using Pooled Lentiviral CRISPR Libraries (McDade et al., 2016).

Browse Libraries: CRISPR Pooled Libraries

Using catalytically inactive Cas9 (dCas9) fused to a fluorescent marker like GFP, researchers have turned dCas9 into a customizable DNA labeler compatible with fluorescence microscopy in living cells. Alternatively, gRNAs can be fused to protein-interacting RNA aptamers, which recruit specific RNA-binding proteins (RBPs) tagged with fluorescent proteins to visualize targeted genomic loci.

CRISPR imaging has numerous advantages over other imaging techniques, including that it is easy to implement due to the simplicity of gRNA design, programmable for different genomic loci, capable of detecting multiple genomic loci, and compatible with live cell imaging. Compared to techniques like fluorescence in situ hybridization (FISH), CRISPR imaging offers a unique method for detecting the chromatin dynamics in living cells.

Multicolor CRISPR imaging allows for simultaneous tracking of multiple genomic loci in living cells. One method uses orthogonal dCas9s (e.g., S. pyogenes dCas9 and S. aureus dCas9) tagged with different fluorescent proteins. Alternatively, one can fuse gRNAs to orthogonal protein-interacting RNA aptamers, which recruit specific orthogonal RNA-binding proteins (RBPs) tagged with different fluorescent proteins, as seen in the popular CRISPRainbow kit.

The fluorescent CRISPR system has been used for dynamic tracking of repetitive and non-repetitive genomic loci, as well as chromosome painting in living cells. Visualizing a specific genomic locus requires recruitment of many copies of labeled proteins to the given region. For example, chromosome-specific repetitive loci can be efficiently visualized in living cells using a single gRNA that has multiple targeted sequences in close proximity. A non-repetitive genomic locus can also be labeled by co-delivering multiple gRNAs that tile the locus. Chromosome painting requires delivery of hundreds of gRNAs with target sites throughout the chromosome.

Browse Plasmids: Label

Identifying molecules associated with a genomic region of interest in vivo is essential to understanding locus function. Using CRISPR, researchers have expanded chromatin immunoprecipitation (ChIP) to allow purification of any genomic sequence specified by a particular gRNA.

In the enChIP (engineered DNA-binding molecule-mediated ChIP) system, catalytically inactive dCas9 is used to purify genomic DNA bound by the gRNA. An epitope tag(s) can be fused to dCas9 or gRNA for efficient purification. Various epitope tags including 3xFLAG-tag, PA, and biotin tags, can be used for enChIP, as well as an anti-Cas9 antibody. Biotin tagging of dCas9 can be achieved by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin ligase, as seen in the CAPTURE system. The locus is subsequently isolated by affinity purification against the epitope tag.

After purification of the locus, molecules associated with the locus can be identified by mass spectrometry (proteins), RNA-sequencing (RNAs), and next-generation sequencing (NGS) (other genomic regions). Compared to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward and enable direct identification of molecules associated with a genomic region of interest in vivo.

Browse Plasmids: Purify

In bacteria, type VI CRISPR systems recognize ssRNA rather than dsDNA. Many type VI enzymes also have the ability to process crRNA precursors to mature crRNAs. Upon ssRNA recognition by the crRNA, the target RNA is degraded. In bacteria, Cas13 enzymes can also cleave RNAs nonspecifically after the initial crRNA-guided cleavage. This promiscuous cleavage activity slows bacterial cell growth and may further protect bacteria from viral pathogens. Non-specific cleavage does not occur in mammalian cells. Similar to Cas9 and Cpf1, Cas13 can be converted to an RNA-binding protein through mutation of its catalytic domain. Learn more about Cas13a.

Browse Plasmids: RNA Targeting

While S. pyogenes Cas9 (SpCas9) is certainly the most commonly used CRISPR endonuclease for genome engineering, it may not be the best endonuclease for every application. For example, the PAM sequence for SpCas9 (5 NGG 3) is abundant throughout the human genome, but an NGG sequence may not be positioned correctly to target your desired genes for modification. This limitation is of particular concern when trying to edit a gene using homology directed repair (HDR), which requires PAM sequences in very close proximity to the region to be edited. Kleinstiver et al. generated synthetic SpCas9-derived variants with non-NGG PAM sequences. Gao et al. subsequently engineered Cpf1 PAM variants. The inclusion of these variants into the CRISPR arsenal effectively doubles the targeting range of CRISPR in the human genome. Read more about Cas9 variants.

Additional Cas9 orthologs from various species bind a variety of PAM sequences. These enzymes may have other characteristics that make them more useful than SpCas9 for specific applications. For example, the relatively large size of SpCas9 (4kb coding sequence) means that plasmids carrying the SpCas9 cDNA cannot be efficiently packaged into adeno-associated virus (AAV). Since the coding sequence for Staphylococcus aureus Cas9 (SaCas9) is 1 kilobase shorter than SpCas9, SaCas9 can be efficiently packaged into AAV. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo.

Another limitation of SpCas9 is the low efficiency of making specific genetic edits via HDR. For specific point edits, CRISPR base editing is a useful alternative to HDR. For larger edits, Cpf1, first described by Zetsche et al., may be a better option. Unlike Cas9 nucleases, which create blunt DSBs, Cpf1-mediated DNA cleavage creates DSBs with a short 3 overhang. Cpf1s staggered cleavage pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 also expands the range of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9.

CRISPR is a powerful system that enables researchers to manipulate the genome like never before. This section will provide a general framework to get you started using CRISPR in your research. Although we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR in other organisms. First, consider the genetic manipulation that is necessary to model your specific disease or process of interest. Do you want to:

Once you have a clear understanding of your experimental goal, you are ready to start navigating the different reagents that are available for your particular experiment.

Different genetic manipulations require different CRISPR components. Selecting a specific genetic manipulation can be a good way to narrow down which reagents are appropriate for a given experiment. Make sure to check whether reagents are available to carry out your experiment in your particular model organism. There may not be a perfect plasmid for your specific application, and in such cases, it may be necessary to customize an existing reagent to suit your needs.

To use CRISPR, you will need both Cas9 and a gRNA expressed in your target cells. For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may be sufficient to express the CRISPR machinery. For more difficult cells (e.g. primary cells), viral delivery of CRISPR reagents may be more appropriate. In cases where off-target editing is a major concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression.

The table below summarizes the major expression systems and variables for using CRISPR in mammalian cells. Some of the variables include:

Once you have selected your CRISPR components and method of delivery, you are ready to select a target sequence and design your gRNA.

When possible, you should sequence the region you are planning to modify prior to designing your gRNA, as sequence variation between your gRNA targeting sequence and target DNA may result in reduced cleavage. The number of alleles for each gene may vary depending on the specific cell line or organism, which may affect the observed efficiency of CRISPR knockout or knockin.

In order to manipulate a given gene using CRISPR, you will have to identify the genomic sequence for the gene you are trying to target. However, the exact region of the gene you target will depend on your specific application. For example:

A PAM sequence is absolutely necessary for Cas9 to bind target DNA. As such, one can start by identifying all PAM sequences within the genetic region to be targeted. If there are no PAM sequences for your chosen enzyme within your desired sequence, you may want to consider alternative Cas enzymes (see Cas9 variants and PAM sequences). Once possible PAM sequences and putative target sites have been identified, it is time to choose which site is likely to result in the most efficient on-target cleavage.

The gRNA target sequence needs to match the target locus, but it is also critical to ensure that the gRNA target sequence does NOT match additional sites within the genome. In a perfect world, your gRNA target sequence would have perfect homology to your target with no homology elsewhere in the genome. Realistically, a given gRNA target sequence will have partial homology to additional sites throughout the genome. These sites are called off-targets and should be examined during gRNA design. In general, off-target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity. To increase specificity, you can also consider using a high-fidelity Cas enzyme.

In addition to off-target activity, it is also important to consider factors that maximize cleavage of the desired target sequence or on-target activity. Two gRNA targeting sequences with 100% homology to their DNA targets may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. For example, gRNA targeting sequences containing a G nucleotide at position 20 (1 bp upstream of the PAM) may be more efficacious than gRNAs containing a C nucleotide at the same position in spite of being a perfect match for the target sequence.

Many gRNA design programs can locate potential PAM and target sequences and rank the associated gRNAs based on their predicted on-target and off-target activity (see gRNA design software). Additionally, many plasmids containing validated gRNAs are now available through Addgene. These plasmids contain gRNAs that have been used successfully in genome engineering experiments. Using validated gRNAs can save your lab valuable time and resources when carrying out CRISPR experiments. Read more about how to design your gRNA.

Browse Plasmids: Validated gRNAs

Once you have selected your target sequences it is time to design your gRNA oligos and clone these oligos into your desired vector. In many cases, targeting oligos are synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector you have chosen, so it is best to review the protocol associated with the specific plasmid in question (see CRISPR protocols from Addgene depositors).

