Japan Set to Allow Gene Editing in Human Embryos


Draft guidelines permit gene-editing tools for research into early human development, but would discourage manipulation of embryos for reproduction.
Japan Set to Allow Gene Editing in Human Embryos

Japan has issued draft guidelines that allow the use of gene-editing tools in human embryos. The proposal was released by an expert panel representing the country’s health and science ministries on 28 September.

Although the country regulates the use of human embryos for research, there have been no specific guidelines on using tools such as CRISPR–Cas9 to make precise modifications in their DNA until now.

Tetsuya Ishii, a bioethicist at Hokkaido University in Sapporo, says that before the draft guidelines were issued, Japan’s position on gene editing in human embryos was neutral. The proposal now encourages this kind of research, he says.

But if adopted, the guidelines would restrict the manipulation of human embryos for reproduction, although this would not be legally binding.

Manipulating DNA in embryos could reveal insights into early human development. Researchers also hope that in the long term, these tools could be used to fix genetic mutations that cause diseases, before they are passed on.

But the editing of genes in human embryos, even for research, has been controversial. Ethicists and many researchers worry that the technique could be used to alter DNA in embryos for non-medical reasons. Many countries ban the practice, allowing gene-editing tools to be used only in non-reproductive adult cells.

Researchers around the world have published at least eight studies on gene editing in human embryos. Some of the work was done in Chinaand the United States, where using the technique does not break any laws if done with private funding; some was done in the United Kingdom, where permission must be granted by a national regulatory body.

Japan’s draft guidelines will be open for public comment from next month and are likely to be implemented in the first half of next year.

Gene editing – and what it really means to rewrite the code of life


We now have a precise way to correct, replace or even delete faulty DNA. Ian Sample explains the science, the risks and what the future may hold

Gene editing has the potential to treat or prevent thousands of forms of human disease.

So what is gene editing?
Scientists liken it to the find and replace feature used to correct misspellings in documents written on a computer. Instead of fixing words, gene editing rewrites DNA, the biological code that makes up the instruction manuals of living organisms. With gene editing, researchers can disable target genes, correct harmful mutations, and change the activity of specific genes in plants and animals, including humans.

What’s the point?
Much of the excitement around gene editing is fuelled by its potential to treat or prevent human diseases. There are thousands of genetic disorders that can be passed on from one generation to the next; many are serious and debilitating. They are not rare: one in 25 children is born with a genetic disease. Among the most common are cystic fibrosis, sickle cell anaemia and muscular dystrophy. Gene editing holds the promise of treating these disorders by rewriting the corrupt DNA in patients’ cells. But it can do far more than mend faulty genes. Gene editing has already been used to modify people’s immune cells to fight cancer or be resistant to HIV infection. It could also be used to fix defective genes in human embryos and so prevent babies from inheriting serious diseases. This is controversial because the genetic changes would affect their sperm or egg cells, meaning the genetic edits and any bad side effects could be passed on to future generations.

What else is it good for?
The agricultural industry has leapt on gene editing for a host of reasons. The procedure is faster, cheaper and more precise than conventional genetic modification, but it also has the benefit of allowing producers to improve crops without adding genes from other organisms – something that has fuelled the backlash against GM crops in some regions. With gene editing, researchers have made seedless tomatoes, gluten-free wheat and mushrooms that don’t turn brown when old. Other branches of medicine have also seized on its potential. Companies working on next-generation antibiotics have developed otherwise harmless viruses that find and attack specific strains of bacteria that cause dangerous infections. Meanwhile, researchers are using gene editing to make pig organs safe to transplant into humans. Gene editing has transformed fundamental research too, allowing scientists to understand precisely how specific genes operate.

So how does it work?
There are many ways to edit genes, but the breakthrough behind the greatest achievements in recent years is a molecular tool called Crispr-Cas9. It uses a guide molecule (the Crispr bit) to find a specific region in an organism’s genetic code – a mutated gene, for example – which is then cut by an enzyme (Cas9). When the cell tries to fix the damage, it often makes a hash of it, and effectively disables the gene. This in itself is useful for turning off harmful genes. But other kinds of repairs are possible. For example, to mend a faulty gene, scientists can cut the mutated DNA and replace it with a healthy strand that is injected alongside the Crispr-Cas9 molecules. Different enzymes can be used instead of Cas9, such as Cpf1, which may help edit DNA more effectively.

Remind me what genes are again?
Genes are the biological templates the body uses to make the structural proteins and enzymes needed to build and maintain tissues and organs. They are made up of strands of genetic code, denoted by the letters G, C, T and A. Humans have about 20,000 genes bundled into 23 pairs of chromosomes all coiled up in the nucleus of nearly every cell in the body. Only about 1.5% of our genetic code, or genome, is made up of genes. Another 10% regulates them, ensuring that genes turn on and off in the right cells at the right time, for example. The rest of our DNA is apparently useless. “The majority of our genome does nothing,” says Gerton Lunter, a geneticist at the University of Oxford. “It’s simply evolutionary detritus.”

What are all those Gs, Cs, Ts and As?
The letters of the genetic code refer to the molecules guanine (G), cytosine (C), thymine (T) and adenine (A). In DNA, these molecules pair up: G with C and T with A. These “base pairs” become the rungs of the familiar DNA double helix. It takes a lot of them to make a gene. The gene damaged in cystic fibrosis contains about 300,000 base pairs, while the one that is mutated in muscular dystrophy has about 2.5m base pairs, making it the largest gene in the human body. Each of us inherits about 60 new mutations from our parents, the majority coming from our father.

But how do you get to the right cells?
This is the big challenge. Most drugs are small molecules that can be ferried around the body in the bloodstream and delivered to organs and tissues on the way. The gene editing molecules are huge by comparison and have trouble getting into cells. But it can be done. One way is to pack the gene editing molecules into harmless viruses that infect particular types of cell. Millions of these are then injected into the bloodstream or directly into affected tissues. Once in the body, the viruses invade the target cells and release the gene editing molecules to do their work. In 2017, scientists in Texas used this approach to treat Duchenne muscular dystrophy in mice. The next step is a clinical trial in humans. Viruses are not the only way to do this, though. Researchers have used fatty nanoparticles to carry Crispr-Cas9 molecules to the liver, and tiny zaps of electricity to open pores in embryos through which gene editing molecules can enter.

