Breakthrough DNA Editor Born of Bacteria


Interest in a powerful DNA editing tool called CRISPR has revealed that bacteria are far more sophisticated than anyone imagined.

Microbes such as E. coli may use CRISPR as a weapon in their millions-year-old struggle against viruses.

 

On a November evening last year, Jennifer Doudna put on a stylish black evening gown and headed to Hangar One, a building at NASA’s Ames Research Center that was constructed in 1932 to house dirigibles. Under the looming arches of the hangar, Doudna mingled with celebrities like Benedict Cumberbatch, Cameron Diaz and Jon Hamm before receiving the 2015 Breakthrough Prize in life sciences, an award sponsored by Mark Zuckerberg and other tech billionaires. Doudna, a biochemist at the University of California, Berkeley, and her collaborator, Emmanuelle Charpentier of the Helmholtz Centre for Infection Research in Germany, each received $3 million for their invention of a potentially revolutionary tool for editing DNA known as CRISPR.

Doudna was not a gray-haired emerita being celebrated for work she did back when dirigibles ruled the sky. It was only in 2012 that Doudna, Charpentier and their colleagues offered the first demonstration of CRISPR’s potential. They crafted molecules that could enter a microbe and precisely snip its DNA at a location of the researchers’ choosing. In January 2013, the scientists went one step further: They cut out a particular piece of DNA in human cells and replaced it with another one.

In the same month, separate teams of scientists at Harvard University and the Broad Institute reported similar success with the gene-editing tool. A scientific stampede commenced, and in just the past two years, researchers have performed hundreds of experiments on CRISPR. Their results hint that the technique may fundamentally change both medicine and agriculture.

Some scientists have repaired defective DNA in mice, for example, curing them of genetic disorders. Plant scientists have used CRISPR to edit genes in crops, raising hopes that they can engineer a better food supply. Some researchers are trying to rewrite the genomes of elephants, with the ultimate goal of re-creating a woolly mammoth. Writing last year in the journal Reproductive Biology and Endocrinology, Motoko Araki and Tetsuya Ishii of Hokkaido University in Japan predicted that doctors will be able to use CRISPR to alter the genes of human embryos “in the immediate future.”

Thanks to the speed of CRISPR research, the accolades have come quickly. Last year MIT Technology Review called CRISPR “the biggest biotech discovery of the century.” The Breakthrough Prize is just one of several prominent awards Doudna has won in recent months for her work on CRISPR; National Public Radio recently reported whispers of a possible Nobel in her future.

Even the pharmaceutical industry, which is often slow to embrace new scientific advances, is rushing to get in on the act. New companies developing CRISPR-based medicine are opening their doors. In January, the pharmaceutical giant Novartis announced that it would be using Doudna’s CRISPR technology for its research into cancer treatments. It plans to edit the genes of immune cells so that they will attack tumors.

But amid all the black-tie galas and patent filings, it’s easy to overlook the most important fact about CRISPR: Nobody actually invented it.

Doudna and other researchers did not pluck the molecules they use for gene editing from thin air. In fact, they stumbled across the CRISPR molecules in nature. Microbes have been using them to edit their own DNA for millions of years, and today they continue to do so all over the planet, from the bottom of the sea to the recesses of our own bodies.

We’ve barely begun to understand how CRISPR works in the natural world. Microbes use it as a sophisticated immune system, allowing them to learn to recognize their enemies. Now scientists are discovering that microbes use CRISPR for other jobs as well. The natural history of CRISPR poses many questions to scientists, for which they don’t have very good answers yet. But it also holds great promise. Doudna and her colleagues harnessed one type of CRISPR, but scientists are finding a vast menagerie of different types. Tapping that diversity could lead to more effective gene editing technology, or open the way to applications no one has thought of yet.

“You can imagine that many labs — including our own — are busily looking at other variants and how they work,” Doudna said. “So stay tuned.”

A Repeat Mystery

The scientists who discovered CRISPR had no way of knowing that they had discovered something so revolutionary. They didn’t even understand what they had found. In 1987, Yoshizumi Ishino and colleagues at Osaka University in Japan published the sequence of a gene called iap belonging to the gut microbe E. coli. To better understand how the gene worked, the scientists also sequenced some of the DNA surrounding it. They hoped to find spots where proteins landed, turning iap on and off. But instead of a switch, the scientists found something incomprehensible.

Near the iap gene lay five identical segments of DNA. DNA is made up of building blocks called bases, and the five segments were each composed of the same 29 bases. These repeat sequences were separated from each other by 32-base blocks of DNA, called spacers. Unlike the repeat sequences, each of the spacers had a unique sequence.

This peculiar genetic sandwich didn’t look like anything biologists had found before. When the Japanese researchers published their results, they could only shrug. “The biological significance of these sequences is not known,” they wrote.

It was hard to know at the time if the sequences were unique to E. coli, because microbiologists only had crude techniques for deciphering DNA. But in the 1990s, technological advances allowed them to speed up their sequencing. By the end of the decade, microbiologists could scoop up seawater or soil and quickly sequence much of the DNA in the sample. This technique — called metagenomics — revealed those strange genetic sandwiches in a staggering number of species of microbes. They became so common that scientists needed a name to talk about them, even if they still didn’t know what the sequences were for. In 2002, Ruud Jansen of Utrecht University in the Netherlands and colleagues dubbed these sandwiches “clustered regularly interspaced short palindromic repeats” — CRISPR for short.

Jansen’s team noticed something else about CRISPR sequences: They were always accompanied by a collection of genes nearby. They called these genes Cas genes, for CRISPR-associated genes. The genes encoded enzymes that could cut DNA, but no one could say why they did so, or why they always sat next to the CRISPR sequence.

Three years later, three teams of scientists independently noticed something odd about CRISPR spacers. They looked a lot like the DNA of viruses.

“And then the whole thing clicked,” said Eugene Koonin.

At the time, Koonin, an evolutionary biologist at the National Center for Biotechnology Information in Bethesda, Md., had been puzzling over CRISPR and Cas genes for a few years. As soon as he learned of the discovery of bits of virus DNA in CRISPR spacers, he realized that microbes were using CRISPR as a weapon against viruses.

Koonin knew that microbes are not passive victims of virus attacks. They have several lines of defense. Koonin thought that CRISPR and Cas enzymes provide one more. In Koonin’s hypothesis, bacteria use Cas enzymes to grab fragments of viral DNA. They then insert the virus fragments into their own CRISPR sequences. Later, when another virus comes along, the bacteria can use the CRISPR sequence as a cheat sheet to recognize the invader.

Scientists didn’t know enough about the function of CRISPR and Cas enzymes for Koonin to make a detailed hypothesis. But his thinking was provocative enough for a microbiologist named Rodolphe Barrangou to test it. To Barrangou, Koonin’s idea was not just fascinating, but potentially a huge deal for his employer at the time, the yogurt maker Danisco. Danisco depended on bacteria to convert milk into yogurt, and sometimes entire cultures would be lost to outbreaks of bacteria-killing viruses. Now Koonin was suggesting that bacteria could use CRISPR as a weapon against these enemies.

To test Koonin’s hypothesis, Barrangou and his colleagues infected the milk-fermenting microbe Streptococcus thermophilus with two strains of viruses. The viruses killed many of the bacteria, but some survived. When those resistant bacteria multiplied, their descendants turned out to be resistant too. Some genetic change had occurred. Barrangou and his colleagues found that the bacteria had stuffed DNA fragments from the two viruses into their spacers. When the scientists chopped out the new spacers, the bacteria lost their resistance.

Barrangou, now an associate professor at North Carolina State University, said that this discovery led many manufacturers to select for customized CRISPR sequences in their cultures, so that the bacteria could withstand virus outbreaks. “If you’ve eaten yogurt or cheese, chances are you’ve eaten CRISPR-ized cells,” he said.

Cut and Paste

As CRISPR started to give up its secrets, Doudna got curious. She had already made a name for herself as an expert on RNA, a single-stranded cousin to DNA. Originally, scientists had seen RNA’s main job as a messenger. Cells would make a copy of a gene using RNA, and then use that messenger RNA as a template for building a protein. But Doudna and other scientists illuminated many other jobs that RNA can do, such as acting as sensors or controlling the activity of genes.

In 2007, Blake Wiedenheft joined Doudna’s lab as a postdoctoral researcher, eager to study the structure of Cas enzymes to understand how they worked. Doudna agreed to the plan — not because she thought CRISPR had any practical value, but just because she thought the chemistry might be cool. “You’re not trying to get to a particular goal, except understanding,” she said.

As Wiedenheft, Doudna and their colleagues figured out the structure of Cas enzymes, they began to see how the molecules worked together as a system. When a virus invades a microbe, the host cell grabs a little of the virus’s genetic material, cuts open its own DNA, and inserts the piece of virus DNA into a spacer.

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

This video illustrates how CRISPR and Cas9 can help microbes fight viruses and how researchers might use that system to edit human genes.

 

As CRISPR’s biology emerged, it began to make other microbial defenses look downright primitive. Using CRISPR, microbes could, in effect, program their enzymes to seek out any short sequence of DNA and attack it exclusively.