Choose a delivery method that is compatible with your experimental system. CRISPR efficiency will vary based on the method of delivery and the cell type. Before proceeding with your experiment, it may be necessary to optimize your delivery conditions. Learn more about CRISPR delivery in mammalian systems.

Once you have successfully delivered the gRNA and Cas enzyme to your target cells, it is time to validate your genome edit. CRISPR editing produces several possible genotypes within the resulting cell population. Some cells may be wild-type due to either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA.

Edited cells may be homozygous or heterozygous for edits at your target locus. Furthermore, in cells containing two mutated alleles, each mutated allele may be different owing to the error-prone nature of NHEJ. In HDR gene editing experiments, most mutated alleles will not contain the desired edit, as a large percentage of DSBs are still repaired by NHEJ.

How do you determine that your desired edit has occurred? The exact method necessary to validate your edit will depend upon your specific application. However, there are several common ways to verify that your cells contain your desired edit, including but not limited to:

More information on each of these techniques can be found in our blog post CRISPR 101: Validating your Genome Edit.

The majority of the CRISPR plasmids in Addgenes collection are from S. pyogenes unless otherwise noted.

Engineered Cpf1 variants with altered PAM specificities. 2017. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F. Nat Biotechnol. 35(8):789-792. PMID: 28581492

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002

Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. 2017. Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR. Sci Adv. 3(8):eaao4774. PMID: 28875174

Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. 2017. Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR. Nat Commun. 8:15790. PMID: 28585549

Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. 2017. Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Nat Biotechnol. 35(4):371-376. PMID: 28191901

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. 2017. Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F. Nat Biotechnol. 35(1):31-34. PMID: 27918548

Nucleic acid detection with CRISPR-Cas13a/C2c2. 2017. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Science. 356(6336):438-442. PMID: 28408723

Programmable base editing of AT to GC in genomic DNA without DNA cleavage. 2017. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Nature. 551(7681):464-471. PMID: 29160308

RNA editing with CRISPR-Cas13. 2017. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. Science. pii: eaaq0180. PMID: 29070703

RNA targeting with CRISPR-Cas13. 2017. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F. . 550(7675):280-284. PMID: 28976959

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. 2016. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. Nature. 529(7587):490-5. PMID: 26735016

Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. 2016. Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T. Nat Biotechnol. . PMID: 27088723

Naturally occurring off-switches for CRISPR-Cas9. 2016. Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR. Cell. 167(7):1829-1838. PMID: 27984730

Practical Considerations for Using Pooled Lentiviral CRISPR Libraries. 2016. McDade JR, Waxmonsky NC, Swanson LE, Fan M. Curr Protoc Mol Biol. 115:31.5.1-31.5.13. PMID: 27366891

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. 2016. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Nature. 533(7603):420-4. PMID: 27096365

Rationally engineered Cas9 nucleases with improved specificity. 2016. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Science. 351(6268):84-8. PMID: 26628643

A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells. 2015. Dalvai M, Loehr J, Jacquet K, Huard CC, Roques C, Herst P, Ct J, Doyon Y. Cell Rep. 13(3):621-33. PMID: 26456817

An updated evolutionary classification of CRISPR-Cas systems. 2015. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. Nat Rev Microbiol. 13(11):722-36. PMID: 26411297

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. 2015. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR. Nat Biotechnol. 33(1):73-80. PMID: 25357182

CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins. 2015. Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts BS, Mackiewicz M, Mendenhall EM, Myers RM. Genome Res. 25(10):1581-9. PMID: 26355004

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What Is CRISPR? – CB Insights

CRISPR. What is it? And why is the scientific community so fascinated by its potential applications? Starting with its definition, we explain how this technology harnesses an ancient bacteria-based defense system and how it will impact the world around us today.

Imagine a future where parents can create bespoke babies, selecting the height and eye color of their yet unborn children.In fact, all traits can be customized to ones preferences: the size of domestic pets, the longevity of plants, etc.

It soundslike the backdrop of a dystopian science fiction novel. Yet some of this isalready happening.

Since its initial discovery in 2012, scientists have marveled at the applications of CRISPR (also known as Cas9 orCRISPR-Cas9).

And with a Jennifer Lopez-produced bio-terror TV drama called C.R.I.S.P.R. on the horizon, CRISPR has reached a new peak in interest from outside the scientific community.

CRISPR may revolutionize howwe tackle some of the worlds biggest problems, like cancer, food shortages, and organ transplant needs.Recent reports even examineits useasa highly efficient disease diagnostics tool. But, as with any new technology, it may also cause new unintended problems.

Changing DNA the code of life will inevitably come with a host ofimportant consequences. But society and industry cant have this conversation without understanding the basics of CRISPR.

In this explainer, we dive into CRISPR, from a simple explanation of what exactly it is to its applications and limitations.

CRISPR is adefining feature of the bacterial genetic code andits immune system,functioningas a defense system that bacteria use to protect themselves against attacks from viruses. Its also used by organisms in the Archaea kingdom (single-celled microorganisms).

The acronym CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. Essentially, it is a series ofshort repeating DNA sequences withspacers sitting in between them.

In short,bacteria usethese geneticsequences to remember each specific virus that attacks them.

They do this byincorporatingthe virus DNA into their own bacterial genome. Thisviral DNA ends up as the spacers in the CRISPR sequence.This method then gives thebacteria protection or immunity when a specific virus tries to attack again.

Accompanying CRISPR are genes that are always located nearby, called Cas (CRISPR-associated) genes.

Once activated, these genes make special proteins known as enzymes that seem to have co-evolved with CRISPR. The significance of these Cas enzymes is their ability to act as molecular scissors that can cut into DNA.

To recap: in nature,when a virus invades bacteria, its unique DNA is integrated into a CRISPR sequence in the bacterial genome. This means that the next time the virus attacks, the bacteria will remember it and sendRNA and Cas to locate and destroy the virus.

While there are other Cas enzymes derived from bacteria that cut out viruses when they attack bacteria, Cas9 is the best enzyme at doing this in animals. The widely-known term CRISPR-Cas9 refers to a Cas variety beingused to cut animal (and human) DNA.

Inharnessing this technology, researchers have added a new step: after DNA is cut by CRISPR-Cas9, a new DNA sequence carrying a fixed version of a gene can nestle into the new space. Alternatively, the cut can altogether knock out ofa particular unwanted gene for example, a gene that causes diseases.

Oneway to think about CRISPR-Cas9 isto compare it to theFind & Replace function in Word: itfinds thegenetic data (or word)you want to correct and replaces it with new material. Or, as CRISPR pioneer Jennifer Doudna puts it in her book A Crack In Creation: Gene Editing and the Unthinkable Power to Control Evolution, CRISPR is likea Swiss army knife, with different functions depending on how we want to use it.

CRISPR research has moved so fast that its already gone beyond basic DNA editing. In December 2017, the Salk Institute designed a handicapped version of the CRISPR-Cas9 system, capable of turninga targeted gene on or off without editing the genome at all. Going forward, this kind of process could ease the concerns surrounding the permanent nature of gene editing.

These are the 3 key players that help theCRISPR-Cas9 tech do its work:

Below, we illustrate how these parts come together to create a potential therapy.

Please click to enlarge.

The guide RNAserves as the GPS coordinates for finding the piece of DNA you want toedit and zeroes in on the targeted part of the gene. Once located, Cas9, the scissors, makes a double stranded break in the DNA, and the DNAyou want to insert takes its place.

The implications for this are vast.

Yes, this technology will disrupt medical treatment. But beyond that, it could also transform everything from the food we eat to the chemicals we use as fuel, since these may be engineered through gene technology as well.

Feng Zhang, PhD, from the Broad Institute of MIT and Harvard, describedCRISPR using a helpful nursery rhyme. We can imagine a certainDNA sequence that is fixed in this way:

Twinkle Twinkle Big Star Twinkle Twinkle Little Star

In this process:

The CRISPR sequence was first discovered in 1987. But its function would not be discovered until 2012.

Keypeople involved in the initial discovery of the bacterial CRISPR-Cas9 systems function include Jennifer Doudna, PhD at University of California, Berkeley, and French scientist Emmanuelle Charpentier, PhD. Through their strategic collaboration, they ushered in a new era of biotechnology.

Another important figure is Feng Zhang, PhD, who was instrumental in figuring out CRISPRs therapeutic applications using mice and human cells in 2013.Harvard geneticist George Church, PhDalso contributed to early CRISPR research with Zhang.