Does it have to be done in the body?
No. In some of the first gene editing trials, scientists collected cells from patients’ blood, made the necessary genetic edits, and then infused the modified cells back into the patients. It’s an approach that looks promising as a treatment for people with HIV. When the virus enters the body, it infects and kills immune cells. But to infect the cells in the first place, HIV must first latch on to specific proteins on the surface of the immune cells. Scientists have collected immune cells from patients’ blood and used gene editing to cut out the DNA that the cells need in order to make these surface proteins. Without the proteins, the HIV virus can no longer gain entry to the cells. A similar approach can be used to fight certain types of cancer: immune cells are collected from patients’ blood and edited so they produce surface proteins that bind to cancer cells and kill them. Having edited the cells to make them cancer-killers, scientists grow masses of them in the lab and infuse them back into the patient. The beauty of modifying cells outside the body is that they can be checked before they are put back to ensure the editing process has not gone awry.

What can go wrong?
Modern gene editing is quite precise but it is not perfect. The procedure can be a bit hit and miss, reaching some cells but not others. Even when Crispr gets where it is needed, the edits can differ from cell to cell, for example mending two copies of a mutated gene in one cell, but only one copy in another. For some genetic diseases this may not matter, but it may if a single mutated gene causes the disorder. Another common problem happens when edits are made at the wrong place in the genome. There can be hundreds of these “off-target” edits that can be dangerous if they disrupt healthy genes or crucial regulatory DNA.

Will it lead to designer babies?
The overwhelming effort in medicine is aimed at mending faulty genes in children and adults. But a handful of studies have shown it should be possible to fix dangerous mutations in embryos too. In 2017, scientists convened by the US National Academy of Sciences and the National Academy of Medicine cautiously endorsed gene editing in human embryos to prevent the most serious diseases, but only once shown to be safe. Any edits made in embryos will affect all of the cells in the person and will be passed on to their children, so it is crucial to avoid harmful mistakes and side effects. Engineering human embryos also raises the uneasy prospect of designer babies, where embryos are altered for social rather than medical reasons; to make a person taller or more intelligent, for example. Traits like these can involve thousands of genes, most of them unknown. So for the time being, designer babies are a distant prospect.

How long before it’s ready for patients?
The race is on to get gene editing therapies into the clinic. A dozen or so Crispr-Cas9 trials are underway or planned, most led by Chinese researchers to combat various forms of cancer. One of the first launched in 2016, when doctors in Sichuan province gave edited immune cells to a patient with advanced lung cancer. More US and European trials are expected in the next few years.

What next?

Base editing
A gentler form a gene editing that doesn’t cut DNA into pieces, but instead uses chemical reactions to change the letters of the genetic code. It looks good so far. In 2017, researchers in China used base editing to mend mutations that cause a serious blood disorder called beta thalassemia in human embryos.

Gene drives
Engineered gene drives have the power to push particular genes through an entire population of organisms. For example, they could be used to make mosquitoes infertile and so reduce the burden of disease they spread. But the technology is highly controversial because it could have massive unintended ecological consequences.

Epigenome editing
Sometimes you don’t want to completely remove or replace a gene, but simply dampen down or ramp up its activity. Scientists are now working on Crispr tools to do this, giving them more control than ever before.

Gene Editing: Report Calls for Caution, No Outright Ban


National Academies panel outlines ‘stringent’ criteria for human germline editing

Every year at this time, MedPage Today‘s writers select a few of the most important stories published earlier in the year and examine what happened afterward. One of those original stories, which first appeared Feb. 14, is republished below; click here to read the follow-up.

WASHINGTON — Clinical trials involving “heritable germline editing” are not yet ready for prime-time, according to a report from the National Academy of Sciencesand the National Academy of Medicine.

However, heritable germline editing — genetic manipulations that can be passed down to offspring — could potentially be allowed in the future for “serious disease or conditions” and with strict oversight, provided certain criteria are met, according to a committee of science, healthcare, and legal experts that made up the Committee on Human Gene Editing.

The report is the result of a year-long examination of the science and policy of human gene editing and its ethical ramifications.

In weighing the benefits and risks of these techniques, the committee decided that “caution is absolutely needed, but being cautious does not mean prohibition,” said R. Alta Charo, JD, committee co-chair and bioethics scholar at the University of Wisconsin Law School in Madison.

According to Charo, the committee agreed to six “strict” and “stringent” criteria under which germline editing could begin to be considered:

  • Lack of reasonable alternatives
  • Limiting to genes “convincingly demonstrated to cause or predispose” one to serious illness
  • “Credible pre-clinical and/or clinical data” regarding the potential risks and benefits
  • Strong and continuous oversight during clinical trials
  • A broad plan for long-term, multi-generational follow-up
  • Extensive and continued review of “health and societal benefits and risks” that involves public engagement

“If those conditions are met, it was the committee’s conclusion that germline heritable editing clinical trials would be permissible — not obligatory, but permissible,” she said, adding that “we are not even close to the amount of research we need before you could actually move forward at a technical level, in terms of the precision and safety, in this particular technique.”

The committee also weighed in on non-heritable clinical trials or the editing of somatic cells.

Basic scientific trials in this category of genome editing are underway, and are in the nascent stages of clinical trials and applications. Treatments that enable “corrected” genes to implant themselves in cells, often using a virus, have shown promise in research studies of cystic fibrosisHIV, and Duchenne muscular dystrophy.

Since these changes cannot be inherited by future generations, they should be allowed to continue only when the research or therapy aims to treat or prevent disease or disability, and not for the purpose of genetic enhancement, according to the committee.

New technologies such as the CRISPR-Cas9 — an enzyme that can slice DNA at targeted points — offer the possibility of altering an individual’s genome, or even a generation’s genome. CRISP-CAS9 is easy, efficient, and relatively cheap, as committee members noted, and with its introduction, the risk of off-target events or “mistaken edits” is shrinking.