“Once we understood it as a programmable DNA-cutting enzyme, there was an interesting transition,” Doudna said. She and her colleagues realized there might be a very practical use for CRISPR. Doudna recalls thinking, “Oh my gosh, this could be a tool.”

It wasn’t the first time a scientist had borrowed a trick from microbes to build a tool. Some microbes defend themselves from invasion by using molecules known as restriction enzymes. The enzymes chop up any DNA that isn’t protected by molecular shields. The microbes shield their own genes, and then attack the naked DNA of viruses and other parasites. In the 1970s, molecular biologists figured out how to use restriction enzymes to cut DNA, giving birth to the modern biotechnology industry.

In the decades that followed, genetic engineering improved tremendously, but it couldn’t escape a fundamental shortcoming: Restriction enzymes did not evolve to make precise cuts — only to shred foreign DNA. As a result, scientists who used restriction enzymes for biotechnology had little control over where their enzymes cut open DNA.

The CRISPR-Cas system, Doudna and her colleagues realized, had already evolved to exert just that sort of control.

To create a DNA-cutting tool, Doudna and her colleagues picked out the CRISPR-Cas system from Streptococcus pyogenes, the bacteria that cause strep throat. It was a system they already understood fairly well, having worked out the function of its main enzyme, called Cas9. Doudna and her colleagues figured out how to supply Cas9 with an RNA molecule that matched a sequence of DNA they wanted to cut. The RNA molecule then guided Cas9 along the DNA to the target site, and then the enzyme made its incision.

Using two Cas9 enzymes, the scientists could make a pair of snips, chopping out any segment of DNA they wanted. They could then coax a cell to stitch a new gene into the open space. Doudna and her colleagues thus invented a biological version of find-and-replace — one that could work in virtually any species they chose to work on.

As important as these results were, microbiologists were also grappling with even more profound implications of CRISPR. It showed them that microbes had capabilities no one had imagined before.

Before the discovery of CRISPR, all the defenses that microbes were known to use against viruses were simple, one-size-fits-all strategies. Restriction enzymes, for example, will destroy any piece of unprotected DNA. Scientists refer to this style of defense as innate immunity. We have innate immunity, too, but on top of that, we also use an entirely different immune system to fight pathogens: one that learns about our enemies.

This so-called adaptive immune system is organized around a special set of immune cells that swallow up pathogens and then present fragments of them, called antigens, to other immune cells. If an immune cell binds tightly to an antigen, the cell multiplies. The process of division adds some random changes to the cell’s antigen receptor genes. In a few cases, the changes alter the receptor in a way that lets it grab the antigen even more tightly. Immune cells with the improved receptor then multiply even more.

This cycle results in an army of immune cells with receptors that can bind quickly and tightly to a particular type of pathogen, making them into precise assassins. Other immune cells produce antibodies that can also grab onto the antigens and help kill the pathogen. It takes a few days for the adaptive immune system to learn to recognize the measles virus, for instance, and wipe it out. But once the infection is over, we can hold onto these immunological memories. A few immune cells tailored to measles stay with us for our lifetime, ready to attack again.

CRISPR, microbiologists realized, is also an adaptive immune system. It lets microbes learn the signatures of new viruses and remember them. And while we need a complex network of different cell types and signals to learn to recognize pathogens, a single-celled microbe has all the equipment necessary to learn the same lesson on its own.

A New Kind of Evolution

CRISPR is an impressive adaptive immune system for another reason: Its lessons can be inherited. People can’t pass down genes for antibodies to their children because only immune cells develop them. There’s no way for that information to get into eggs or sperm. As a result, children have to start learning about their invisible enemies pretty much from scratch.

CRISPR is different. Since microbes are single-celled organisms, the DNA they alter to fight viruses is the same DNA they pass down to their descendants. In other words, the experiences that these organisms have alter their genes, and that change is inherited by future generations.

For students of the history of biology, this kind of heredity echoes a largely discredited theory promoted by the naturalist Jean-Baptiste Lamarck in the early 19th century. Lamarck argued for the inheritance of acquired traits. To illustrate his theory, he had readers imagine a giraffe gaining a long neck by striving to reach high branches to feed on. A nervous fluid, he believed, stretched out its neck, making it easier for the giraffe to reach the branches. It then passed down its lengthened neck to its descendants.

The advent of genetics seemed to crush this idea. There didn’t appear to be any way for experiences to alter the genes that organisms passed down to their offspring. But CRISPR revealed that microbes rewrite their DNA with information about their enemies — information that Barrangou showed could make the difference between life and death for their descendants.

Did this mean that CRISPR meets the requirements for Lamarckian inheritance? “In my humble opinion, it does,” said Koonin.

But how did microbes develop these abilities? Ever since microbiologists began discovering CRISPR-Cas systems in different species, Koonin and his colleagues have been reconstructing the systems’ evolution. CRISPR-Cas systems use a huge number of different enzymes, but all of them have one enzyme in common, called Cas1. The job of this universal enzyme is to grab incoming virus DNA and insert it in CRISPR spacers. Recently, Koonin and his colleagues discovered what may be the origin of Cas1 enzymes.

Along with their own genes, microbes carry stretches of DNA called mobile elements that act like parasites. The mobile elements contain genes for enzymes that exist solely to make new copies of their own DNA, cut open their host’s genome, and insert the new copy. Sometimes mobile elements can jump from one host to another, either by hitching a ride with a virus or by other means, and spread through their new host’s genome.

Koonin and his colleagues discovered that one group of mobile elements, called casposons, makes enzymes that are pretty much identical to Cas1. In a new paper in Nature Reviews Genetics, Koonin and Mart Krupovic of the Pasteur Institute in Paris argue that the CRISPR-Cas system got its start when mutations transformed casposons from enemies into friends. Their DNA-cutting enzymes became domesticated, taking on a new function: to store captured virus DNA as part of an immune defense.

While CRISPR may have had a single origin, it has blossomed into a tremendous diversity of molecules. Koonin is convinced that viruses are responsible for this. Once they faced CRISPR’s powerful, precise defense, the viruses evolved evasions. Their genes changed sequence so that CRISPR couldn’t latch onto them easily. And the viruses also evolved molecules that could block the Cas enzymes. The microbes responded by evolving in their turn. They acquired new strategies for using CRISPR that the viruses couldn’t fight. Over many thousands of years, in other words, evolution behaved like a natural laboratory, coming up with new recipes for altering DNA.

The Hidden Truth

To Konstantin Severinov, who holds joint appointments at Rutgers University and the Skolkovo Institute of Science and Technology in Russia, these explanations for CRISPR may turn out to be true, but they barely begin to account for its full mystery. In fact, Severinov questions whether fighting viruses is the chief function of CRISPR. “The immune function may be a red herring,” he said.

Severinov’s doubts stem from his research on the spacers of E. coli. He and other researchers have amassed a database of tens of thousands of E. coli spacers, but only a handful of them match any virus known to infect E. coli. You can’t blame this dearth on our ignorance of E. coli or its viruses, Severinov argues, because they’ve been the workhorses of molecular biology for a century. “That’s kind of mind-boggling,” he said.

It’s possible that the spacers came from viruses, but viruses that disappeared thousands of years ago. The microbes kept holding onto the spacers even when they no longer had to face these enemies. Instead, they used CRISPR for other tasks. Severinov speculates that a CRISPR sequence might act as a kind of genetic bar code. Bacteria that shared the same bar code could recognize each other as relatives and cooperate, while fighting off unrelated populations of bacteria.

But Severinov wouldn’t be surprised if CRISPR also carries out other jobs. Recent experiments have shown that some bacteria use CRISPR to silence their own genes, instead of seeking out the genes of enemies. By silencing their genes, the bacteria stop making molecules on their surface that are easily detected by our immune system. Without this CRISPR cloaking system, the bacteria would blow their cover and get killed.

“This is a fairly versatile system that can be used for different things,” Severinov said, and the balance of all those things may differ from system to system and from species to species.

If scientists can get a better understanding of how CRISPR works in nature, they may gather more of the raw ingredients for technological innovations. To create a new way to edit DNA, Doudna and her colleagues exploited the CRISPR-Cas system from a single species of bacteria, Streptococcus pyogenes. There’s no reason to assume that it’s the best system for that application. At Editas, a company based in Cambridge, Massachusetts, scientists have been investigating the Cas9 enzyme made by another species of bacteria, Staphylococcus aureus. In January, Editas scientists reported that it’s about as efficient at cutting DNA as Cas9 from Streptococcus pyogenes. But it also has some potential advantages, including its small size, which may make it easier to deliver into cells.

To Koonin, these discoveries are just baby steps into the ocean of CRISPR diversity. Scientists are now working out the structure of distantly related versions of Cas9 that seem to behave very differently from the ones we’re now familiar with. “Who knows whether this thing could become even a better tool?” Koonin said.

And as scientists discover more tasks that CRISPR accomplishes in nature, they may be able to mimic those functions, too. Doudna is curious about using CRISPR as a diagnostic tool, searching cells for cancerous mutations, for example. “It’s seek and detect, not seek and destroy,” she said. But having been surprised by CRISPR before, Doudna expects the biggest benefits from these molecules to surprise us yet again. “It makes you wonder what else is out there,” she said.