All four researchers went on to play crucial roles in setting up someof the most well-funded CRISPR therapeutic startups, includingEditas Medicine, CRISPR Therapeutics, and Intellia Therapeutics.All 3 of these companiesIPOed in 2016 and are in the drug discovery/pre-clinical stage of testing their respective CRISPR therapeutic candidates for various human diseases.

Before CRISPR was heralded asthegene editing method, two other gene-editing techniques dominated the field: Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). Research efforts using these tools are still ongoing.

Like CRISPR, these toolscan each cut DNA. Thought they are generally more difficult to make and use, these tools do offer their own advantages:

Each also has vital therapeutic applications.

Biotech companyCellectis uses the TALEN gene editing technology to make CAR-T therapies for leukemia, whileSangamo BioSciencesmakes ZFNs that can disable a gene known to be key in HIV infection.Notably, each of these companies hold key IP rights to these specific gene-editing methods, which could make it difficult for other biotech companiesto use these tools.

Meanwhile, CRISPR has certainly stolen the spotlight as of late, due to its efficiency, flexibility, and cheap price tag. Itsplausible that CRISPR could face similar IP issues and there are already some IP controversies going on but with such vast applications for this system, research on multiple fronts seems to be moving forward fast.

Every industry can harnessCRISPR as a tool: itcan create new drug therapies for human diseases, help farmers grow pathogen-resistant crops, create new species of plants and animals and maybe even bring back old ones.

Since the initial discovery of CRISPR as a gene-editing mechanism, the list of applications has grown rapidly. Though still in early stages, animal models (i.e. lab animals) have provided key insights into how we may be able to manipulate CRISPR.

Mice have been especially telling when it comes to CRISPRs therapeutic potential. As mammals sharing more than 90% of our human genes, mice have been used as ideal test subjects.

Experiments on mice haveshown that CRISPR can disable a defective gene associated with Duchenne muscular dystrophy (DMD), inhibit the formation of deadly proteins involved in Huntingtons disease, and eliminate HIV infection.

In 2015, Chinese scientists created two super muscular beagles by disabling the myostatin gene, which directs normal muscle development. In the absence of thegene, the beagles displayed muscular hypertrophy, creating dogs which were visibly much more muscular than non-genetically modified ones.

Other CRISPR animal trials haveranged from genetically engineering long-haired goats for higher production of cashmere to breeding hornless cows to avoid the painful process of shearing horns off.

Compared to research involving animals, CRISPR trialsthat edit human DNA have movedmore slowly, largely due to the ethical and regulatory issues at play.

Given the permanent nature of altering a humans genome, the FDA is approaching CRISPR cautiously. Some scientists have even proposed a moratorium on CRISPR trials untilwe have more information on the potentialimpact on humans.

In the US and Europe, 2018 will be the year for CRISPR human trials.

Currently (as of 2/13/18),the University of Pennsylvania is awaiting the FDAs final approval to start a study that would evaluate the safety of using CRISPR for patients with multiple myeloma, melanoma, and sarcoma.

Europe may also see its first human CRISPR study in 2018 withCRISPR Therapeutics study focused on a blood disorder known as beta-thalassemia,which results in abnormal red blood cell production.

While clinical trials involving patient participation are still awaiting regulatory approval, CRISPR has already been applied to both viable and non-viable human embryos.

For example, in August 2017, a team lead by reproductive biologist Shoukhrat Mitalipov of Oregon Health and Science University received private funding to use CRISPR-Cas9 to target a mutation in viable human embryos that causes the thickening of heart muscles. The altered embryos came back 72% mutation-free in the lab (higher than theusual 50% chance of inheritance).

Some critics say the gene editing of embryos is unethical, even if the edited embryos are not destined for transfer and implantation. This type of testing currently does not receive federal funding, but instead relies on private donor funding.

On the other side of the world, Chinese researchersoperate under a different regulatory framework. Some hospital ethics committees can approve studies in as little as one day, with no need to seek approval from a federal agency.

Since 2015, China has been conductinghuman trials using CRISPRto combat various cancers, HIV, and HPV. It is the only country in the world toconduct human trials thus far.

According to ClinicalTrials.Gov, there are 10 active or upcoming CRISPR therapy trials in China, targeting advanced cancers like stage 4 gastric and nasopharyngeal carcinomas. So far results are only anecdotal, and while some participants tumors shrank, no formal results have been made available.

Although possible long-term side effectsarent fully understood,CRISPR is already an option for some patients in China who have exhausted all of the conventional treatments.

Potential high impact industries for CRISPR include medicine, food, agriculture, and the industrial biotech space. BecausetheCRISPR-Cas9 gene-editing system issoeasy to make and use, researchers from a range of scientific disciplines can access it to genetically engineer the organism of their choice.

The future of medicine will be written with CRISPR.

The current drug discovery process is long, given the need to ensure patientsafety and gain a thorough understanding of unintended effects.Moreover, current US regulatory policies often result in a decades-long development process.

However, teamsusing CRISPR can bringcustomized therapies to market more quickly than was previously dreamed, speeding upthe traditional drug discovery process.

Timeline of drug development. Credit: PhRMA

CRISPRscheap price tag and flexibilityallows accurate and fast identification of potential gene targets for efficient pre-clinical testing. Because itcan be used to knock out different genes, CRISPR givesresearchers a faster and more affordableway to study hundreds of thousands of genes to see which ones are affected by a particular disease.

Of course, alongwith providing a more streamlined drug development process, CRISPR offers the possibility of new ways to treat patients.

For example,monogenic diseasesdiseases caused by a mutation ina single gene present an attractive starting point for CRISPR trials. The nature of these illnesses provides an exact target for the treatment: the problematic mutation on a single gene.

Blood-based, single-gene diseases like beta-thalassemia or sickle cell are alsogreat candidates for CRISPR therapy, because of their ability to be treated outside of the body (known as ex-vivo therapy). A patients blood cells can be taken out, treated with the CRISPR system, then put back into the body.

An earlyapplication of CRISPR was pioneered by yogurt company Danisco in the 2000s, when scientists used an early version of CRISPR to combat a key bacterium found inmilk and yogurt cultures (streptococcus thermophilus) that kept getting infected by viruses. At that point, the ins and outs of CRISPRwere still unclear.

Fast forward to today, when climate change will further increasethe need to use CRISPR to protect the food and agriculture industries against new bacteria.For example, cacao is becoming difficult to farm as growing regions get hotter and drier. This environmental change will further exacerbate the damage done by pathogens.

If youve eaten yogurt or cheese, chances are youve eaten CRISPR-ized cells.

Rodolphe Barrangou, former Daniscoscientist & Editor-in-Chief of The CRISPR Journal

To combat this issue, the Innovative Genomics Institute (IGI) at UC Berkeley is applying CRISPR to create disease-resistant cacao. Leading chocolate supplier MARS Inc. is supporting this effort.Gene editing can make farming more efficient. It can curb global food shortages for staple crops like potatoes and tomatoes. And it can create resilient crops, impervious to droughts and other environmental impacts.Regulators have shown little resistanceto gene-edited crops, and the United States Department of Agriculture (USDA) in particular is not regulating the space. This is largely because when CRISPR is applied to crops, theres no foreign DNA being added: CRISPR is simply used to edit a crops own genetics to select for desirable traits.In 2016, the white button mushroom, modified to beresistant to browning, became the first CRISPR-edited organism to bypass USDA. In October 2017, it was announced that agriculture company DuPont Pioneer and the Broad Institute would collaborate for agriculture researchusing their CRISPR-Cas9 intellectual property.

InSeptember 2017, biotech company Yield10 Bioscience got approval for its CRISPR-edited plantCamelina sativa (false flax), which hasenhanced omega-3 oil and is used to make vegetable oil and animal feed.

These are indicationsthat newbreeds of crops could reachmarketsmuch faster than previously thought. Without USDA oversight, these items and other food products could go into production relatively quickly.

This will impact the food we eat, as food items are edited tocarry more nutrients or to last longer on grocery shelves.

Another area currently generating buzz isthe production of leaner livestock.

In October 2017, scientists at the Chinese Academy of Sciences in Beijing used CRISPR to genetically engineer pig meat that had 24% less body fat.

Researchersdid this by inserting a mouse gene into pig cellsin order tobetter regulate body temperature.Although this example technically makes the result a GMO product, it may not be too long before pigs genes are used for the same purpose.

Future versionsof this technology applied to human nutrition will be one area to look out for.

Another key, but less obvious, use of CRISPR lies is in the industrial biotech space. By re-engineering microbes using CRISPR,researcher can create new materials.

How is this relevant to society at large?

From an industrial standpoint, this is big for modifying and creating new chemical products. We can alter microbes to increase diversity, create new bio-based materials, and make more efficient biofuels.From active chemicals in fragrances to those involved in industrial cleaning, CRISPR could have agreat impact here by creating new and more efficientbiological materials.