Instead of worrying “it’s too risky,” stakeholders are now beginning to shift their focus to the ethical ramifications of germline editing, said committee Jeffrey Kahn, PhD, MPH, director of the Johns Hopkins Berman Institute of Bioethics in Baltimore.

Rather than fully opening a door that was previously closed for germline editing, Kahn told MedPage Today that the report is more “like a knock on the door. The door’s not open yet.”

He pointed out that the committee’s criteria are “pretty rigid,” and not necessarily easy to meet. In addition, in the U.S., the NIH Recombinant DNA Advisory Committee and the FDA also have regulations regarding germline editing.

The NIH committee previously stated that it will “not entertain proposals for germline alterations,” so those restrictions still need to be relaxed, Kahn noted, describing the NIH stance as “more than a door — that’s a locked door.”

Rules about germline editing are not necessarily as strict in other countries. For instance, in 2015, scientists at Sun Yat-sen University in Guangzhou, China, were the first to use CRISPR-CAS9 on human embryos, according to Nature.

While the embryos were defective and could not have led to a live birth, the experiment was likely tied to a call for a moratorium by an international group of scientists on gene editing that could cause “inheritable changes to the genome,” according to The New York Times.

Former Director of National Intelligence James Clapper expressed concern that CRISPR could be used as a weapon of mass destruction, according toScience.

In the current report, the committee issued guiding principles “that should be used by any nation in governing human genome editing research or applications.” These are:

  • Promote well-being
  • Transparency
  • Due care
  • Responsible science
  • Respect for persons
  • Fairness
  • Transnational cooperation

“These overarching principles, and the responsibilities that flow from them, should be reflected in each nation’s scientific community and regulatory processes,” said committee co-chair Richard Hynes, PhD, of Massachusetts Institute of Technology in Boston, in a press release.

Charo noted that the guidelines set forth in the report, including its criteria, might be weighted differently in different jurisdictions.

“In some countries, [germline editing] is entirely illegal,” she noted, adding that some states have made embryo research unlawful.

“The bottom line is that there is no planetary government with enforcement power, but the goal of the human genome initiative [and] the goal of this study committee is to help develop international norms that will be influential, with the policymakers, with physicians, with researchers, with patient groups … so that to the greatest extent possible, there is some global agreement” on a set of guiding principles that aim toward beneficial purposes, Charo stated.

The Progress and Promise of Gene Editing


Earlier this year, a report prepared for the National Academies urged caution in developing the gene-editing technology known as CRISPR-Cas9, but stopped short of calling for an outright ban. Click here to read MedPage Today’s original report on the Academies’ position. In this follow-up, we review further developments with CRISPR and its regulation.

New technologies such as the CRISPR-Cas9 offer the possibility of altering an individual’s genome, or even a generation’s genome.

Jennifer Doudna, PhD, a geneticist and professor at the University of California Berkeley and the Howard Hughes Medical Institute, created CRISPR in collaboration with Emmanuelle Charpentier, PhD, of Umea University in Sweden, in 2012.

Out of fear the technology could be misused, Doudna advocated a worldwide moratorium on gene editing that involved heritable changes.

Thus far, no researchers have publicly stated that they have made germline alterations in a human embryo with the intent of nurturing it to birth. But over the past year they have inched closer.

In August, researchers at Oregon Health & Science University in Portland, Oregon, led by biologist Shoukhrat Mitalipov, PhD, for the first time in the U.S. demonstrated the potential to edit human embryo DNA to prevent a congenital heart condition known as hypertrophic cardiomyopathy, which may cause heart failure or sudden death.

Then in October, the New Scientist reported that the CRISPR method was showing promise across a range of diseases in animal studies, including in muscular dystrophy and liver disease.

 Most of the research involved ex vivoexperiments — removing cells, editing them in a lab and then replacing them.

While this process is “relatively easy” for immune cells or blood stem cells, “this isn’t possible with most bodily tissues,” noted the New Scientist’s Michael LePage.

Matthew Porteus, MD, PhD, associate professor of pediatrics at Stanford University and an NAM committee member, told a Senate, Health Education, Labor and Pensions Committee in November that the best approach for other conditions such as congenital blindness and muscular dystrophy likely involves in vivo gene editing.

Regarding other conditions studied through ex vivo experiments, Porteus said his lab developed a method for correcting mutations of sickle cells in patients’ stem cells. If a cure is found, it might take only a few “tweaks” to then find a cure for other illnesses, such as severe combined immunodeficiency, he noted.

He anticipates seeing multiple CRISPR-Cas9 clinical trials in the U.S. or Europe in the next 12-18 months, Porteus added.

What’s Next for Gene Editing?

Asked about the most notable breakthroughs in the field right now, R. Alta Charo, JD, co-chair of the Committee on Human Gene Editing and a professor at the University of Wisconsin in Madison, spoke of “the developing capacity to do epigenetic editing,” speaking on her own behalf, in an email to MedPage Today.

“[I]t offers the prospect of making beneficial changes that, because they are reversible, in many cases will pose fewer risks,” she said.

As another benefit, this form of gene editing could be used to respond to conditions that stem from a “constellation of genetic factors” rather than a single mutation, reported Wired.

Researchers have already begun testing epigenetic editing in mice for diseases such as diabetes, acute kidney disease, and muscular dystrophy, Wired noted.

“Successful somatic gene therapy” and the OHSU study “pending confirmation by the scientific community” are the most notable breakthroughs of the year, said Jeffrey Kahn, PhD, MPH, director of the Johns Hopkins Berman Institute of Bioethics in Baltimore, and a member of the NAM committee, in an email to MedPage Today.

However, he noted that pre-implantation genetic diagnosis could have replaced gene editing in the OHSU study. In other words, the researchers ignored one of the NAM committee’s key criteria for heritable gene editing: lack of a reasonable alternative.

Others disagreed.