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Doudna’s Confidence in CRISPR’s Research Potential Burns Bright


Jennifer Doudna, one of CRISPR’s primary innovators, stays optimistic about how the gene-editing tool will continue to empower basic biological understanding.
 

Portrait of Jennifer Doudna

Jennifer Doudna, one of the inventors of CRISPR genome editing technology, outside her laboratory and the Innovative Genomics Institute she founded at the University of California, Berkeley. “If you had told me 10 years ago that bacteria had evolved proteins that could be programmed to find and cut any DNA sequence,” she said, “I would have just laughed.”

Jana Ašenbrennerová for Quanta Magazine

No one needs to remind Jennifer Doudna about the power of CRISPR, the precision genome-editing technology she codeveloped. CRISPR “gives us a way to ultimately control the evolution of any organism — including ourselves. It is a profound thing. Human beings have now learned enough about our own genetic code that we can change it at will,” she said. “It’s kind of crazy to think about.”

That’s why when reports emerged last November that the scientist He Jiankui of the Southern University of Science and Technology in Shenzhen, China, had used CRISPR to alter the DNA of twin baby girls — crossing a line that genetic engineers had respected for decades and reaffirmed in 2015 — Doudna was quick to speak out. Describing herself to the media as “horrified and stunned,” she criticized his actions as risky, premature and unnecessary, given the absence of pressing medical need for the children to be modified experimentally. She encouraged the international scientific community to develop better guidelines for permissible genome editing in humans.

CRISPR technology makes genome editing temptingly simple because it allows scientists to cut and edit sequences of DNA in any species, including humans, at will. It was inspired by a long-overlooked defense mechanism with which many bacteria fend off viruses: By inserting fragments of viral DNA into specialized structures in their own genome (the “clustered regularly interspaced short palindromic repeats” that give CRISPR its name), bacteria provide their daughter cells with a way to recognize and quickly rebuff future invasions by similar viruses. Doudna and Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin showed in 2012 that the bacterial system could be adapted as an editing tool. (Several other scientists, including Feng Zhang of the Broad Institute and Virginijus Šikšnys of Vilnius University are also credited with contributing to CRISPR’s development, and multiple patent lawsuits surround the ownership of the intellectual property.)

But although Doudna argues for caution when contemplating changes that could be passed down for generations, she remains a forceful advocate for the potential of CRISPR in basic research, as well as its medical and biotech applications. “I think when you understand how things work, you can apply them more effectively. And once you apply them, you invariably uncover things that you didn’t understand about the fundamental biology of that system,” Doudna said. “I love that kind of interplay.”

Quanta Magazine sat down with Doudna, a professor of chemistry and molecular and cell biology at the University of California, Berkeley, to discuss how CRISPR is furthering basic biology research. That interview and subsequent exchanges have been condensed and edited for clarity. 

When CRISPR is discussed in the public, it’s often in regard to how it will be used to cure and treat disease. How do you think CRISPR has and will further basic biology research?

I’ll give you two examples that I think are fun because they illustrate some of the creative things people are doing now that weren’t possible in the past. One of them is a project to investigate the origins of bipedalism. This project involves comparing the genetics of two types of rodents — a standard quadruped mouse and a rodent called a jerboa that hops on its hind legs, so it’s bipedal.

What if I start putting genes from the bipedal rodent into the other rodent? Can I eventually make a bipedal field mouse? This is the kind of project that’s possible now with CRISPR.

The other CRISPR experiment I want to highlight explores the content of the Neanderthal genome. A lot of us now know that we have a little bit of Neanderthal DNA in our background, but what were the real differences between modern humans and Neanderthals? Why did Neanderthals go extinct, and what can that tell us about our own evolution?

PHOTO: Doudna holding a crispr model

Doudna holds a model of a CRISPR-Cas9 complex beginning to edit a stretch of DNA (blue). The CRISPR’s guide RNA (orange) binds to the defined DNA target; the Cas9 enzyme (white) cuts the DNA.

Jana Ašenbrennerová for Quanta Magazine

How do you explore that?

An experiment that’s now underway is taking genes from the Neanderthal genome and putting them into human cells that are cultured in the lab in the form of brain organoids. Organoids are balls of tissue that form organlike structures in a laboratory dish. I don’t want to say they’re like little brains, but these organoids have some properties of collections of neurons found in the brain.

The question then is to ask, if you start introducing Neanderthal specific genes into these human brain organoids, what happens? What kinds of changes do we see physiologically in those cells and in those balls of tissue? Can we learn something about the genetics of neuronal development in Neanderthals that might be different from what happens in humans?

It’s the early days of this research, and an organoid is not the same thing as a brain, so there’s going to have to be some interpretations of what the data mean, but I think that’s the kind of experiment that couldn’t have been done before.

Why not?

For the most part, we didn’t have a way to introduce changes to genomes precisely. The way that gene therapy was done originally was using viruses that integrate into human DNA, but the viruses integrate where they want to go, not necessarily where you want to make a change to the DNA.

Earlier technologies for gene editing were also very difficult to use in many settings. They required a lot of legwork to develop, like engineering particular proteins for each desired change to a genome. Certainly, it was difficult with those technologies to make more than one change at a time. With CRISPR, experimenters can change multiple genes in a genome in one shot.

Do you think that the medical applications of CRISPR in themselves can inform basic science?

For sure. CRISPR technology has been widely adopted by all kinds of scientists, including people like me. I was never doing anything with genome editing before CRISPR came along.

In my lab we’ve had a project over the last few years working on Huntington’s chorea, a degenerative neurological disease. The mutation that causes the disease is a single codon — three base pairs in the DNA — that gets repeated many times. If the codon gets repeated too many times, it leads to a defective protein that causes this disease. That’s been known for a long time, but the challenge was, how do you fix it?

We’ve been working on a way to deliver the CRISPR into mouse neuronal cells to make the necessary edits. But one of the curious things that’s come out of that line of work is that we found that only neuronal cells in the mouse brain were getting edited, not [the supportive glial] cells called astrocytes.

These cells are a lot smaller, so it could be that they don’t have enough surface area to take up the CRISPR protein efficiently. Or maybe they don’t respond to DNA cutting and editing in the same way as other cells.

Jennifer Doudna, one of the coinventors of CRISPR technology, discusses how her work on bacterial defenses against viruses helped lead to a discovery with a revolutionary impact on biological research.

Video: Jennifer Doudna, one of the coinventors of CRISPR technology, discusses how her work on bacterial defenses against viruses helped lead to a discovery with a revolutionary impact on biological research.

Jana Ašenbrennerová for Quanta Magazine

So not all the cells in the brain respond to the CRISPR treatment the same way.

We also found that, when we inject CRISPR molecules into one place in the mouse brain, we see cells that are a fair distance away from the injection site also get edited. That was a surprise because it suggests there’s some way of trafficking molecules through the brain to areas that are not right next door to where the needle goes in.

Is there some mechanism for molecular trafficking in the brain that hasn’t been appreciated? That’s a very fundamental question in biology. That takes us back to asking, “Gee, how does the brain work?” Now we’re exploring answers to these questions.

I have to say that this example from my lab is emblematic of what’s going on in a variety of labs. CRISPR enables very applied experiments, but these experiments raise very fundamental questions that you have to go back and address.

How pervasive do you think the use of CRISPR will become in biology? It already seems to be everywhere in biomedical labs. Do you think there might be applications in fields like ecology, for example?

Absolutely. It depends on your definition of ecology, but some of the early adopters of the CRISPR technology were people who were trying to understand the genetics of butterfly development. Having a tool that allowed the manipulation of genes in non-model organisms — organisms that scientists haven’t been working on for decades in the laboratory but instead collected in the wild — opens up the possibility of real experimentation in a way that previously was not possible.

Effectively any organism becomes a model organism — a genetically tractable system for doing experiments. We joke in the lab that we’re turning humans into yeast: In the past, you had to do experiments in yeast or fruit flies and then infer what those data meant about human cells. You couldn’t easily manipulate human cells genetically to understand genetic function. Now, with CRISPR, you can.

CRISPR was first discovered as a defense mechanism that bacteria use against viruses. Do you think other new research tools might come from other discoveries about bacteria?

I suspect so. If you look at the technologies that have come along over the last several decades — polymerase chain reaction for amplifying DNA, molecular cloning — they all came about from studying how microbes fight off viruses or respond to stimuli.

We also know very little about the bacterial world right now. There’s a huge number of organisms that have never been identified by scientists or cultivated in the laboratory, and they surely have interesting biology associated with their lifestyles. As more of those organisms are studied and identified, I have no doubt that we’ll find things that will lend themselves to new technologies.

PHOTO: Doudna walks in her lab

Doudna walks through her lab in the Energy Biosciences building on the Berkeley campus.

Jana Ašenbrennerová for Quanta Magazine

Are there any particular enigmas in bacteria that you have a hunch might lead to some sort of research tool?