Jennifer Doudnas first CRISPR startup, Caribou Biosciences, was founded in 2011 for non-therapeutic research purposes across industries. It is one of the key companies providing various industries with the tools to use CRISPR fora range of purposes.

CRISPRs list of potential benefits is a long one. But the technology also brings with it a number of limitations.

Possible unintended effects and all the unknown variables are some of the drawbacks to this newtechnology, while newethicalquestions and controversies are also emerging as human trials near.

When using CRISPRfor human therapies, safety is the biggest issue. As with any new form of technology, researchers are unsure of the entire range of CRISPRs effects.Off-target activity is the main concern here. A single gene editcould cause unintended activity somewhere else in the genome. A possible consequence of this is abnormal growth of tissues, leading to cancer. As more research uncovers new details, this could result in more refined, precise gene targeting.

Another issue is the possibility of mosaic generation.After a CRISPR treatment, a patient could have a mix of both edited and unedited cells a mosaic. As cells continue to divide and replicate, some cells may get repaired, while others wont.

Finally, immune systemcomplications mean that these interventions and therapies may trigger an undesired response froma patients immune system.Early research shows theimmune system may dispose of Cas enzymes before they achieve their purpose, or may have an averse reaction resulting in side effects like inflammation. (In 1999, a patient in the US died of a severe immune reaction, instilling more caution in researchers when it comes to CRISPR trials.)

However, all three of these limitations have some possible solutions.

Different enzymes (molecular scissors) or more precise delivery vehicles can reduce off-target activity. If modified stem cells in egg or sperm (i.e. cells that can become every cell in the human body) are edited, mosaics can be avoided.

With the immune system issue, researchers can isolate different Cas proteins from more obscure bacterial strains that humans dont already have an adaptive immunity to in order to circumvent an unwanted immune response. Meanwhile, ex-vivo therapies, wherescientists take a patients blood cells out of the body and treat them before infusing them back in, can also helpbypass the immune system.

One potential big limitation for CRISPR isthat CRISPR-Cas9 system lacks surgical precision. The Cas enzyme cuts both strands of the DNA double helix, and this double-stranded breakcreates worries over the precision of the cut.

Repairing a defective gene would be like finding a needle in a haystack and then removing that needle without disturbing a single strand of hay in the process.-Jennifer Doudna

While currently the Cas9 enzyme gets the most attentionas the enzyme doing the cutting, scientists are actively pursuing alternatives to find better candidates.

Alternative options include asmaller version of Cas9, or a different enzyme entirely: Cpf1, whichhas become popular due to its easy transport to the targeted DNA location.

Besides using other Cas enzymes, alternate delivery vehiclesfor therapeutic genes are another option. Harmless engineered viruses can carry therapeutic genes to the site of mutation, while lipid nanoparticles can avoid immune system detection, avoiding an immune reaction. Both options present promising avenues of research.

Whentechnology can alter the code of life, its implications are far-reaching as are its controversies. Here we outlinea few of the main controversies surroundingCRISPR.

Originally posted here:
What Is CRISPR? – CB Insights

Crisprs Epic Patent Fight Changed the Course of Biology | WIRED

After three bitter years and tens of millions of dollars in legal fees, the epic battle over who owns one of the most common methods for editing the DNA in any living thing is finally drawing to a close. On Monday, the US Court of Appeals for the Federal Circuit issued a decisive ruling on the rights to Crispr-Cas9 gene editingawarding crucial intellectual property spoils to scientists at the Broad Institute of Cambridge, Massachusetts.

The fight for Crispr-Cas9which divided the research community and triggered an uncomfortable discussion about science for personal profit versus public goodhas dramatically shaped how biology research turns into real-world products. But its long-term legacy is not what happened in the courtroom, but what took place in the labs: A wealth of innovation that is now threatening to make Cas9 obsolete.

This latest legal decision, which upholds a 2017 ruling by the US Patent and Trademark Office, was an expected one, given how rarely such rulings are overturned. And it more or less seals defeat for researchers at the University of California Berkeley, who also have claims to invention of the world-remaking technology.

The Broad celebrated the win while calling for a cease-fire, saying it was time to work together to ensure wide, open access to this transformative technology. UCs general counsel, Charles F. Robinson, struck a less conciliatory note, saying in a statement that the university was evaluating further litigation options. Those could include a rehearing from the same court or appeal to the Supreme Court.

But legal experts say the chances of either happening are vanishingly slim. It is very possible that there is no path forward for Berkeley in regards to broad patents covering Crispr-Cas9 at this point , says Jacob Sherkow a scholar of patent law at New York Law School who has closely followed the case. In addition to the Broad Institutes claims, UC-Berkeley also has to contend with another foundational patent for Crispr-Cas9 gene editing filed before anyone else in March 2012, by Virginijus iknys, a Lithuanian scientist who shares the prestigious Kavli Prize with Berkeleys Jennifer Doudna and The University of Viennas Emmanuelle Charpentier for their early work on Crispr. The USPTO has since granted his patent. UC didnt know about it at the time of its own filing because of an 18-month secrecy statute surrounding new applications. If this was a choose-your-own-adventure book, they just turned all the wrong pages, says Sherkow.

The University of California isnt the only loser here; the companies that already placed bets on it being the patent victor must now tread a difficult though not impassable IP landscape. That includes Intellia and Crispr Therapeuticscompanies cofounded by Doudna and Charpentier respectivelywhich are both developing Crispr treatments for human disease. The two firms released a joint statement Monday afternoon underscoring their faith in the strength and scope of UCs foundational IP. A spokesperson for Intellia also said in an email that the Federal Circuit decision will not impact the companys freedom to operate going forward.

For all the ferocity that fueled the fight from its outset, Mondays decision was met with muted interest from inside the halls of science to the crowded trading floors of Wall Street. Thats because a lot has changed since the first gene editing pioneers filed the original Crispr-Cas9 patents. In 2012, Cas9 was the entire Crispr universe. That little enzyme powered all the promise of Crispr gene editing, and the stakes for owning it couldnt have been higher. Scientists didnt yet know that biology would prove to be more creative than patent lawyers. They still had no notion of the vast constellations of constructs and enzymes that could be engineered, evolved in a lab, or harvested from the wild to replace Cas9.

Since then though, the fast-moving field of Crispr biology has yielded more than just alternative pairs of molecular scissors. Researchers have updated the Crispr system to manipulate the code of life in myriad novel waysfrom swapping out individual DNA letters to temporarily flipping genes on and off to detecting dangerous infections. And theyve unearthed dozens of Crispr enzymes of still unknown functions that might one day solve problems scientists havent even thought of yet.

The rush of discoveries and inventions has led to a full-blown patent race, says Sherkow, with anyone who found any new variation racing to file IP protections. The irony is that as the universe of Crispr expands, owning a part of it becomes less and less valuable. Twenty years from now, when the umpteenth drug gets approved using Crispr and some nuclease named Cas132013, people are going to look back on this patent battle and think, what a godawful waste of money, says Sherkow.

He expects that the field will eventually reach a point where the value of each new Crispr patent is so low that researchers dont bother going through all the paperwork and spending the thousands of dollars necessary to file an application. Already, biotechnologists are beginning to learn this lesson in adjacent fields; a land grab for patents is not the only way to go.

The Biobricks Foundation is a nonprofit dedicated to supporting the development of an open-source biotechnology commons. In 2015, it created a legal framework for scientists to put their discoveries in the public domain, safeguarding them from being patented elsewhere, and ensuring that anyone can access them. So far, the organization has begun to stockpile gene sequences for useful tools like fluorescent proteins. Linda Kahl, Biobricks senior counsel and a director there, says theyre still waiting for a group to design an open-source Crispr system. Thats a gauntlet thats in front of researchers, she says. With the ashes of the patent fight still glowing, it might be too soon to expect anyone to give a Crispr tool away for free just yet. But it probably wont take long.

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Crisprs Epic Patent Fight Changed the Course of Biology | WIRED

CRISPR, one of the biggest science stories of the decade …

One of the biggest and most important science stories of the past few years will probably also be one of the biggest science stories of the next few years. So this is as good a time as any to get acquainted with the powerful new gene editing technology known as CRISPR.

If you havent heard of CRISPR yet, the short explanation goes like this: In the past six years, scientists have figured out how to exploit a quirk in the immune systems of bacteria to edit genes in other organisms plants, mice, even humans. With CRISPR, they can now make these edits quickly and cheaply, in days rather than weeks or months. (The technology is often known as CRISPR/Cas9, but well stick with CRISPR, pronounced crisper.)

Let that sink in. Were talking about a powerful new tool to control which genes get expressed in plants, animals, and even humans; the ability to delete undesirable traits and, potentially, add desirable traits with more precision than ever before.