Because the study did not involve a pregnancy or birth “it constitutes purely laboratory research” and would be “permissible” under committee guidelines, said Charo.

“An emerging area in gene editing is harnessing these new precision engineering tools to edit regions of the genome outside of genes,” said Neville Sanjana, PhD, a core faculty member at the New York Genome Center and a professor at New York University, in an email, responding to the same question.

The ‘Dark Genome’ Emerges as Target

Gene editing tools can help to translate these regulatory and noncoding variants, those outside of the genes — less than 2% are actually in the genes themselves.

This area is sometimes referred to as the “dark genome.”

“Most of our genome is actually this ‘noncoding’ DNA and not in genes (less than 2% is in genes). We understand very little about how this noncoding DNA works and how changes in the sequence (primary sequence — not epigenome) results in changes in gene expression and disease,” said Sanjana.

Sanjana also highlighted the approval of “a multitude” of new gene therapies by the FDA for conditions such as congenital blindness, spinal muscular atrophy and different hemophilias, which he said has also generated a lot of excitement.

Although some gene therapies have been around since the 1990s, not all involve gene editing. However, the approvals represent progress, he noted.

“It is clear that this new modality of therapy — adding back a missing or damaged gene — will open new avenues of medicine,” said Sanjana.

“[I]t is a matter of time before gene editing tools are also part of the gene therapy arsenal to aid in curing disease for which we currently have no therapies,” he added.

As always, oversight will remain important to this process.

“The challenge is to find the ‘just right’ regulatory approach for what are new, emerging, and controversial biotechnologies such as gene editing tools. That often requires some tweaking to get right, and I hope that there is willingness to engage in the discussion necessary to find the appropriate balance of control with a path for innovation,” wrote Kahn.

Should Gene Editing Be a Human Right?


IN BRIEF
  • With technology like CRISPR making gene editing easier than ever before, society is divided on the ethical implications of using the tech to alter simply “unwanted” genes.
  • Given the potential of gene editing to drastically change humanity, it’s good that we’re having this debate on what and who it should be used for right now.

GENETIC EDITING FOR ALL

We are all subject to the genetic lottery. That’s how it’s always been, and for a while, we thought that was how it would always be.

Then, in 2014, a gene-editing technology called CRISPR was introduced. With CRISPR, geneticists could edit sections of the genome to alter, add, or remove parts of the DNA sequence. To date, it is by far the easiest way we’ve found to manipulate the genetic code, and it is already paving the way for more efficient and effective treatments of conditions with a genetic component. However, the technology brings with it the potential to manipulate and remove simply “unwanted” genes.
The potential to change someone’s DNA even before they are born has led to claims that CRISPR will be used to create “designer babies.” Detractors were appalled at the hubris of science being used to engineer the human race. Supporters, on the other hand, are saying this ability should be a human right.While most of the proposed CRISPR applications are focused on editing somatic (non-reproductive) cells, altering germline (reproductive) cells is also a very real possibility. This prospect of editing germline cells and making changes that would be passed on from generation to generation has sparked a heated ethical debate.

RIGGING THE GAME

To be fair, most advocates of genetic editing aren’t rallying for support so CRISPR can be used to create a superior human race. Rather, they believe people should have free access to technology that is capable of curing diseases. It’s not about rigging the genetic game — it’s about putting the technique to good use while following a set of ethical recommendations.

To that end, a panel made up of experts chosen by the National Academy of Sciences and the National Academy of Medicine released a series of guidelines that essentially gives gene editing a “yellow light.” These guidelines supports gene editing on the premise that it follows a set of stringent rules and is conducted with proper oversight and precaution.

Obviously, genetic enhancement would not be supported under these guidelines, which leaves some proponents miffed. Josiah Zaynor, whose online company The ODIN sells kits allowing people to conduct simple genetic engineering experiments at home, is among those who are adamant that gene editing should be a human right. He expressed his views on the subject in an interview with The Outline:

We are at the first time in the history of humanity where we can no longer be stuck with the genes we are dealt. As a society we have begun to see how choice is a right, but for some reason when it comes to genetics, some people think we shouldn’t have a choice. I can be smart and attractive, but everyone else should be ugly, fat, and short because those are the genes they were dealt and they should just deal with it.

However, scientific institutions continue to caution against such lax views of genetic editing’s implications. Apart from the ethical questions it raises, CRISPR also faces opposition from various religious sects and legal concerns regarding the technology. Governments seem divided on the issue, with nations like China advancing research, while countries like the U.K., Germany, and the U.S. seem more concerned about regulating it.

The immense potential of gene editing to change humanity means the technology will continue to be plagued by ethical and philosophical concerns. Given the pace of advancement, however, it’s good that we’re having this debate on what and who it should be used for right now.

Gene Editing is Leading to a New Age in Human Health and Longevity


IN BRIEF
  • Scientific advances are allowing us to personalize the medical revolution — but how many lives can we actually save with gene therapy?
  • Gene-editing therapy was successfully used to treat a leukemia patient, and now scientists are hoping it will help other patients, including those with diseases other than cancer.

THE MAGIC OF GENE EDITING

Advances in medicine and technology are revolutionizing what it means to be human. By providing us with gene editing tools, such as CRISPR, we’re well on our way to personalizing the medical revolution.

CRISPR provides a way for us to alter gene expression in particular cells, based on need. Up until recently, the process was very difficult to execute. It took many years to develop precision when altering gene expression. With the latest technology, the desired precision can be obtained in just a few weeks. CRISPR, and other technologies like it, are shaping the future of medicine.

When all possible treatments had failed, the parents of Layla, a 1-year-old with leukemia, sought help from new technology developed by Cellectis — a biopharmaceutical company based in Paris, France. The gene editing therapy, which was still experimental, had been utilized once before in a patient with HIV.

Layla and her parents have immunologist Waseem Qasim and his team to thank. While the treatment was approved for Layla under her particular circumstance, the Great Ormond Street Hospital for Children NHS Trust in London intends to continue the trial in 10 to 12 patients in the upcoming year. Several months after the procedure, Qasim notes Layla is doing well.