That’s always hard to predict. That being said, I’ll give you an example of an interesting phenomenon: the discovery of this new category of bacteria that are incredibly small. It’s a whole new phylum of organisms — they’re currently called the candidate phyla radiation (CPR) bacteria. They almost challenge the notion of what’s a cell and what’s a virus.

A lot of these organisms probably grow symbiotically with other bugs, sharing important molecules, maybe even the building blocks of DNA, RNA and proteins. But how do they import molecules? How do they control their environment so that other kinds of bacteria don’t overgrow and crowd them out?

These are all unanswered questions. We don’t understand anything about their fundamental biology in a molecular sense. Will answers to these questions lead to a new technology? I don’t know, but it’s certainly going to lead to interesting biology.

So, the place to look for new research tools might be organisms that are atypical, so to speak?

But how do you define atypical? There’s this old Steve Forbert song: “It’s often said that life is strange … but compared to what?”

These tiny CPR bacteria are the ones in which you and Jillian Banfield of the University of California, Berkeley recently found new Cas enzymes [for cutting strands of DNA] that could be used with CRISPR technology, aren’t they? What makes those Cas enzymes potentially so interesting and useful?

One of the new enzymes we identified is called “CasX.” It’s particularly interesting because it seems to work quite differently from its cousin Cas9, the enzyme that many conventional bacteria use in their CRISPR defenses and that’s commonly used in CRISPR technology. But a few core ingredients are the same. This gives us insight into the basic recipe for CRISPR cutting proteins. The more we understand these proteins, the better we can engineer them. CasX is also appealing because it’s much smaller than Cas9, which might make it easier to slip into cells for therapeutic genome editing.

There have also been other new spinoff technologies developing out of CRISPR-Cas9, like CRISPR-GO, DNA imaging and anti-CRISPR. How might they help basic biology?

So let’s just go through those. CRISPR-GO is this clever way of using CRISPR enzymes to bring particular parts of the genome into physical proximity. There’s evidence that when genes are being expressed together in cells, they’re often brought together physically to the same location in the cells, and that can fundamentally affect the levels of proteins that are produced from certain genes. What CRISPR-GO does is provide a technology for doing that kind of physical tethering, except now the scientists can control it rather than the cell controlling it. I think that creates an opportunity to start dissecting the relationship between the 3D architecture of the genome and the communication between genes, and the resulting levels of proteins or RNA molecules that are made from those genes. So that’s exciting. It’s something that, again, really hasn’t been possible before, to control the 3D architecture of chromosomes and ask how that affects the output from the genome.

You mentioned DNA imaging. The idea there is what’s being referred to as “chromosome painting,” where you can program the CRISPR-Cas9 protein to bind and basically sit for extended periods of time at certain places in the DNA. You can decorate the CRISPR-Cas9 protein with different colors of dyes to light up a particular gene or section of a genome, even an entire chromosome, by just tiling it with these little RNA-protein complexes. So it’s a method for imaging.

In the case of anti-CRISPR, these are teeny tiny natural proteins involved in regulating CRISPR systems. You can imagine that in bacteria that are getting infected by viruses, over time viruses have evolved ways of avoiding being taken out by CRISPRs, and one of the ways they do that is using these little inhibitors called anti-CRISPRs. There’s interest in these because of the potential to control gene-editing outcomes — using these kinds of proteins to turn off gene-editing proteins in cells to protect the genome from being modified in unintended ways. There’s a whole line of research now that’s taken off to look at natural regulators and inhibitors of CRISPR pathways and ask whether those can be harnessed for technology purposes.

Could the development of anti-CRISPR quell fears about genome editing in humans or other organisms, if we had an off switch to throw if CRISPR-Cas9 wasn’t working as intended?

That’s exactly what people are thinking about. In fact, there’s a whole program funded by DARPA (the U.S. Defense Advanced Research Projects Agency), that has the title “Safe Genes,” that’s about safe ways of manipulating genes and genomes. And one of the strategies that groups are using to do that is using these anti-CRISPRs.

Do you think that CRISPR helps us get closer to understanding how all the pieces in cells are working together rather than just separately?

I think it will increasingly play that kind of a role in the future.

Let’s go back to neuroscience, because there’s a case where CRISPR has come to the fore in studies of the development of the brain. Researchers haven’t been able to figure out how many different types of cells are in the brain. We also don’t know how the brain develops in the sense of its 3D architecture. If you start with a stem cell or a few stem cells, how does that develop into an entire brain, and what’s the map of the brain?

There’s a lot of interest right now in using CRISPR to do what’s called lineage mapping. If you have a population of cells that develop from a single cell or a small collection of cells, you can track how cells from that starting population give rise to their progeny by introducing a little edit to their DNA to mark them.

Several research teams are using CRISPR this way to figure out where these daughter cells end up in the brain and even what kinds of cells they become. I think these kinds of experiments will lead to a more fundamental understanding of tissue development — in particular, in the brain — that hasn’t been possible before.

That does sound promising.

I’ll give you another example. There are interesting cases — and we’re finding more and more of these as people get their DNA sequenced — of families in which everybody has a certain allele, a certain DNA sequence of a gene, but only some of them have a disease that’s associated with that allele. The others don’t. So you know that there’s something in the DNA of the people who are unaffected that suppresses a negative impact of that gene and makes them not susceptible to cancer or whatever other disease they would otherwise succumb to. What are those suppressors?

I think understanding those kinds of genetic interactions is going to be incredibly powerful going forward. Up until now, we haven’t really had a way to do it because, first of all, people weren’t widely going around sequencing their genomes. That’s starting to happen more and more, with companies that offer this and the cost coming down. Then there’s also having a technology that allows genetic manipulation of patient-derived cells. So if you have somebody that comes into a clinic and they have a disease that gets diagnosed, you can take cells from that person and you can cultivate them in the lab. That’s been possible for a while, but what wasn’t possible previously was to do genetics on those cells. Now we can, in living cells that relate to an actual patient.

That sounds like an unexpected benefit of the sequencing technology.

I always like to point out that there’s a certain serendipity to science. It’s wonderful, but it also means that you can’t predict outcomes. CRISPR technology is a great example of that. If you had told me 10 years ago that bacteria had evolved proteins that could be programmed to find and cut any DNA sequence, I would have just laughed. I would have been like, “Yeah, that’s definitely science fiction.”

I think it’s important for people to appreciate that this is how a lot of science happens.

CRISPR: Crispy Fries Your DNA


Story at-a-glance

  • CRISPR gene-editing technology may have significant unintended consequences to your DNA, including large deletions and complex rearrangements
  • The DNA deletions could end up activating genes that should stay “off,” such as cancer-causing genes, as well as silencing those that should be “on”
  • The deletions detected were at a scale of “thousands of bases,” which is more than previously thought and enough to affect adjacent genes
  • As a result of CRISPR-Cas9, DNA may be rearranged, previously distant DNA sequences may become attached, or unrelated sections could be incorporated into the chromosome

By Dr. Mercola

CRISPR gene-editing technology brought science fiction to life with its ability to cut and paste DNA fragments, potentially eliminating serious inherited diseases. CRISPR-Cas9, in particular, has gotten scientists excited because,1 by modifying an enzyme called Cas9, the gene-editing capabilities are significantly improved. That’s not to say they’re perfect, however, as evidenced by a recent study that showed CRISPR may have significant unintended consequences to your DNA, including large deletions and complex rearrangements.2

Many of the concerns to date regarding CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeat, technology have centered on off-target mutations. The featured study, published in Nature Biotechnology, looked at on-target mutations at the site of the “cuts,” revealing potentially dangerous changes that could increase the risk of chronic diseases like cancer.

Is CRISPR Scrambling DNA?

Researchers at the U.K.’s Wellcome Sanger Institute systematically studied mutations from CRISPR-Cas9 in mouse and human cells, focusing on the gene-editing target site. Large genetic rearrangements were observed, including DNA deletions and insertions, that were spotted near the target site.

They were far enough away, however, that standard tests looking for CRISPR-related DNA damage would miss them. The DNA deletions could end up activating genes that should stay “off,” such as cancer-causing genes, as well as silencing those that should be “on.” One of the study’s authors, professor Allan Bradley, said in a statement:3

“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 deletions detected were at a scale of “thousands of bases,” which is more than previously thought and enough to affect adjacent genes. For instance, deletions equivalent to thousands of DNA letters were revealed. “In one case, genomes in about two-thirds of the CRISPR’d cells showed the expected small-scale inadvertent havoc, but 21 percent had DNA deletions of more than 250 bases and up to 6,000 bases long,” Scientific American reported.4

The cells targeted by CRISPR try to “stitch things back together,” according to Bradley, “But it doesn’t really know what bits of DNA lie adjacent to each other.” As a result, the DNA may be rearranged, previously distant DNA sequences may become attached, or unrelated sections could be incorporated into the chromosome.5

Cas9, a bacteria enzyme that acts as the “scissors” in CRISPR, actually remains in the body for a period of hours to weeks. Even after the initial DNA segment had been cut out and a new section “pasted” into the gap to repair it, Cas9 continued to make cuts into the DNA. “[T]he scissors continued to cut the DNA over and over again. They found significant areas near the cut site where DNA had been removed, rearranged or inverted,” The Conversation reported.6

Does This Mean CRISPR Isn’t Safe?