In 2017 alone, researchers reported in Nature that theyd successfully used CRISPR in human embryos to fix a mutation that causes a terrible heart muscle disorder called hypertrophic cardiomyopathy. (Other researchers have since called some of the conclusions into question.) Another team used it to reduce the severity of genetic deafness in mice, suggesting it could one day be used to treat the same type of hearing loss in people.

Meanwhile, researchers at the Broad Institute of MIT and Harvard launched a coordinated blitz with two big studies that move CRISPR in that safer and more precise direction. A paper published in Science describes an entirely new CRISPR-based gene editing tool that targets RNA, DNAs sister, allowing for transient changes to genetic material. In Nature, scientists published on a more refined type of CRISPR gene editing that can alter a single bit of DNA without cutting it increasing the tools precision and efficiency.

And these are just a few of the astounding things researchers have recently shown CRISPR can do. Weve already learned that it can help us create mushrooms that dont brown easily and edit bone marrow cells in mice to treat sickle-cell anemia. Down the road, CRISPR might help us develop drought-tolerant crops and create powerful new antibiotics. CRISPR could one day even allow us to wipe out entire populations of malaria-spreading mosquitoes or resurrect once-extinct species like the passenger pigeon.

But there are real limits to what CRISPR can do, at least right now. Scientists have recently learned that the approach to gene editing can inadvertently wipe out and rearrange large swaths of DNA in ways that may imperil human health. That follows recent studies showing that CRISPR-edited cells can inadvertently trigger cancer.

As scientists work to overcome these limitations, much of the hype around CRISPR has focused on whether we might engineer humans with specific genetic traits (like heightened intelligence). But in some ways, thats a sideshow. Designer babies are still far off, and there are enormous obstacles to making those sorts of complex genetic modifications. The stuff thats closer at hand from new therapies to fighting malaria is whats most exciting. So heres a basic guide to what CRISPR is and what it can do.

If we want to understand CRISPR, we should go back to 1987, when Japanese scientists studying E. coli first came across some unusual repeating sequences in the bacterias DNA. The biological significance of these sequences, they wrote, is unknown. Over time, other researchers found similar clusters in the DNA of other bacteria (and archaea). They gave these sequences a name: Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR.

Yet these CRISPR sequences were mostly a mystery until 2007, when food scientists studying the Streptococcus bacteria used to make yogurt showed how these odd clusters actually served a vital function: Theyre part of the bacterias immune system.

See, bacteria are under constant assault from viruses and produce enzymes to fight off viral infections. Whenever the bacterias enzymes manage to kill off an invading virus, other little enzymes will come along, scoop up the remains of the viruss genetic code, cut it into little bits, and then store it in those CRISPR spaces.

Now comes the clever part: The bacteria use the genetic information stored in these CRISPR spaces to fend off future attacks. When a new infection occurs, the bacteria produce special attack enzymes, known as Cas9, that carry around those stored bits of viral genetic code like a mug shot. When these Cas9 enzymes come across a virus, they see if the viruss RNA matches whats in the mug shot. If theres a match, the Cas9 enzyme starts chopping up the viruss DNA to neutralize the threat. It looks a little like this:

So thats what CRISPR/Cas9 does. For a while, these discoveries werent of much interest to anyone except microbiologists until a series of further breakthroughs occurred.

In 2011, Jennifer Doudna of the University of California Berkeley and Emmanuelle Charpentier of Ume University in Sweden were puzzling over how the CRISPR/Cas9 system actually worked. How did the Cas9 enzyme match the RNA in the mug shots with that in the viruses? How did the enzymes know when to start chopping?

The scientists soon discovered they could fool the Cas9 protein by feeding it artificial RNA a fake mug shot. When they did that, the enzyme would search for anything with that same code, not just viruses, and start chopping. In a landmark 2012 paper, Doudna, Charpentier, and Martin Jinek showed they could use this CRISPR/Cas9 system to cut up any genome at any place they wanted.

While the technique had only been demonstrated on molecules in test tubes at that point, the implications were breathtaking.

Further advances followed. Feng Zhang, a scientist at the Broad Institute in Boston, co-authored a paper in Science in February 2013 showing that CRISPR/Cas9 could be used to edit the genomes of cultured mouse cells or human cells. In the same issue of Science, Harvards George Church and his team showed how a different CRISPR technique could be used to edit human cells.

Since then, researchers have found that CRISPR/Cas9 is ridiculously versatile. Not only can scientists use CRISPR to silence genes by snipping them out, they can also harness repair enzymes to substitute desired genes into the hole left by the snippers (though this latter technique is trickier to pull off). So, for instance, scientists could tell the Cas9 enzyme to snip out a gene that causes Huntingtons disease and insert a good gene to replace it.

Gene editing itself isnt new. Various techniques to knock out genes have been around for years. What makes CRISPR so revolutionary is that its incredibly precise: The Cas9 enzyme mostly goes wherever you tell it to. And its incredibly cheap and easy: In the past, it might have cost thousands of dollars and weeks or months of fiddling to alter a gene. Now it might cost just $75 and only take a few hours. And this technique has worked on every organism its been tried on.

This has become one of the hottest fields around. In 2011, there were fewer than 100 published papers on CRISPR. In 2017, there were more than 14,000 and counting, with new refinements to CRISPR, new techniques for manipulating genes, improvements in precision, and more. This has become such a fast-moving field that I even have trouble keeping up now, says Doudna. Were getting to the point where the efficiencies of gene editing are at levels that are clearly going to be useful therapeutically as well as a vast number of other applications.

Theres been an intense legal battle over who exactly should get credit for this CRISPR technology was Doudnas 2012 paper the breakthrough, or was Zhangs 2013 paper the key advance? Ultimately, a court ruled in February that the patent should go to Zhang and the Broad Institute, Harvard, and MIT. In the July, the University of California and others on Doudnas side said they were launching an appeal of the decision. But the important thing is that CRISPR has arrived.

So many things. Paul Knoepfler, an associate professor at UC Davis School of Medicine, told Vox that CRISPR makes him feel like a kid in a candy store.

At the most basic level, CRISPR can make it much easier for researchers to figure out what different genes in different organisms actually do by, for instance, knocking out individual genes and seeing which traits are affected. This is important: While weve had a complete map of the human genome since 2003, we dont really know what function all those genes serve. CRISPR can help speed up genome screening, and genetics research could advance massively as a result.

Researchers have also discovered there are numerous CRISPRs. So CRISPR is actually a pretty broad term. Its like the term fruit it describes a whole category, said the Broads Zhang. When people talk about CRISPR, they are usually referring to the CRISPR/Cas9 system weve been talking about here. But in recent years, researchers like Zhang have found other types of CRISPR proteins that also work as gene editors. Cas13, for example, can edit DNAs sister, RNA. Cas9 and Cas13 are like apples and bananas, Zhang added.

The real fun and, potentially, the real risks could come from using CRISPRs to edit various plants and animals. A recent paper in Nature Biotechnology by Rodolphe Barrangou and Doudna listed a flurry of potential future applications:

1) Edit crops to be more nutritious: Crop scientists are already looking to use CRISPR to edit the genes of various crops to make them tastier or more nutritious or better survivors of heat and stress. They could potentially use CRISPR to snip out the allergens in peanuts. Korean researchers are looking to see if CRISPR could help bananas survive a deadly fungal disease. Some scientists have shown that CRISPR can create hornless dairy cows a huge advance for animal welfare.

Recently, major companies like Monsanto and DuPont have begun licensing CRISPR technology, hoping to develop valuable new crop varieties. While this technique wont entirely replace traditional GMO techniques, which can transplant genes from one organism to another, CRISPR is a versatile new tool that can help identify genes associated with desired crop traits much more quickly. It could also allow scientists to insert desired traits into crops more precisely than traditional breeding, which is a much messier way of swapping in genes.

With genome editing, we can absolutely do things we couldnt do before, says Pamela Ronald, a plant geneticist at the University of California Davis. That said, she cautions that its only one of many tools for crop modification out there and successfully breeding new varieties could still take years of testing.

Its also possible that these new tools could attract controversy. Foods that have had a few genes knocked out via CRISPR are currently regulated more lightly than traditional GMOs. Policymakers in Washington, DC, are currently debating whether it might make sense to rethink regulations here. This piece for Ensia by Maywa Montenegro delves into some of the debates CRISPR raises in agriculture.

2) New tools to stop genetic diseases: As the new Nature paper shows, scientists are now using CRISPR/Cas9 to edit the human genome and try to knock out genetic diseases like hypertrophic cardiomyopathy. Theyre also looking at using it on mutations that cause Huntingtons disease or cystic fibrosis, and are talking about trying it on the BRCA-1 and 2 mutations linked to breast and ovarian cancers. Scientists have even shown that CRISPR can knock HIV infections out of T cells.