HOW GENE EDITING WORKS

While Layla’s doctors believe she is in remission, only time will tell if this was a “one-off” fix or a case that may need revisiting. Additional trials are needed so physicians and scientists can better understand how gene-editing can benefit patients, and treat diseases other than cancer.Physicians and scientists worked together to give Layla immune cells from a healthy donor that had been modified with a gene editing tool. In this case, TALEN — a DNA-cutting enzyme — was utilized to modify the donor T-cells so that they would not attack Layla’s own cells. In order for the treatment to work, a patient’s immune system is essentially destroyed and replaced with the modified cells. However, this is not a permanent fix: it’s just a temporary solution until a matching T-cell donor can be found.

Treating cancer isn’t the full extent of gene editing by any means: we can halt the spread of malaria by looking at mosquitos, bring back species gone extinct by the unforgiving hand of human industrialization, or even restore vision in patients.

With the gene editing, it seems the possibilities are only the beginning.

CRISPR: gene editing is just the beginning


The real power of the biological tool lies in exploring how genomes work.

Molecular biologists are riding a wave of new technologies made possible by CRISPR.

Whenever a paper about CRISPR–Cas9 hits the press, the staff at Addgene quickly find out. The non-profit company is where study authors often deposit molecular tools that they used in their work, and where other scientists immediately turn to get them. It is also where other scientists immediately turn to get their hands on these reagents. “We get calls within minutes of a hot paper publishing,” says Joanne Kamens, executive director of the company in Cambridge, Massachusetts.

Addgene’s phones have been ringing a lot since early 2013, when researchers first reported1, 2, 3 that they had used the CRISPR–Cas9 system to slice the genome in human cells at sites of their choosing. “It was all hands on deck,” Kamens says. Since then, molecular biologists have rushed to adopt the technique, which can be used to alter the genome of almost any organism with unprecedented ease and finesse. Addgene has sent 60,000 CRISPR-related molecular tools — about 17% of its total shipments — to researchers in 83 countries, and the company’s CRISPR-related pages were viewed more than one million times in 2015.

Much of the conversation about CRISPR–Cas9 has revolved around its potential for treating disease or editing the genes of human embryos, but researchers say that the real revolution right now is in the lab. What CRISPR offers, and biologists desire, is specificity: the ability to target and study particular DNA sequences in the vast expanse of a genome. And editing DNA is just one trick that it can be used for. Scientists are hacking the tools so that they can send proteins to precise DNA targets to toggle genes on or off, and even engineer entire biological circuits — with the long-term goal of understanding cellular systems and disease.

“For the humble molecular biologist, it’s really an extraordinarily powerful way to understand how the genome works,” says Daniel Bauer, a haematologist at the Boston Children’s Hospital in Massachusetts. “It’s really opened the number of questions you can address,” adds Peggy Farnham, a molecular biologist at the University of Southern California, Los Angeles. “It’s just so fun.”

Here, Nature examines five ways in which CRISPR–Cas9 is changing how biologists can tinker with cells.

Broken scissors

There are two chief ingredients in the CRISPR–Cas9 system: a Cas9 enzyme that snips through DNA like a pair of molecular scissors, and a small RNA molecule that directs the scissors to a specific sequence of DNA to make the cut. The cell’s native DNA repair machinery generally mends the cut — but often makes mistakes.

That alone is a boon to scientists who want to disrupt a gene to learn about what it does. The genetic code is merciless: a minor error introduced during repair can completely alter the sequence of the protein it encodes, or halt its production altogether. As a result, scientists can study what happens to cells or organisms when the protein or gene is hobbled.

But there is also a different repair pathway that sometimes mends the cut according to a DNA template. If researchers provide the template, they can edit the genome with nearly any sequence they desire at nearly any site of their choosing.

In 2012, as laboratories were racing to demonstrate how well these gene-editing tools could cut human DNA, one team decided to take a different approach. “The first thing we did: we broke the scissors,” says Jonathan Weissman, a systems biologist at the University of California, San Francisco (UCSF).

Weissman learned about the approach from Stanley Qi, a synthetic biologist now at Stanford University in California, who mutated the Cas9 enzyme so that it still bound DNA at the site that matched its guide RNA, but no longer sliced it. Instead, the enzyme stalled there and blocked other proteins from transcribing that DNA into RNA. The hacked system allowed them to turn a gene off, but without altering the DNA sequence4.

The team then took its ‘dead’ Cas9 and tried something new: the researchers tethered it to part of another protein, one that activates gene expression. With a few other tweaks, they had built a way to turn genes on and off at will5.

Several labs have since published variations on this method; many more are racing to harness it for their research6 (see ‘Hacking CRISPR’). One popular application is to rapidly generate hundreds of different cell lines, each containing a different guide RNA that targets a particular gene. Martin Kampmann, another systems biologist at UCSF, hopes to screen such cells to learn whether flipping certain genes on or off affects the survival of neurons exposed to toxic protein aggregates — a mechanism that is thought to underlie several neurodegenerative conditions, including Alzheimer’s disease. Kampmann had been carrying out a similar screen with RNA interference (RNAi), a technique that also silences genes and can process lots of molecules at once, but which has its drawbacks. “RNAi is a shotgun with well-known off-target effects,” he says. “CRISPR is the scalpel that allows you to be more specific.”

Nik Spencer/Nature

Weissman and his colleagues, including UCSF systems biologist Wendell Lim, further tweaked the method so that it relied on a longer guide RNA, with motifs that bound to different proteins. This allowed them to activate or inhibit genes at three different sites all in one experiment7. Lim thinks that the system can handle up to five operations at once. The limit, he says, may be in how many guide RNAs and proteins can be stuffed into a cell. “Ultimately, it’s about payload.”

That combinatorial power has drawn Ron Weiss, a synthetic biologist at the Massachusetts Institute of Technology (MIT) in Cambridge, into the CRISPR–Cas9 frenzy. Weiss and his colleagues have also created multiple gene tweaks in a single experiment8, making it faster and easier to build complicated biological circuits that could, for example, convert a cell’s metabolic machinery into a biofuel factory. “The most important goal of synthetic biology is to be able to program complex behaviour via the creation of these sophisticated circuits,” he says.