It’s too soon to say what the long-term effects of gene-editing technology will be, and there are many variables to the safety equation. The findings likely only apply to CRISPR-Cas9, which cuts through the DNA’s double strand. Other CRISPR technologies exist that may alter only a single strand or not involve cutting at all, instead swapping DNA letters.

There are also CRISPR systems that target RNA instead of DNA and those that could potentially involve only cells isolated from the body, such as white blood cells, which could then be analyzed for potential mutations before being put back into the body.7

The Nature study did make waves in the industry, though, such that within the first 20 minutes of the results being made public three CRISPR companies lost more than $300 million in value.8

Some companies using CRISPR have said they’re already on the lookout for large and small DNA deletions (including one company using the technology to make pig organs that could be transplanted into humans). One company also claims it hasn’t found large deletions in their work on cells that do not divide often (the Nature study used actively dividing cells).9

The researchers are standing by their findings, however, which the journal took one year to publish. During that time, Bradley says, he was asked to conduct additional experiments and “the results all held up.”10 Past studies have also found unexpected mutations, including one based on a study that used CRISPR-Cas9 to restore sight in blind mice by correcting a genetic mutation.

The researchers sequenced the entire genome of the CRISPR-edited mice to search for mutations. In addition to the intended genetic edit, they found more than 100 additional deletions and insertions along with more than 1,500 single-nucleotide mutations.11 The study was later retracted, however, due to insufficient data and a need for more research to confirm the results.12

CRISPR-Edited Cells Could Cause Cancer

Revealing the many complexities of gene editing, CRISPR-Cas9 also leads to the activation of the p53 gene, which works to either repair the DNA break or kill off the CRISPR-edited cell.13

CRISPR actually has a low efficacy rate for this reason, and CRISPR-edited cells that survive are able to do so because of a dysfunctional p53. The problem is that p53 dysfunction is also linked to cancer (including close to half of ovarian and colorectal cancers and a sizable portion of lung, pancreatic, stomach, breast and liver cancers as well).14

In one recent study, researchers were able to boost average insertion or deletion efficiency to greater than 80 percent, but that was because of a dysfunctional p53 gene,15 which would mean the cells could be predisposed to cancer. The researchers noted, ” … it will be critical to ensure that [CRISPR-edited cells] have a functional p53 before and after engineering.”16

A second study, this one by the Karolinska Institute in Sweden, found similar results and concluded, ” … p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.”17

Some have suggested that if CRISPR could cure one chronic or terminal disease at the “cost” of an increased cancer risk later,18 it could still be a beneficial technology, but most agree that more work is needed and caution warranted.

A CRISPR clinical trial in people with cancer is already underway in China, and the technology has been used to edit human embryos made from sperm from men carrying inherited disease mutations. The researchers successfully altered the DNA in a way that would eliminate or correct the genes causing the inherited disease.19

If the embryos were implanted into a womb and allowed to grow, the process, which is known as germline engineering, would result in the first genetically modified children — and any engineered changes would be passed on to their own children. A February 2017 report issued by the U.S. National Academies of Sciences (NAS) basically set the stage for allowing research on germline modification (such as embryos, eggs and sperm) and CRISPR, but only for the purpose of eliminating serious diseases.

In the U.S., a first of its kind human trial involving CRISPR is currently recruiting participants with certain types of cancer. The trial is going to attempt to use CRISPR to modify immune cells to make them attack tumor cells more effectively. As far as risks from potential mutations, it’s anyone’s guess, but lead researcher Dr. Edward Stadtmauer of the University of Pennsylvania told Scientific American, “We are doing extensive testing of the final cellular product as well as the cells within the patient.”20

Are ‘Designer Babies’ Next?

It’s easy to argue for the merits of CRISPR when you put it in the context of curing deafness, inherited diseases or cancer, and at least 17 clinical trials using gene-editing technologies to tackle everything from gastrointestinal cancer to tumors of the central nervous system to sickle cell disease have been registered in the U.S.21 Another use of the technology entirely is the creation of “designer babies” with a certain eye color or increased intelligence.

About 40 countries have already banned the genetic engineering of human embryos and 15 of 22 European countries prohibit germ line modification.22 In the U.S., the NAS report specifically said research into CRISPR and germline modification could not be for “enhancing traits or abilities beyond ordinary health.” Still, using gene editing to create designer babies is a question of when, not if, with some experts saying it could occur in a matter of decades.23

There are both safety and ethical considerations to think about. With some proponents saying it would be unethical not to use the technology. For instance, Julian Savulescu, an ethicist at the University of Oxford, told Science News he believes parents would be morally obligated to use gene-editing technology to keep their children healthy.

“If CRISPR could … improve impulse control and give a child a greater range of opportunities, then I’d have to say we have the same moral obligation to use CRISPR as we do to provide education, to provide an adequate diet …”24 Others have suggested CRISPR could represent a new form of eugenics, especially since it can only be done via in vitro fertilization (IVF), putting it out of reach of many people financially and potentially expanding inequality gaps.

On the other hand, some argue that countries with national health care could provide free coverage for gene editing, possibly helping to reduce inequalities.25 It’s questions like these that make determining the safety of CRISPR and other gene-editing technology more important now than ever before.

What Does a CRISPR-Enabled Future Hold?

We’ve already entered the era of genetic engineering and CRISPR represents just one piece of the puzzle. It’s an exciting time that could lead to major advances in diseases such as sickle-cell anemia, certain forms of blindness, muscular dystrophy, HIV and cancer, but also one that brings the potential for serious harm. In addition to work in human and animal cells, gene-edited crops, in which DNA is tweaked or snipped out at a precise location, have already been created — and eaten.

To date, the technology has been used to produce soybeans with altered fatty acid profiles, potatoes that take longer to turn brown and potatoes that remain fresher longer and do not produce carcinogens when fried. The latter could be sold as early as 2019.

The gene-editing science, in both plants and animals, is progressing far faster than long-term effects can be fully realized or understood. There are many opportunities for advancement to be had, but they must come with the understanding that unintended mutations with potentially irreversible effects could be part of the package.

Watch the video. URL:https://youtu.be/faSoxyiAAPE

CRISPR: Crispy Fries Your DNA


Story at-a-glance

  • CRISPR gene-editing technology may have significant unintended consequences to your DNA, including large deletions and complex rearrangements
  • The DNA deletions could end up activating genes that should stay “off,” such as cancer-causing genes, as well as silencing those that should be “on”
  • The deletions detected were at a scale of “thousands of bases,” which is more than previously thought and enough to affect adjacent genes
  • As a result of CRISPR-Cas9, DNA may be rearranged, previously distant DNA sequences may become attached, or unrelated sections could be incorporated into the chromosome

By Dr. Mercola

CRISPR gene-editing technology brought science fiction to life with its ability to cut and paste DNA fragments, potentially eliminating serious inherited diseases. CRISPR-Cas9, in particular, has gotten scientists excited because,1 by modifying an enzyme called Cas9, the gene-editing capabilities are significantly improved. That’s not to say they’re perfect, however, as evidenced by a recent study that showed CRISPR may have significant unintended consequences to your DNA, including large deletions and complex rearrangements.2

Many of the concerns to date regarding CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeat, technology have centered on off-target mutations. The featured study, published in Nature Biotechnology, looked at on-target mutations at the site of the “cuts,” revealing potentially dangerous changes that could increase the risk of chronic diseases like cancer.

Is CRISPR Scrambling DNA?

Researchers at the U.K.’s Wellcome Sanger Institute systematically studied mutations from CRISPR-Cas9 in mouse and human cells, focusing on the gene-editing target site. Large genetic rearrangements were observed, including DNA deletions and insertions, that were spotted near the target site.

They were far enough away, however, that standard tests looking for CRISPR-related DNA damage would miss them. The DNA deletions could end up activating genes that should stay “off,” such as cancer-causing genes, as well as silencing those that should be “on.” One of the study’s authors, professor Allan Bradley, said in a statement:3

“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 deletions detected were at a scale of “thousands of bases,” which is more than previously thought and enough to affect adjacent genes. For instance, deletions equivalent to thousands of DNA letters were revealed. “In one case, genomes in about two-thirds of the CRISPR’d cells showed the expected small-scale inadvertent havoc, but 21 percent had DNA deletions of more than 250 bases and up to 6,000 bases long,” Scientific American reported.4

The cells targeted by CRISPR try to “stitch things back together,” according to Bradley, “But it doesn’t really know what bits of DNA lie adjacent to each other.” As a result, the DNA may be rearranged, previously distant DNA sequences may become attached, or unrelated sections could be incorporated into the chromosome.5

Cas9, a bacteria enzyme that acts as the “scissors” in CRISPR, actually remains in the body for a period of hours to weeks. Even after the initial DNA segment had been cut out and a new section “pasted” into the gap to repair it, Cas9 continued to make cuts into the DNA. “[T]he scissors continued to cut the DNA over and over again. They found significant areas near the cut site where DNA had been removed, rearranged or inverted,” The Conversation reported.6

Does This Mean CRISPR Isn’t Safe?