So far, however, scientists have only tested this on cells in the lab. There are still a few hurdles to overcome before anyone starts clinical trials on actual humans. For example, the Cas9 enzymes can occasionally misfire and edit DNA in unexpected places, which in human cells might lead to cancer or even create new diseases. As geneticist Allan Bradley, of Englands Wellcome Sanger Institute, told STAT, CRISPRs ability to wreak havoc on DNA has been seriously underestimated.

And while there have also been major advances in improving CRISPR precision and reducing these off-target effects, scientists are urging caution on human testing. Theres also plenty of work to be done on actually delivering the editing molecules to particular cells a major challenge going forward.

3) Powerful new antibiotics and antivirals: One of the most frightening public health facts around is that we are running low on effective antibiotics as bacteria evolve resistance to them. Currently, its difficult and costly to develop fresh antibiotics for deadly infections. But CRISPR/Cas9 systems could, in theory, be developed to eradicate certain bacteria more precisely than ever (though, again, figuring out delivery mechanisms will be a challenge). Other researchers are working on CRISPR systems that target viruses such as HIV and herpes.

4) Gene drives that could alter entire species: Scientists have also demonstrated that CRISPR could be used, in theory, to modify not just a single organism but an entire species. Its an unnerving concept called gene drive.

It works like this: Normally, whenever an organism like a fruit fly mates, theres a 50-50 chance that it will pass on any given gene to its offspring. But using CRISPR, scientists can alter these odds so that theres a nearly 100 percent chance that a particular gene gets passed on. Using this gene drive, scientists could ensure that an altered gene propagates throughout an entire population in short order:

By harnessing this technique, scientists could, say, genetically modify mosquitoes to only produce male offspring and then use a gene drive to push that trait through an entire population. Over time, the population would go extinct. Or you could just add a gene making them resistant to the malaria parasite, preventing its transmission to humans, Voxs Dylan Matthews explains in his story on CRISPR gene drives for malaria.

Suffice to say, there are also hurdles to overcome before this technology is rolled out en masse and not necessarily the ones youd expect. The problem of malaria gene drives is rapidly becoming a problem of politics and governance more than it is a problem of biology, Matthews writes. Regulators will need to figure out how to handle this technology, and ethicists will need to grapple with knotty questions about its fairness.

5) Creating designer babies: This is the one that gets the most attention. Its not entirely far-fetched to think we might one day use CRISPR to edit the human genome to eliminate disease, or to select for athleticism or superior intelligence.

That said, scientists arent even close to being able to do this. Were not even close to the point where scientists could safely make the complex changes needed to, for instance, improve intelligence, in part because it involves so many genes. So dont go dreaming of Gattaca just yet.

I think the reality is we dont understand enough yet about the human genome, how genes interact, which genes give rise to certain traits, in most cases, to enable editing for enhancement today, Doudna said in 2015. Still, she added: Thatll change over time.

Given all the fraught issues associated with gene editing, many scientists are advocating a slow approach here. They are also trying to keep the conversation about this technology open and transparent, build public trust, and avoid some of the mistakes that were made with GMOs.

In February 2017, a report from the National Academy of Sciences said that clinical trials could be greenlit in the future for serious conditions under stringent oversight. But it also made clear that genome editing for enhancement should not be allowed at this time.

Society still needs to grapple with all the ethical considerations at play here. For example, if we edited a germline, future generations wouldnt be able to opt out. Genetic changes might be difficult to undo. Even this stance has worried some researchers, like Francis Collins of the National Institutes of Health, who has said the US government will not fund any genomic editing of human embryos.

In the meantime, researchers in the US who can drum up their own funding, along with others in the UK, Sweden, and China, are moving forward with their own experiments.

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CRISPR, one of the biggest science stories of the decade …

CRISPR safety calls for cautious approach – washingtonpost.com

In the movie Rampage, the character played by Dwayne Johnson uses a genetic engineering technology called CRISPR to transform a gorilla into a flying dragon-monster with gigantic teeth. Although this is science fiction, not to mention impossible, the movie captures the recent interest and fascination with one of the newest scientific technologies.

CRISPR which stands for clustered regularly interspaced short palindromic repeats was originally seen as part of a bacterial defense system that evolved to destroy foreign DNA that entered a bacterium. But this system is also capable of editing DNA and now geneticists have honed the technology to alter DNA sequences that we specify.

This has generated enormous excitement and great expectations about the possibility of using CRISPR to alter genetic sequences to improve our health, to treat diseases, to improve the quality and quantity of our food supplies, and to tackle environmental pollution.

Using genome editing to treat human diseases is very tantalizing. Correcting inherited genetic defects that cause human disease just as one edits a sentence is the obvious application. This strategy has been successful in tests on animals.

But a few recent scientific papers suggest that CRISPR is not without its problems. The research reveals that CRISPR can damage DNA located far from the target DNA we are trying to correct. As a cancer biologist at the University of Pittsburgh School of Medicine, I use CRISPR in my lab to study human cancers and develop ways to kill cancer cells.

Although the new findings appear significant, I dont think that these revelations rule out using the technology in a clinical setting; rather, they suggest we take additional cautionary measures as we implement these strategies.

Treating human diseases

In the United States and Europe, clinical trials have been planned for several human diseases. Most notably, a gene-editing Phase I/II trial is planned in Europe for beta-thalassemia, a hereditary blood disorder that causes anemia that requires lifelong blood transfusions. This year, a CRISPR trial for sickle cell anemia, another inherited blood disorder caused by a mutation that deforms the red blood cells, is planned in the United States.

For both of these trials, the gene editing is done ex vivo meaning outside the patients body. Hematopoietic blood cells the stem cells that generate red blood cells are taken from the patient and edited in the lab. The cells are then reintroduced into the same patients after the mutations have been corrected. The expectation is that by correcting the stem cells, the cells they produce will be normal, curing the disease.

The ex vivo approach has also been used in China to test treatments against an array of human cancers. There, researchers take immune cells called T cells from cancer patients and use CRISPR to stop these cells from producing a protein called PD-1 (program cell death-1). Normally, PD-1 prevents T cells from attacking ones own tissues. However, cancer cells exploit this protective mechanism to evade the bodys defense system. Removing PD-1 allows T cells to attack cancer cells vigorously. The initial results from clinical trials using gene-edited T cells appear mixed.

In my lab, we have recently been focusing on chromosome rearrangement, a genetic defect where a segment of chromosome skips and joins distant parts of the same or a different chromosome. A scrambled chromosome is a defining characteristic of most cancers. The most famous example of such an alteration is the Philadelphia Chromosome in which Chromosome 9 is connected to Chromosome 22 which causes acute myeloid leukemia.

My team has used CRISPR in animal models to insert a suicide gene to specifically target liver and prostate cancer cells that harbor such rearrangements. Since these chromosome rearrangements occur only in cancer cells but not normal cells, we can target the cancer without collateral damage to healthy cells.

CRISPR concerns

Despite all the excitement surrounding CRISPR editing, researchers have urged caution about moving too fast. Two recent studies have raised concerns that CRISPR may not be as effective as previously thought, and in some cases it may produce unwanted side effects.

The first study showed that when the Cas9 protein part of the CRISPR system that snips the DNA before correcting the mutation cuts the DNA of stem cells, it causes them to become stressed and stops them from being edited. While some cells can recover after their DNA has been corrected, other cells could die.

The second study showed that a protein called p53, which is well known for guarding against tumors, is activated by cellular stress. The protein then inhibits CRISPR from editing. Since CRISPR activity causes stress, the editing process may be thwarted before it even accomplishes its task.

Another study over the past year has revealed an additional potential issue with using CRISPR in humans. Because CRISPR is a bacterial protein, a significant portion of the human population may have been exposed to it during common bacterial infections. In these cases, the immune systems of these people may have developed immune defense against the protein, which means a persons body could attack the CRISPR machinery, just as it would attack an invading bacterium or virus, preventing the cell from the benefits of CRISPR-based therapy.

Additionally, like most technologies, not all editing is accurate. Occasionally, CRISPR targets the wrong sites in the DNA and makes changes that researchers fear could cause disease. A recent study showed that CRISPR caused large chunks of the chromosome to rearrange near the site of genome editing in mouse embryonic stem cells, although this effect isnt always observed in the other cell systems. Most published results indicate that off-target rates range from 1 to 5 percent. Even if the off-target rate is relatively low, we dont yet understand the long-term consequences.

Dangers have been hyped

The studies referenced above have led to a glut of media reports about the potential negative effect of CRISPR, many citing potential cancer risk. More often than not, these involve a far-fetched extrapolation of actual results. As far as I am aware, no animals treated with the CRISPR-Cas9 system have been shown to develop cancers.