CRISPR epigenetics

When geneticist Marianne Rots began her career, she wanted to unearth new medical cures. She studied gene therapy, which targets genes mutated in disease. But after a few years, she decided to change tack. “I reasoned that many more diseases are due to disturbed gene-expression profiles, not so much the single genetic mutations I had been focused on,” says Rots, at the University Medical Center Groningen in the Netherlands. The best way to control gene activity, she thought, was to adjust the epigenome, rather than the genome itself.

The epigenome is the constellation of chemical compounds tacked onto DNA and the DNA-packaging proteins called histones. These can govern access to DNA, opening it up or closing it off to the proteins needed for gene expression. The marks change over time: they are added and removed as an organism develops and its environment shifts.

In the past few years, millions of dollars have been poured into cataloguing these epigenetic marks in different human cells, and their patterns have been correlated with everything from brain activity to tumour growth. But without the ability to alter the marks at specific sites, researchers are unable to determine whether they cause biological changes. “The field has met a lot of resistance because we haven’t had the kinds of tools that geneticists have had, where they can go in and directly test the function of a gene,” says Jeremy Day, a neuroscientist at the University of Alabama at Birmingham.

CRISPR–Cas9 could turn things around. In April 2015, Charles Gersbach, a bioengineer at Duke University in Durham, North Carolina, and his colleagues published9 a system for adding acetyl groups — one type of epigenetic mark — to histones using the broken scissors to carry enzymes to specific spots in the genome.

The team found that adding acetyl groups to proteins that associate with DNA was enough to send the expression of targeted genes soaring, confirming that the system worked and that, at this location, the epigenetic marks had an effect. When he published the work, Gersbach deposited his enzyme with Addgene so that other research groups could use it — and they quickly did. Gersbach predicts that a wave of upcoming papers will show a synergistic effect when multiple epigenetic markers are manipulated at once.

The tools need to be refined. Dozens of enzymes can create or erase an epigenetic mark on DNA, and not all of them have been amenable to the broken-scissors approach. “It turned out to be harder than a lot of people were expecting,” says Gersbach. “You attach a lot of things to a dead Cas9 and they don’t happen to work.” Sometimes it is difficult to work out whether an unexpected result arose because a method did not work well, or because the epigenetic mark simply doesn’t matter in that particular cell or environment.

Rots has explored the function of epigenetic marks on cancer-related genes using older editing tools called zinc-finger proteins, and is now adopting CRISPR–Cas9. The new tools have democratized the field, she says, and that has already had a broad impact. People used to say that the correlations were coincidental, Rots says — that if you rewrite the epigenetics it will have no effect on gene expression. “But now that it’s not that difficult to test, a lot of people are joining the field.”

CRISPR code cracking

Epigenetic marks on DNA are not the only genomic code that is yet to be broken. More than 98% of the human genome does not code for proteins. But researchers think that a fair chunk of this DNA is doing something important, and they are adopting CRISPR–Cas9 to work out what that is.

Some of it codes for RNA molecules — such as microRNAs and long non-coding RNAs — that are thought to have functions apart from making proteins. Other sequences are ‘enhancers’ that amplify the expression of the genes under their command. Most of the DNA sequences linked to the risk of common diseases lie in regions of the genome that contain non-coding RNA and enhancers. But before CRISPR–Cas9, it was difficult for researchers to work out what those sequences do. “We didn’t have a good way to functionally annotate the non-coding genome,” says Bauer. “Now our experiments are much more sophisticated.”

Farnham and her colleagues are using CRISPR–Cas9 to delete enhancer regions that are found to be mutated in genomic studies of prostate and colon cancer. The results have sometimes surprised her. In one unpublished experiment, her team deleted an enhancer that was thought to be important, yet no gene within one million bases of it changed expression. “How we normally classify the strength of a regulatory element is not corresponding with what happens when you delete that element,” she says.

“I wish I had had this technology sooner. My postdoc would have been a lot shorter.”

More surprises may be in store as researchers harness CRISPR–Cas9 to probe large stretches of regulatory DNA. Groups led by geneticists David Gifford at MIT and Richard Sherwood at the Brigham and Women’s Hospital in Boston used the technique to create mutations across a 40,000-letter sequence, and then examined whether each change had an effect on the activity of a nearby gene that made a fluorescent protein10. The result was a map of DNA sequences that enhanced gene expression, including several that had not been predicted on the basis of gene regulatory features such as chromatin modifications.

Delving into this dark matter has its challenges, even with CRISPR–Cas9. The Cas9 enzyme will cut where the guide RNA tells it to, but only if a specific but common DNA sequence is present near the cut site. This poses little difficulty for researchers who want to silence a gene, because the key sequences almost always exist somewhere within it. But for those who want to make very specific changes to short, non-coding RNAs, the options can be limited. “We cannot take just any sequence,” says Reuven Agami, a researcher at the Netherlands Cancer Institute in Amsterdam.

Researchers are scouring the bacterial kingdom for relatives of the Cas9 enzyme that recognize different sequences. Last year, the lab of Feng Zhang, a bioengineer at the Broad Institute of MIT and Harvard in Cambridge, characterized a family of enzymes called Cpf1 that work similarly to Cas9 and could expand sequence options11. But Agami notes that few alternative enzymes found so far work as well as the most popular Cas9. In the future, he hopes to have a whole collection of enzymes that can be targeted to any site in the genome. “We’re not there yet,” he says.

CRISPR sees the light

Gersbach’s lab is using gene-editing tools as part of an effort to understand cell fate and how to manipulate it: the team hopes one day to grow tissues in a dish for drug screening and cell therapies. But CRISPR–Cas9’s effects are permanent, and Gersbach’s team needed to turn genes on and off transiently, and in very specific locations in the tissue. “Patterning a blood vessel demands a high degree of control,” he says.