It’s too soon to say what the long-term effects of gene-editing technology will be, and there are many variables to the safety equation. The findings likely only apply to CRISPR-Cas9, which cuts through the DNA’s double strand. Other CRISPR technologies exist that may alter only a single strand or not involve cutting at all, instead swapping DNA letters.

There are also CRISPR systems that target RNA instead of DNA and those that could potentially involve only cells isolated from the body, such as white blood cells, which could then be analyzed for potential mutations before being put back into the body.7

The Nature study did make waves in the industry, though, such that within the first 20 minutes of the results being made public three CRISPR companies lost more than $300 million in value.8

Some companies using CRISPR have said they’re already on the lookout for large and small DNA deletions (including one company using the technology to make pig organs that could be transplanted into humans). One company also claims it hasn’t found large deletions in their work on cells that do not divide often (the Nature study used actively dividing cells).9

The researchers are standing by their findings, however, which the journal took one year to publish. During that time, Bradley says, he was asked to conduct additional experiments and “the results all held up.”10 Past studies have also found unexpected mutations, including one based on a study that used CRISPR-Cas9 to restore sight in blind mice by correcting a genetic mutation.

The researchers sequenced the entire genome of the CRISPR-edited mice to search for mutations. In addition to the intended genetic edit, they found more than 100 additional deletions and insertions along with more than 1,500 single-nucleotide mutations.11 The study was later retracted, however, due to insufficient data and a need for more research to confirm the results.12

CRISPR-Edited Cells Could Cause Cancer

Revealing the many complexities of gene editing, CRISPR-Cas9 also leads to the activation of the p53 gene, which works to either repair the DNA break or kill off the CRISPR-edited cell.13

CRISPR actually has a low efficacy rate for this reason, and CRISPR-edited cells that survive are able to do so because of a dysfunctional p53. The problem is that p53 dysfunction is also linked to cancer (including close to half of ovarian and colorectal cancers and a sizable portion of lung, pancreatic, stomach, breast and liver cancers as well).14

In one recent study, researchers were able to boost average insertion or deletion efficiency to greater than 80 percent, but that was because of a dysfunctional p53 gene,15 which would mean the cells could be predisposed to cancer. The researchers noted, ” … it will be critical to ensure that [CRISPR-edited cells] have a functional p53 before and after engineering.”16

A second study, this one by the Karolinska Institute in Sweden, found similar results and concluded, ” … p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.”17

Some have suggested that if CRISPR could cure one chronic or terminal disease at the “cost” of an increased cancer risk later,18 it could still be a beneficial technology, but most agree that more work is needed and caution warranted.

A CRISPR clinical trial in people with cancer is already underway in China, and the technology has been used to edit human embryos made from sperm from men carrying inherited disease mutations. The researchers successfully altered the DNA in a way that would eliminate or correct the genes causing the inherited disease.19

If the embryos were implanted into a womb and allowed to grow, the process, which is known as germline engineering, would result in the first genetically modified children — and any engineered changes would be passed on to their own children. A February 2017 report issued by the U.S. National Academies of Sciences (NAS) basically set the stage for allowing research on germline modification (such as embryos, eggs and sperm) and CRISPR, but only for the purpose of eliminating serious diseases.

In the U.S., a first of its kind human trial involving CRISPR is currently recruiting participants with certain types of cancer. The trial is going to attempt to use CRISPR to modify immune cells to make them attack tumor cells more effectively. As far as risks from potential mutations, it’s anyone’s guess, but lead researcher Dr. Edward Stadtmauer of the University of Pennsylvania told Scientific American, “We are doing extensive testing of the final cellular product as well as the cells within the patient.”20

Are ‘Designer Babies’ Next?

It’s easy to argue for the merits of CRISPR when you put it in the context of curing deafness, inherited diseases or cancer, and at least 17 clinical trials using gene-editing technologies to tackle everything from gastrointestinal cancer to tumors of the central nervous system to sickle cell disease have been registered in the U.S.21 Another use of the technology entirely is the creation of “designer babies” with a certain eye color or increased intelligence.

About 40 countries have already banned the genetic engineering of human embryos and 15 of 22 European countries prohibit germ line modification.22 In the U.S., the NAS report specifically said research into CRISPR and germline modification could not be for “enhancing traits or abilities beyond ordinary health.” Still, using gene editing to create designer babies is a question of when, not if, with some experts saying it could occur in a matter of decades.23

There are both safety and ethical considerations to think about. With some proponents saying it would be unethical not to use the technology. For instance, Julian Savulescu, an ethicist at the University of Oxford, told Science News he believes parents would be morally obligated to use gene-editing technology to keep their children healthy.

“If CRISPR could … improve impulse control and give a child a greater range of opportunities, then I’d have to say we have the same moral obligation to use CRISPR as we do to provide education, to provide an adequate diet …”24 Others have suggested CRISPR could represent a new form of eugenics, especially since it can only be done via in vitro fertilization (IVF), putting it out of reach of many people financially and potentially expanding inequality gaps.

On the other hand, some argue that countries with national health care could provide free coverage for gene editing, possibly helping to reduce inequalities.25 It’s questions like these that make determining the safety of CRISPR and other gene-editing technology more important now than ever before.

What Does a CRISPR-Enabled Future Hold?

We’ve already entered the era of genetic engineering and CRISPR represents just one piece of the puzzle. It’s an exciting time that could lead to major advances in diseases such as sickle-cell anemia, certain forms of blindness, muscular dystrophy, HIV and cancer, but also one that brings the potential for serious harm. In addition to work in human and animal cells, gene-edited crops, in which DNA is tweaked or snipped out at a precise location, have already been created — and eaten.

To date, the technology has been used to produce soybeans with altered fatty acid profiles, potatoes that take longer to turn brown and potatoes that remain fresher longer and do not produce carcinogens when fried. The latter could be sold as early as 2019.

The gene-editing science, in both plants and animals, is progressing far faster than long-term effects can be fully realized or understood. There are many opportunities for advancement to be had, but they must come with the understanding that unintended mutations with potentially irreversible effects could be part of the package.

Watch the video.URL:https://youtu.be/faSoxyiAAPE

Scientists Who Said CRISPR Is Dangerous Can’t Even Replicate Their Own Results


An alarming study that claimed the gene-editing technique CRISPR could produce hundreds of unexpected mutations in edited genomes has now been followed up by its authors, who say they cannot replicate their controversial result.

main article image

The acknowledgment – which comes in a report of new mice experiments that didn’t introduce such mutations – isn’t technically a retraction of their earlier findings, but it goes a long way to showing that the alarm bells should probably never have been sounded in the first place.

In the new research, the team conducted whole-genome sequencing on two mouse lines that had undergone a CRISPR-editing procedure.

In their original study, they performed the same analysis – and it was the first time whole-genome sequencing had ever been run on a living organism subjected to CRISPR gene-editing.

But unlike the original results, in the new experiments, no unintended gene variants showed up after the genetic alterations.

This contrasts starkly with the team’s first study, in which they found that the two CRISPR-edited mice had sustained over 1,500 single-nucleotide mutations, along with more than 100 larger deletions and insertions that weren’t intended.

These variations showed up in ‘off-target’ portions of the animals’ genomes, suggesting that while CRISPR editing could alter genetic code to fix certain abnormalities, it could also introduce unwanted mutations elsewhere in the genome.

“We feel it’s critical that the scientific community consider the potential hazards of all off-target mutations caused by CRISPR,” one of the team, cell biologist Stephen Tsang from Columbia University said at the time.

That’s a valid concern to have, and it’s something we certainly should be on the lookout for.

But the problems other scientists had with these alarming findings weren’t with the team’s ‘big picture’ approach, but with shortcomings in their method.

Soon after publication, a critique of the original paper by another team pointed out that the two gene-edited mice in the experiment were genetically more closely related to each other than to the third, ‘control’ mice.

The implication was that the ‘unexpected mutations’ Tsang’s team had detected weren’t the result of CRISPR, but simply due to the pre-existing genetics of the mice selected for the study.

And since their sample of animals in the experiment was so small, the results weren’t just unreliable, they were misleading – especially since the researchers were vocal about how this kind of analysis hadn’t been done before, implying it revealed dangerous shortcomings about CRISPR.

Due to the level of controversy and concern over the original study, the editors of Nature Methods – the journal in which the paper was published – formally stated they were concerned about the veracity of the findings, given an “alternative proposed interpretation is that the observed changes are due to normal genetic variation”.

While Tsang’s team did not share those concerns, they nonetheless cared enough to revisit the matter in their new research, and their Corrigendum (correction) analysis is a vindication for CRISPR, acknowledging the ‘unexpected mutations’ hypothesis was, as far as we call tell, a mistake and nothing more.

While defending their “reasonable concern” about such unintended mutations, the authors nonetheless conclude the new results “support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations”.

All this is a good thing. It’s the scientific method at work, revising our interpretation based on new information, and while some are arguing the original paper should finally be retracted, that hasn’t happened yet.