Studies have shown CRISPR-based genome editing works more efficiently in cancer cells than normal cells. Indeed, the resistance of normal cells to CRISPR editing actually makes it more appealing for cancer treatment since there would be less potential collateral damage to normal tissues, a conclusion that is supported by research in our lab.

Looking forward, it is obvious that the technology has great potential to treat human diseases. The recent studies have revealed new aspects of how CRISPR works that may have implications for the ways in which these therapies are developed. However, the long-term effect of genome editing can only be assessed after CRISPR has been used widely to treat human diseases.

health-science@washpost.com

Luo is a professor of pathology at the University of Pittsburgh. This article was originally published on theconversation.com.

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CRISPR safety calls for cautious approach – washingtonpost.com

What is CRISPR? – YouTube

In this video Paul Andersen explains how the CRISPR/Cas immune system was identified in bacteria and how the CRISPR/Cas9 system was developed to edit genomes.

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All of the images are licensed under creative commons and public domain licensing:Adenosine. (2009). English: Artistic rendering of a T4 bacteriophage. The colours grey and orange do not signify anything, they are just used to illustrate structure. Created for Wikipedia. Retrieved from https://commons.wikimedia.org/wiki/Fi…E. coli Bacteria. (n.d.). Retrieved February 17, 2016, from https://www.flickr.com/photos/niaid/1…Fioretti, B. F. Hallbauer &. (2015). English: Director, Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology. Visiting professor The Laboratory for Molecular Infection Medicine Sweden MIMS; http://www.mpiib-berlin.mpg.de/resear…. Retrieved from https://commons.wikimedia.org/wiki/Fi…Foresman, P. S. ([object HTMLTableCellElement]). English: Line art drawing of a chimera. Retrieved from https://commons.wikimedia.org/wiki/Fi…Magladem96. (2014). English: Picture of DNA Base Flipping. Retrieved from https://commons.wikimedia.org/wiki/Fi…project, C. wiki. (2014). English: Crystal Structure of Cas9 bound to DNA based on the Anders et al 2014 Nature paper. Rendition was performed using UCSFs chimera software. Retrieved from https://commons.wikimedia.org/wiki/Fi…Providers, P. C. (1979). English: Photomicrograph of Streptococcus pyogenes bacteria, 900x Mag. A pus specimen, viewed using Pappenheims stain. Last century, infections by S. pyogenes claimed many lives especially since the organism was the most important cause of puerperal fever and scarlet fever. Streptococci. Retrieved from https://commons.wikimedia.org/wiki/Fi…RRZEicons. (2010). English: zipper, open, close. Retrieved from https://commons.wikimedia.org/wiki/Fi…UC Berkeley. (n.d.). Gene editing with CRISPR-Cas9. Retrieved from https://www.youtube.com/watch?v=avM1Y…

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What is CRISPR? – YouTube

With Embryo Base Editing, China Gets Another Crispr First

Scientists in the US may be out in front developing the next generation of Crispr-based genetic tools, but its China thats pushing those techniques toward human therapies the fastest. Chinese researchers were the first to Crispr monkeys, and non-viable embryos, and to stick Crisprd cells into a real live human. And now, a team of scientists in China have used a cutting-edge Crispr technique, known as base editing, to repair a disease-causing mutation in viable human embryos.

Published last week in the journal Molecular Therapy, and reported first by Stat, the study represents significant progress over previous attempts to remodel the DNA of human embryos. Thats in part because the editing worked so well, and in part because that editing took place in embryos created by a standard in-vitro fertilization technique.

So-called germline editing, the contentious technology that can permanently change the code in every cell in the human body, has been gaining acceptance in the last few years as research has pushed forward, illuminating the possibilities of Crispr. Immediately following those first reports of embryonic gene-editing in China in 2015, an international summit convened by the US National Academy of Sciences concluded that actually trying to produce a human pregnancy from such modified germlines was irresponsible, given ongoing safety concerns and lack of societal consensus. Two years later, a report from the NAS and the National Academy of Medicine stated that clinical trials for editing out heritable diseases could be permitted in the future, but only for serious conditions under stringent oversight.

Attitudes may be slowly changing: Last month, the United Kingdoms Nuffield Council on Bioethics went so far as to say that heritable genome editing could be ethically acceptable in some circumstances. A Pew Research Council study released at the end of July found that 72 percent of Americans think changing an unborn babys DNA to treat a serious disease would be an appropriate use of gene-editing technology.

In the study published in Molecular Therapy, the Chinese scientists corrected a mutation that causes Marfan syndrome, an incurable connective tissue disorder that affects about 1 in 5,000 people. A single letter mistake in the gene for FBN1, which codes for the fibrillin protein, can cause a ripple effect of problemsfrom loose joints to weak vision to life-threatening tears in the hearts walls. Starting with healthy eggs and sperm donated by a Marfan syndrome patient, the team of researchers from Shanghai Tech University and Guangzhou Medical University used an IVF technique to make viable human embryos. Then they injected the embryos with a Crispr construct known as a base editor, which swaps out a single DNA nucleotide for anotherin this case, removing a C and replacing it with a T.1 They kept the embryos alive for another two days in the lab, long enough to run tests to see how well the editing worked.

Sequencing revealed that all 18 embryos had been edited, with 16 of the embryos bearing only the corrected version of the FBN1 gene. In two of the embryos, additional unwanted edits had also taken place. Previously, the most successful demonstration of gene editing in the human germline was the correction of a mutation that causes a hereditary heart condition in 42 out of 58 embryos. That study, which was published last year, used standard Crispr cut-and-paste technology.

Its a nice demonstration of the use of base editors to correct a well-known point mutation that causes a human genetic disease in a setting that may become therapeutically relevant, says David Liu, whose lab at Harvard developed the base editor used to correct the Marfan mutation, though he was not involved in the study.

Rather than breaking the double-stranded DNA molecule and allowing the cell to repair itself with a healthy gene template, these newer versions of Crispr change just a single letter. If Crispr is a pair of molecular scissors, Lius base editors are more like a pencil with a squeaky new eraser. While the hope is that such precise gene-writing implements wont cause the kind of sloppy chaos that Crispr 1.0 is capable of, Liu says its too early to make any general statements about their relative risks as a therapeutic. Despite more than 50 publications using base editors from laboratories around the world, the entire field of base editing is only about two years old, and additional studies are needed to assess as many possible consequences of base editing as can be reasonably detected.

Some of those studies are being conducted at Beam Therapeutics, the startup that Liu co-founded earlier this year with fellow Crispr pioneer Feng Zhang. Beams first license agreement with Harvard covers Lius C base editor, which makes programmable G-to-A or C-to-T edits, like the one used to correct the Marfan mutation. The second is the A base editor, which can do T-to-C as well as A-to-G edits. But dont expect Beam to be erasing genetic diseases from the germline any time soon. The company is focused on using base editing to treat serious diseases in children and adults only, not on embryo editing, says CEO John Evans. More consideration would be needed before society is ready to consider embryo editing, and we look forward to participating in the discussion.

In the meantime, Beam will be just one of many US companies looking at an increasingly streamlined path for genetic medicines. In July, FDA Commissioner Scott Gottlieb announced a new regulatory framework for gene therapies to treat rare diseases. The agency issued a suite of six guidance documents updating the approval process. And on August 17, the FDA along with the National Institutes of Health proposed changes in the way the agencies together assess the safety of gene-therapy human trials.

Specifically, the proposals will eliminate review by the NIHs Recombinant DNA Advisory Committee, which was established in 1974 to advise on emerging genetic technologies. In a New England Journal of Medicine editorial describing the changes, Gottlieb and NIH Director Francis Collins wrote it was their view that there is no longer sufficient evidence to claim that the risks of gene therapy are entirely unique and unpredictableor that the field still requires special oversight that falls outside our existing framework for ensuring safety. A more streamlined approval process may help the US move faster in the long-run, though probably not enough to catch Chinas head start. But when it comes to gene editing’s most controversial applications, theres nothing wrong with being slow.

1Correction appended 8-27-2018, 10:45 EDT. The researchers changed a cytosine to a thymine, not an adenine to guanine, as previously stated.

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With Embryo Base Editing, China Gets Another Crispr First

CRISPR | Genome Editing, DNA Repair

Cas9 and Cpf1 can be reprogrammed to different sites or multiple sites using multiple gRNAs. The availability of the different engineered variants of Cas9 and Cpf1 allows for different types of cuts for genome editing, which include the following:

Cut & Revise and Cut & Remove typically result in disruption of a problematic gene or elimination of a mutation. These approaches leverage the cell’s natural DNA repair mechanisms known as non-homologous end joining, or NHEJ, to complete the edit.