Gersbach and his colleagues took their broken, modified scissors — the Cas9 that could now activate genes — and added proteins that are activated by blue light. The resulting system triggers gene expression when cells are exposed to the light, and stops it when the light is flicked off12. A group led by chemical biologist Moritoshi Sato of the University of Tokyo rigged a similar system13, and also made an active Cas9 that edited the genome only after it was hit with blue light14.

Others have achieved similar ends by combining CRISPR with a chemical switch. Lukas Dow, a cancer geneticist at Weill Cornell Medical College in New York City, wanted to mutate cancer-related genes in adult mice, to reproduce mutations that have been identified in human colorectal cancers. His team engineered a CRISPR–Cas9 system in which a dose of the compound doxycycline activates Cas9, allowing it to cut its targets15.

The tools are another step towards gaining fine control over genome editing. Gersbach’s team has not patterned its blood vessels just yet: for now, the researchers are working on making their light-inducible system more efficient. “It’s a first-generation tool,” says Gersbach.

Model CRISPR

Cancer researcher Wen Xue spent the first years of his postdoc career making a transgenic mouse that bore a mutation found in some human liver cancers. He slogged away, making the tools necessary for gene targeting, injecting them into embryonic stem cells and then trying to derive mice with the mutation. The cost: a year and US$20,000. “It was the rate-limiting step in studying disease genes,” he says.

A few years later, just as he was about to embark on another transgenic-mouse experiment, his mentor suggested that he give CRISPR–Cas9 a try. This time, Xue just ordered the tools, injected them into single-celled mouse embryos and, a few weeks later — voilá. “We had the mouse in one month,” says Xue. “I wish I had had this technology sooner. My postdoc would have been a lot shorter.”

Researchers who study everything from cancer to neurodegeneration are embracing CRISPR-Cas9 to create animal models of the diseases. It lets them engineer more animals, in more complex ways, and in a wider range of species. Xue, who now runs his own lab at the University of Massachusetts Medical School in Worcester, is systematically sifting through data from tumour genomes, using CRISPR–Cas9 to model the mutations in cells grown in culture and in animals.

Researchers are hoping to mix and match the new CRISPR–Cas9 tools to precisely manipulate the genome and epigenome in animal models. “The real power is going to be the integration of those systems,” says Dow. This may allow scientists to capture and understand some of the complexity of common human diseases.

Take tumours, which can bear dozens of mutations that potentially contribute to cancer development. “They’re probably not all important in terms of modelling a tumour,” says Dow. “But it’s very clear that you’re going to need two or three or four mutations to really model aggressive disease and get closer to modelling human cancer.” Introducing all of those mutations into a mouse the old-fashioned way would have been costly and time-consuming, he adds.

Bioengineer Patrick Hsu started his lab at the Salk Institute for Biological Studies in La Jolla, California, in 2015; he aims to use gene editing to model neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease in cell cultures and marmoset monkeys. That could recapitulate human behaviours and progression of disease more effectively than mouse models, but would have been unthinkably expensive and slow before CRISPR–Cas9.

Even as he designs experiments to genetically engineer his first CRISPR–Cas9 marmosets, Hsu is aware that this approach may be only a stepping stone to the next. “Technologies come and go. You can’t get married to one,” he says. “You need to always think about what biological problems need to be solved.”

Gene editing can drive science to openness


The emergence of gene-drive systems — which spread engineered mutations quickly through populations — means that a single released organism could eventually alter most of its local population, and quite possibly all populations of the species throughout the world. Any accidental release, even if there was no ecological damage, would surely damage public trust and prompt harsh restrictions on research.

The US National Academy of Sciences released guidelines this week for the responsible conduct of gene-drive research. The report comes almost two years after the first published description of how the CRISPR–Cas9 genome-editing technology could enable gene drives in many different organisms. That’s a fast turnaround for the academy, but an eternity for the field: in that time, scientists have demonstrated CRISPR-based gene-drive systems in four species.

The report makes some sensible suggestions, such as phased testing and ecological-risk assessments, but if we’re going to develop proper safeguards for gene drives or other powerful technologies, we need to fix a greater problem: the closed-door nature of science.

No one would rationally design the current scientific enterprise. It is wasteful and inefficient. Researchers repeatedly run into the same problems and unknowingly duplicate efforts. It stunts collaboration: we never learn who has the other piece of a puzzle unless we run into them at a conference. It wastes time on endless grant-writing. It’s terrible for researcher well-being: competitive pressure ruins playful discovery and creation.

And it’s unsafe. Regulation will always be too slow. Science is too vast for researchers to reliably foresee the consequences of their work. The problem was neatly summarized by atom-bomb pioneer Robert Oppenheimer: “When you see something that is technically sweet, you go ahead and do it, and you argue about what to do about it only after you have had your technical success.”

Some technical successes are not to be pursued. But others are desperately needed. How can we hope to tell the difference when science is done behind closed doors?

There are signs of progress. My colleagues and I publicly discussed the probable consequences of a CRISPR-based gene drive before doing any experiments. And many gene-drive researchers have already worked together to improve safety and call for transparency. But this has been done on an informal basis. For example, my group saw a gene-drive paper by another laboratory and was able to suggest changes — the need for extra safeguards to prevent an accidental release — but only because we received an in-press copy of the publication from a journalist.

“Open and responsive science flies in the face of current incentives.”

Sadly, open and responsive science flies in the face of current incentives. Scientists who disclose their ideas are often ‘rewarded’ by being scooped by another lab, rather than by being recognized for their creativity. It is a prisoner’s dilemma. The benefits come from cooperation by every­one. But by participating you risk being exploited by people who steal your idea, get it working before you do, and claim the credit.

Gene-drive research offers a way out. The field is new and small, and many of us have already worked together to publish a joint recommendation calling for future experiments to use multiple stringent confinement strategies. Several groups already disclose proposed and ongoing gene-drive research and invite feedback, and active discussions between researchers and funders seek ways to ensure that everyone will be similarly forthcoming.