Many are probably still angry the original paper was published at all. But for now at least, new data have come to light, and there are still important things we have learned from this research.

A Crack in Creation review – Jennifer Doudna, Crispr and a great scientific breakthrough


This is an invaluable account, by Doudna and Samuel Sternberg, of their role in the revolution that is genome editing

Scientific zeal … Jennifer Doudna.

It began with the kind of research the Trump administration wants to unfund: fiddling about with tiny obscure creatures. And there had been US Republican hostility to science before Trump, of course, when Sarah Palinobjected to federal funding of fruit fly research (“Fruit flies – I kid you not,” she said). The fruit fly has been a vital workhorse of genetics for 100 years. Jennifer Doudna’s work began with organisms even further out on the Palin scale: bacteriophages, tiny viruses that prey on bacteria.

Yoghurt manufacturers knew they were important, not least because bacteriophages can destroy yoghurt cultures. Research on the mechanism of this process began in the labs of Danisco (now part of the giant DuPont) in the early 2000s, before spreading through the university biotech labs. In 2012 Doudna and Samuel Sternberg’s team at Berkeley (they are co-authors of the book but it’s written solely in Doudna’s voice) came up with probably the greatest biological breakthrough since that of Francis Crick, James Watson and Rosalind Franklin.

Biologists had become intrigued by a curiosity in the genome of some bacteria: they had repeat patterns interspersed always by 20 bases of DNA, which turned out to match sequences found in the phages (as bacteriophages are always known) that prey on them. They had stumbled on a bacterial immune system, now known as Crispr (Clustered regularly interspaced short palindromic repeats) – a sequence reading the same forwards and backwards.

An astonishing story of molecular countermeasures against phage invasion was revealed; these enable the bacterium to recognise the phage next time it invades. More than that, Crispr guides a killer enzyme to cut the phage’s DNA at the point where the 20‑base sequence is found. Doudna then demonstrated that bacterial Crispr can be reprogrammed to cut any DNA from any organism. This is what has been sought for more than 30 years: an accurate (or almost accurate) way of editing DNA. And there has never been a better example of the unforeseen benefits of pure research because no one guessed that a technique of such power and universality would emerge from what appeared to be a fascinating but arcane corner of biology.

The Jurassic Park fantasy is kept alive by Crispr.
 The ‘Jurassic Park fantasy’ is kept alive by Crispr. 

Crispr is not just a triumph for unfettered scientific curiosity, it’s also a reminder that the secret of life lies in tiny things. The visible world can be beautiful but we are gulled into thinking it must be more important than what we can’t see. People have been making that mistake for a long time. In The Citizen of the World (1762), Oliver Goldsmith mocked the supposed pedantry of all who study the tiny creatures revealed by the microscope: “Their fields of vision are too contracted to take in the whole … Thus they proceed, laborious in trifles, constant in experiment, without one single abstraction, by which alone knowledge may be properly said to increase.” But, of course, it is precisely being able to “see” small things that has unlocked the biological treasure trove.

Very soon after Doudna’s paper on the technique appeared in 2012, labs all over the world tried it and found it was surpassingly easy to use; a gold rush began. It’s always difficult when something like this happens to sort the hope from the hype, but anticipation is now intense. Doudna does, though, sound many notes of caution. Yes, Crispr is the most accurate form of gene editing so far, but it isn’t perfect. There are 3bn bases in the human genome so there is always a chance of a stray 20-base match and a fatal cut in the wrong place. A debate is taking place on whether to allow gene edits only outside the body (with the edited cells reinserted) or to allow editing of eggs and sperm, which changes that germline forever. Doudna comes down cautiously for germline editing, pointing out that mitochondrial replacement therapy, which also leads to permanent genetic alteration, is already a reality in the UK.

For now the most exciting potential medical application is in single gene diseases, such as cystic fibrosis, sickle-cell anaemia and muscular dystrophy. This is the simplest possible task for Crispr. Just one base has to be corrected out of the 3bn and it’s not a needle in a haystack: Crispr can find and cut and repair it. Sickle-cell anaemia is caused by a faulty haemoglobin gene, so blood can easily be withdrawn from the body, the gene edited and returned to the body. But this approach demands extreme caution. Genes often have multiple effects and the sickle-cell gene is known to protect against malaria. So if you fixed the sickle-cell gene in the African population (where it is prevalent) there would be many new cases of malaria. But then Crispr can probably fix that, too; other researchers, with Gates Foundation funding, are urgently tackling that problem. There is hardly an area of medicine that could not benefit from Crispr, and on the fringe there is the Jurassic Park fantasy, kept tenuously alive by the work of Crispr’s other great name, George Church at Harvard, who is editing the elephant genome to create a creature more like a woolly mammoth.

If medical ethics loom large in debates around Crispr, money and patents loom even larger. Now that this apparently unpromising research has blossomed, the venture capitalists are gathering. Doudna recounts how, so soon after her triumph, “colleagues became rivals; papers were pored over for future patent battles”. The patent battle in question came to fruition after the book was completed. Doudna’s team lost this round, and it’s not clear what the future holds for Crispr’s intellectual property rights. It is unlikely that medical progress will be delayed but there will be some bruised participants and money spent along the way.

CRISPR ISN’T ENOUGH ANY MORE. GET READY FOR GENE EDITING 2.0


IN FEWER THAN five years, the gene-editing technology known as Crispr has revolutionized the face and pace of modern biology. Since its ability to find, remove, and replace genetic material was first reported in 2012, scientists have published more than 5,000 papers mentioning Crispr. Biomedical researchers are embracing it to create better models of disease. And countless companies have spun up to commercialize new drugs, therapies, foods, chemicals, and materials based on the technology.

Usually, when we’ve referred to Crispr, we’ve really meant Crispr/Cas9—a riboprotein complex composed of a short strand of RNA and an efficient DNA-cutting enzyme. It did for biology and medicine what the Model T did for manufacturing and transportation; democratizing access to a revolutionary technology and disrupting the status quo in the process. Crispr has already been used to treat cancer in humans, and it could be in clinical trials to cure genetic diseases like sickle cell anemia and beta thalassemia as soon as next year.

But like the Model T, Crispr Classic is somewhat clunky, unreliable, and a bit dangerous. It can’t bind to just any place in the genome. It sometimes cuts in the wrong places.And it has no off-switch. If the Model T was prone to overheating, Crispr Classic is prone to overeating.

Even with these limitations, Crispr Classic will continue to be a workhorse for science in 2018 and beyond. But this year, newer, flashier gene editing tools began rolling off the production line, promising to outshine their first-generation cousin. So if you were just getting your head around Crispr, buckle up. Because gene-editing 2.0 is here.

Power Steering

Crispr’s targeted cutting action is its defining feature. But when Cas9 slices through the two strands of an organism’s DNA, the gene-editor introduces an element of risk. Cells can make mistakes when they repair such a drastic genetic injury. Which is why scientists have been designing ways to achieve the same effects in safer ways.

One approach is to mutate the Cas9 enzyme so it can still bind to DNA, but its scissors don’t work. Then other proteins—like ones that activate gene expression—can be combined with the crippled Cas9, letting them toggle genes on and off (sometimes with light or chemical signals) without altering the DNA sequence. This kind of “epigenetic editing” could be used to tackle conditions that arise from a constellation of genetic factors, as opposed to the straightforward single mutation-based disorders most well-suited to Crispr Classic. (Earlier this month, researchers at the Salk Institute used one such system to treat several diseases in mice, including diabetes, acute kidney disease, and muscular dystrophy.)

Other scientists at Harvard and the Broad Institute have been working on an even more daring tweak to the Crispr system: editing individual base pairs, one at a time. To do so, they had to design a brand-new enzyme—one not found in nature—that could chemically convert an A-T nucleotide pairing to a G-C one. It’s a small change with potentially huge implications. David Liu, the Harvard chemist whose lab did the work, estimates that about half of the 32,000 known pathogenic point mutations in humans could be fixed by that single swap.

“I don’t want the public to come away with the erroneous idea that we can change any piece of DNA to any other piece of DNA in any human or any animal or even any cell in a dish,” says Liu. “But even being where we are now comes with a lot of responsibility. The big question is how much more capable will this age get? And how quickly will we be able to translate these technological advances into benefits for society?”

Putting On The Brakes

Crispr evolved in bacteria as a primitive defense mechanism. Its job? To find enemy viral DNA and cut it up until there was none left. It’s all accelerator, no brake, and that can make it dangerous, especially for clinical applications. The longer Crispr stays in a cell, the more chances it has to find something that sort of looks like its target gene and make a cut.

To minimize these off-target effects, scientists have been developing a number of new tools to more tightly control Crispr activity.

So far, researchers have identified 21 unique families of naturally occurring anti-Crispr proteins—small molecules that turn off the gene-editor. But they only know how a handful of them work. Some bind directly to Cas9, preventing it from attaching to DNA. Others turn on enzymes that outjostle Cas9 for space on the genome. Right now, researchers at UC Berkeley, UCSF, Harvard, the Broad, and the University of Toronto are hard at work figuring out how to turn these natural off-switches into programmable toggles.