When a cell repairs a DNA cut by NHEJ, it leaves small insertions and deletions at the cut site, collectively referred to as indels. NHEJ can be used to either cut and revise the targeted gene or to cut and remove a segment of DNA. In the ”cut and revise” process, a single cut is made. In the ”cut and remove” process, two cuts are made, which results in the removal of the intervening segment of DNA. This approach could be used to delete either a small or a large segment of DNA depending on the type of repair desired.

The second mechanism our Cut & Replace approach leverages a different DNA repair mechanism known as homology directed repair, or HDR. In this approach, a DNA template is also provided, one that is similar to the DNA that has been cut. The cell can use the template to construct reparative DNA, resulting in the replacement of a defective genetic sequence with the correct one.

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CRISPR | Genome Editing, DNA Repair

Addgene: CRISPR References and Information

This table lists gRNA sequences that have been experimentally validated for use in CRISPR experiments.

gRNA design tool with extensive selection of eukaryotic pathogen genomes (200+) that can predict gRNA targets in gene families, HDR oligonucleotide design, and batch processing for designing genome-wide gRNA libraries. PubMed PMID 28348817.

This tool helps design (10 different prediction scores), clone (primer design), and evaluate gRNAs, as well as predict off-targets, for CRISPR in 180+ genomes. PubMed PMID: 27380939.

sgRNA Scorer 2.0From the Church Lab: a tool that identifies putative target sites for S. pyogenes Cas9, S. thermophilus Cas9, or Cpf from your input sequence or list of sequences.

Quilt Universal guide RNA designerSearch for gRNAs via gene name or by genomic location. Database includes gRNAs from popular CRISPR libraries and from more than two million DNAse hypersensitive sites for intergenic guide RNAs in hg19, filtered for off-target effects.

From the Kim Lab, Cas-OFFinder identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans, mouse, human, rat, cow, dog, pig, Thale cress, rice (Oryza sativa), tomato, corn, monkey (macaca mulatta).

Cas-Designer searches for targets that maximize knockout efficiency while having a a low probability of off-target effects. Cas-Designer integrates information from the Kim Lab’s Cas-OFFinder and Microhomology predictor.

From the Qi Lab, a sgRNA design tool for genome editing, as well as gene regulation (repression and activation). Genome support for bacteria (E. coli, B. subtilis), yeast (S. cerevisiae), worm (C. elegans), fruit fly, zebrafish, mouse, rat, and human.

Identifies candidate sgRNA target sites by off-target quality. Validated for gene inactivation, NHEJ, and HDR. Reference genomes include Arabidopsis, C. elegans , sea squirt, cavefish, Chinese hamster, fruit fly, human, rice fish, mouse, silk worm, stickleback, tobacco, tomato, frog (X. laevis and X. tropicalis), and zebrafish.

Program for designing optimal gRNAs. Provides feedback on number of potential off-targets, target’s genomic location, and genome annotation. Available genomes are human (hg19 & hg38), mouse (mm10), and yeast (strain w303).

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Addgene: CRISPR References and Information

Here’s Why CRISPR Therapeutics Lost 18.8% in July — The …

What happened

Shares of CRISPR Therapeutics (NASDAQ:CRSP) fell nearly 19% last month, according to data provided by S&P Global Market Intelligence, after yet another study reminded Wall Street and investors that there’s still much for scientists to understand about the use of CRISPR gene-editing tools in human cells. Previously, in June, two studies surfaced that suggested certain uses of CRISPR could trigger faulty DNA repair mechanisms to activate and turn a cell cancerous.

That was followed up last month by a new study suggesting that certain uses of CRISPR tools “seriously underestimated” the number of off-target changes made to a genome. CRISPR Therapeuticssaid to Reutersthat “[w]e do not use the methods described in this Nature Biotech paper … nevertheless, in our work, we do not see similar findings.” While that wasn’t enough to appease Wall Street in July, shareholders have still enjoyed a year-to-date gain of 103%.

Image source: Getty Images.

The study published last month came from researchers at the prestigious Wellcome Sanger Institute, an affiliation that helped the results to be taken more seriously. But considering CRISPR Therapeutics says it doesn’t use the specific techniques identified, investors may be wondering why the company’s shares were impacted at all. Well, it has to do with increasing uncertainty over an important part of using certain gene-editing tools.

More specifically, the most recent study detailing off-target changes to DNA and those identifying the potential to activate cancerous mutations already present in cells all seem to imply the same thing: Scientists may have gotten a little ahead of themselves by assuming DNA repair mechanisms would work in a simple fashion. While CRISPR tools intend to fix genetic defects by cutting one or both strands of human DNA, all rely on DNA repair mechanisms already present in a cell to stitch the genome back together. If those fail, then CRISPR tools might be less effective or could even end up having significant unintended effects.

Right now, it appears that the most troubling side effects are observed when CRISPR tools cut both strands of DNA (a “double-strand break”). The lead drug candidates of all three major CRISPR companies deploying the technology for medical applications avoid that headache, although all companies are exploring preclinical therapeutics that will have to navigate that obstacle eventually.

Investors can likely expect gene-editing stocks such as CRISPR Therapeutics to experience a higher-than-normal amount of volatility. The technology has received an incredible amount of attention in the media and even popular culture, and the potential to cure diseases has Wall Street understandably excited. Those forces have combined to hand the pioneering companies premium market valuations, but it’s important to remember that CRISPR is a relatively new technology. Investors in it for the long haul will simply need to buckle up and remain patient as results from the first clinical trials (yet to get started) begin to trickle in within the next few years.

Maxx Chatsko has no position in any of the stocks mentioned. The Motley Fool owns shares of CRISPR Therapeutics. The Motley Fool has a disclosure policy.

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Here’s Why CRISPR Therapeutics Lost 18.8% in July — The …

Here’s Why CRISPR Stocks Are Down as Much as 11.4% Today …

What happened

Shares of the three leading companies developing human therapeutics based on CRISPR gene-editing technology fell as much as 11.4% today. There was no new news that could be interpreted as detrimental to CRISPR Therapeutics (NASDAQ:CRSP), Editas Medicine (NASDAQ:EDIT), or Intellia Therapeutics (NASDAQ:NTLA). But on July 23, many media outlets published stories commenting on a study released one week earlier.

While the stock moves may seem to be based on the rehashing of old news, there are good reasons investors shouldn’t be too quick to dismiss the concerns. As of 2:44 p.m. EDT, CRISPR Therapeutics stock had settled to a 9.5% loss, while Editas Medicine shares were down 10.4%, and Intellia Therapeutics stock had sunk by 7.2%.

Image source: Getty Images.

On July 16, scientists published a study in Nature Biotechnology demonstrating that using CRISPR tools to edit faulty DNA sequences can lead to unintended deletions and rearrangements of genetic material. The lead author, Dr. Allan Bradley, issued a cautious summary of the study:

This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects.

The researchers, who hail from the prestigious Wellcome Sanger Institute, found that some of the genetic changes occurred far away from where CRISPR tools cut a genome, locations which would elude existing diagnostic tools used to gauge off-target effects. In other words, the field has “seriously underestimated” the potential for unintended genetic alterations because it hasn’t been looking in the right places.

All three companies made statements to Reuters last week regarding the study. CRISPR Therapeutics commented: “We do not use the methods described in this Nature Biotech paper … nevertheless, in our work, we do not see similar findings.” Editas Medicine said it was “not specifically concerned.” Intellia Therapeutics said it didn’t think the findings would affect the future of CRISPR-based therapies.

While it’s important for investors not to panic over the latest study showing potentially unintended consequences of using gene-editing tools, it is worth noting that most of the recent uncertainty injected into CRISPR stocks has come from observations of DNA repair mechanisms — one thing gene-editing tools have little to no control over. While companies focus on developing safe and effective ways to cut a genome, they must rely on natural cellular processes to stitch up the genome afterwards.

For instance, in June, investors worried over two studies showing that CRISPR tools could activate a faulty DNA repair mechanism and result in cancerous cells. The latest study from the Wellcome Sanger Institute was not concerned with the same question, but demonstrated that researchers may be overlooking the details of how genomes get stitched back up.

An open-minded approach to investing in CRISPR Therapeutics, Editas Medicine, and Intellia Therapeutics would nod to the awesome potential of the technology while acknowledging the risks of an early-stage investment. Recent stock moves hint that the hype may need to come back down to earth, as there’s much left to understand about using CRISPR tools in human cells. To date researchers have focused mostly on the ability to cut DNA, but it may be time to start paying closer attention to what happens after that.

Maxx Chatsko has no position in any of the stocks mentioned. The Motley Fool owns shares of CRISPR Therapeutics. The Motley Fool recommends Editas Medicine. The Motley Fool has a disclosure policy.

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Here’s Why CRISPR Stocks Are Down as Much as 11.4% Today …

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