My group and others will soon launch the Responsive Science Project to enable gene-drive scientists to share their plans and research with one another and with interested communities. We hope that it will become a central repository of ideas and information relevant to gene-drive research that will permit open assessment and critique before experiments begin.

Journals could help by offering incentives to persuade scientists to share their proposals. When a paper is published by authors who didn’t play by the new rules (to share what they’re doing and collaborate with the people who first shared the key ideas), journals could check the repository to identify scientists who deserve a share of the credit and invite them to write an accompanying piece. Similarly, all funders should require immediate public disclosure of proposals involving gene drives, as well as regular public updates on the status of funded research.

If this attempt at open science works for one field, it could expand to encompass research on other shared-impact technologies and to fields beyond. That alone is reason enough to try the approach. But gene-drive technology is also unique in that its very nature demands a new approach.

Because the consequences of mistakes involving gene-drive organisms could affect communities outside the laboratory, scientists have an obligation to openly share their plans, invite suggestions and concerns, disclose experimental results as soon as possible, and redesign the technology as needed. Applied to gene drives, such an approach will also have a greater chance of earning popular support for applications that could save millions of human lives and rescue numerous species from extinction.

We should ensure that gene-drive research is open and responsive — then drive those changes through the scientific ecosystem.

Controlling CRISPR: Researchers May Have Found An “Off-Switch” for Gene-Editing


IN BRIEF
  • Researchers have uncovered two distinct anti-CRISPR proteins — AcrIIA2 and AcrIIA4 — that worked to inhibit the ability of SpyCas9 to target specific genomes in bacteria.
  • The discovery could lead to an “off switch” for CRISPR that would give scientists greater control over the gene editing tool when using it in human subjects.

CRISPR DISCOVERY

As it stands, CRISPR is a rather impressive gene editing tool and already the most precise method we have available for genetic manipulation. Studies like this recent one from UC Berkeley are helping us refine the CRISPR-Cas9 system, and now a new study published in Cell from UC San Francisco (UCSF) is offering a way to deal with some of its greatest remaining downsides.

Researchers discovered a way to switch off this gene-editing system using recently identified proteins discovered in the lab of Joseph Bondy-Denomy from UCSF’s Department of Microbiology and Immunology. These anti-CRISPR proteins could eventually improve the safety and accuracy of already very accurate CRISPR applications, and the researchers relied on a nifty little trick to discover them.

anticrispr graphic

“Just as CRISPR technology was developed from the natural anti-viral defense systems in bacteria, we can also take advantage of the anti-CRISPR proteins that viruses have sculpted to get around those bacterial defenses,” explains the leader of the study, Benjamin Rauch.

In their research, the team looked for bacterial strains that had been infected by a known virus. They reasoned that their existence would be evidence that a bacteria’s Cas9 was not functioning properly.

“Cas9 isn’t very smart,” according to Bondy-Denomy. “It’s not able to avoid cutting the bacterium’s own DNA if it is programmed to do so. So we looked for strains of bacteria where the CRISPR-Cas9 system ought to be targeting its own genome — the fact that the cells do not self-destruct was a clue that the whole CRISPR system was inactivated.”

After examining nearly 300 strains of Listeria using Rauch’s bioinformatic approach, the team found three strains that showed this evidence. From those, they isolated four distinct anti-CRISPR proteins, and of these four, test showed that two — dubbed AcrIIA2 and AcrIIA4 — worked to inhibit the ability of SpyCas9 to target specific genomes.

A BETTER SYSTEM

“These natural Cas9-specific ‘anti-CRISPRs’ present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9,” the researchers write. They believe that with these proteins, it’s possible to avoid unintended or “off-target” gene modifications caused by keeping CRISPR’s machinery active in the body.

Of course, the next step would be to see how these proteins function in human cells. “We also want to understand exactly how the inhibitor proteins block Cas9’s gene targeting abilities, and continue the search for more and better CRISPR inhibitors in other bacteria,” Raunch explained.

Ultimately, this “off-switch” for CRISPR could prove almost as important as the system itself. “Researchers and the public are reasonably concerned about CRISPR being so powerful that it potentially gets put to dangerous uses,” Bondy-Denomy said. “These inhibitors provide a mechanism to block nefarious or out-of-control CRISPR applications, making it safer to explore all the ways this technology can be used to help people.”

Gene Editing Can Now Change An Entire Species Forever


In Brief

CRISPR has opened up limitless avenues for genetic modification. From disease prevention to invasive species control, Jennifer Kahn discusses the discover, application, and implications of gene drives.

Jennifer Kahn, a science journalist for the New York Times, recently did a TED Talk in which she discussed the discovery, application, and implications of a CRISPR gene drive used to make mosquitoes resistant to malaria and other diseases like chikungunya, and Zika.

Watch the talk in the video below, and learn how geneticists are achieving the (seemingly) impossible:

In pioneering experiments, biologist Anthony James became enamored with the idea of using genetic modification to make the genus of mosquito (Anopheles) that is responsible for transmitting malaria incapable of carrying the disease causing parasite. James was able to create the resistant mosquitoes, but he ran into a roadblock when trying to perpetuate the trait into future generations.

Kahn states that it would take introducing a number of engineered mosquitoes ten times the population of native mosquitoes. This would likely not go over well with the local humans.

In Janurary of 2016, James received word from biologist Ethan Bier that Bier and his grad student Valentino Gantz discovered a means in which biologists can ensure the inheritance of an engineered trait as well as the rapid proliferation of that trait from generation to generation. Prior to these experiments, it had been deemed impossible, due to Mendelian genetics, for an entire generation to inherit a specific trait.

Against conventional understanding, this is exactly what happened.

Two mosquitoes engineered to have red eyes were introduced to 30 white eyed mosquitoes. Two generations later, there were 3,800 new mosquitoes, all of which had red eyes.

This discovery opens up a world of possibilities outside of disease prevention such as invasive species elimination among countless others. These possibilities present some serious bioethical questions for the use of, or abstinence from, this form of modification. Kahn uses Asian carp as an example. While Asian carp is an invasive species in the Great Lakes, the species could go extinct if modified carp were introduced to their natural habitat.

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