Beyond medical applications, these will be crucial for the continued development of gene drives—a gene-editing technology that quickly spreads a desired modification through a population. Being able to nudge evolution one way or the other would be a powerful tool for combating everything from disease to climate change. They’re being considered for wiping out malaria-causing mosquitoes,and eradicating harmful invasive species. But out in the wild, they have the potential to spread out of control, with perhaps dire consequences. Just this year Darpa poured $65 million toward finding safer gene drive designs, including anti-Crispr off-switches.

Step On The Cas

Despite decades of advances, there’s still so much scientists don’t understand about how bugs in your DNA can cause human disease. Even if they know what genes are coded into a cell’s marching orders, it’s a lot harder to know where those orders get delivered, and how they get translated (or mistranslated) along the way. Which is why groups at Harvard and the Broad led by Crispr co-discoverer Feng Zhang are working with a new class of Cas enzymes that target RNA instead of DNA.

Since those are the instructions that a cell’s machinery reads to build proteins, they carry more information about the genetic underpinnings of specific diseases. And because RNA comes and goes, making changes to it would be useful for treating short-term problems like acute inflammation or wounds. The system, which they’re calling Repair, for RNA Editing for Programmable A to I Replacement, so far only works for one nucleotide conversion. The next step is to figure out how to do the other 11 possible combinations.

And scientists are finding new Cas enzymes all the time. Teams at the Broad have also been working to characterize cpf1—a version of Cas that leaves sticky ends instead of blunt ones when it cuts DNA. In February, a group from UC Berkeley discovered CasY and CasX, the most compact Crispr systems yet. And researchers expect to turn up many more in the coming months and years.

Only time will tell if Crispr-Cas9 was the best of these, or merely the first that captured the imagination of a generation of scientists. “We don’t know what’s going to wind up working best for different applications,” says Megan Hochstrasser, who did her PhD in Crispr co-discoverer Jennifer Doudna’s lab and now works at the Innovative Genomics Institute. “So for now I think it makes sense for everyone to be pushing on all these tools all at once.”

It will take many more years of work for this generation of gene-editors to find their way out of the lab into human patients, rows of vegetables, and disease-carrying pests. That is, if gene-editing 3.0 doesn’t make them all obsolete first.

Science and Morality


Science and Morality

science doesn’t give us a script for what to value or believe in, but it helps us write that script.

I am a faithful book buyer and an omnivorous reader, but one with a precocious streak—I like to look up authors and email them with questions about their books. Since penning a book about the CRISPR-Cas9 gene-modification system, readers are now writing to me with all sorts of middle-of-the-night thoughts. Many people think of science as a good thing—STEM has cachet, synonymous with our goodness—but the advance of the life sciences unnerves some people.

Matthew Endrizzi, a biology teacher in New Hampshire, suggested recombinant DNA research—including CRISPR—was dangerous enough in theory that he has proposed to move it all to the moon (he has not yet secured the funding or political will to do this). Margalit Laufer, a therapist in the Netherlands, has started a grassroots campaign to stop the application of CRISPR, a motivation which is linked to her views on the divinity of nature.

Science can discredit our speculations, folk science and illusions about how the world works and what to be afraid of; but the opposite, science as a positive script for what to value or believe has its limitations. Robert Oppenheimer was painfully aware of this when he concluded that “science is not all of the life of reason; it is a part of it.”

CRISPR may indeed be used to create bioweapons through the engineering of microbes, or create pathological strains through unscrupulous genetic manipulation. But the unleashing of dangerous microbes has been a concern at least since the 1970s when recombinant DNA first emerged, not to mention giving rise to films such as the Andromeda Strain and The Stand.

In fact, a temporary moratorium on gene engineering was tried in the 1970s, but many scientists already thought the risks of biohazard were overblown. British microbiologist Ephraim Anderson titled one paper Indiscriminant use of antibiotics has exerted more pressure on the bacterial population than could be wielded by all the research workers in the world put together. We cannot rule-out the prospect that a genetically modified microbe could cause a global threat to humans. But the risks are minute and simply worth enduring, most academics have concluded.

The argument that genes embody a sort of sacrosanct character that should not be interfered with is not too compelling, since artifacts of viruses are burrowed in our genomes, and genes undergo mutations with each passing generation. Even so, the principle that all life has inherent dignity is hardly a bad thought and provides a necessary counterbalance to the impulse to use in vitro techniques and CRISPR to alter any gene variant to reduce risk or enhance features, none of which are more or less perfect but variations in human evolution.

Indeed, the question of dignity is thornier than we might imagine, since science tends to challenge the belief in abstract or enduring concepts of value. How to uphold beliefs or a sense of dignity seems ever confusing and appears to throw us up against an age of radical nihilism as scientists today are using the gene editing tool CRISPR to do things such as tinker with the color of butterfly wingsgenetically alter pigs, even humans. If science is a method of truth-seeking, technology its mode of power and CRISPR is a means to the commodification of life. It also raises the possibility this power can erode societal trust.

In 2008, the President’s Council on Bioethics released a 555-page report, titled Human Dignity and Bioethics, which fielded essays by wide array of thinkers including the progressive philosopher Daniel Dennett and conservatives such as Leon Kass. As Dennett put the problem, “When we start treating living bodies as motherboards on which to assemble cyborgs, or as spare parts collections to be sold to the highest bidder, where will it all end?” The solution of rescuing human dignity from the commercial forces of science, Dennett noted, cannot involve resorting to “traditional myths” since this “will backfire” but instead concepts of human dignity should be based on our sovereign right to “belief in the belief that something matters.”

Dennett argues that faith is important in an everyday sense, such as most people have faith in democracy even as “we are often conflicted, eager to point to flaws that ought to be repaired, while just as eager to reassure people that the flaws are not that bad, that democracy can police itself, so their faith in it is not misplaced.” The point is also true about science, “since the belief in the integrity of scientific procedures is almost as important as the actual integrity.” In fact, we engage in a sort of “belief maintenance” insofar that “this idea that there are myths we live by, myths that must not be disturbed at any cost, is always in conflict with our ideal of truth-seeking” and even as we commit to ideas in public or just in our hearts, “a strange dynamic process is brought into being, in which the original commitment gets buried” in layers of internal dialog and counterargument. “Personal rules are a recursive mechanism; they continually take their own pulse, and if they feel it falter, that very fact will cause further faltering,” the psychiatrist George Ainslie wrote in the Breakdown of Will. If science can challenge beliefs, dignity is more primal—it is the right to hold beliefs, make use of science, and exercise belief maintenance.

Dignity is tricky to defend against the explication and engineering of human life by means of chemical processes, and it is complicated by the reality that many people increasingly look to science to shape their view and moral direction, as we are living in a new age of resurgent scientism—an assumption that science encodes values. A century ago, scientism appeared to be all but dead. The modernist break caused rupture between the moral and cultural commitments and sheer existence—hence it led to existentialism and the struggle over defining our commitments.

Whatever it meant to life a good life, it couldn’t be predefined by culture or science. In Anton Chekhov’s 1889 short story, “A Boring Story,” Nikolai Stepanovich, an internationally recognized scientist and professor of medicine, slips into melancholy near the end of his life. Despite his incredible success, his life seems evermore ambiguous, as the modernist movement comes to displace his authority. Katja, a young girl, a representative of the new generation, comes to him asking for advice and guidance, but Nikolai knows he has no way to tell her how to live. The irony is that freedom invoked a melancholy. His physician friend Mikhail Fyodorovich confides in Nikolai, “Science, God knows, has become obsolete. Its song has sung. Yes… Humanity has already begun to feel the need of replacing it with something else.”

In fact, we may be in the midst of a rebound to this break, whereby a resurgent scientism defines the moral directive, and data science is used to shape the arc of our decisions. Scientists can appeal to a mythos of bringing us closer to reality, as if peering into neuroimagery or analyzing the genome gives us information that is more true that life as we experience it. To some extent we learn bits and pieces of what makes us who we are. But ironically, it can weaken our sense of reality due to the obsession with statistical signals, which are often taken out of context, algorithms which speciously shape societal decisions, datingdecisions, or pick the next president—much of which fails us. More time and data is going to vastly improve the ability of science to regulate our lives, quite the opposite. This is because the life of the mind often involves the toggling between two opposing ideas, where there are no right decisions. As economist Thomas Sowell put it, “The march of science and technology does not imply growing intellectual complexity in the lives of most people. It often means the opposite.”

In his essay “The Virtue of Scientific Thinking” in the Boston Review, Harvard science historian Steven Shapin, who has also written on how much of our belief in science and the world is based on trust in the written word, has argued that trust in science has a critical role in morality, and that science, say climate science, can indeed be useful to shape values and direct policy decisions. But there are also obvious pitfalls to resurgent scientism. In recent decades, the free inquiry of science has been linked to technology, and thus to modes of institutional power, and monetization.

Therefore, scientific inquiry can be in jeopardy to the extent that it becomes put to the extreme uses of capitalization of the life sciences. Science, once a challenge to institutional authority, has increasingly been defined by status, finance and what look like hierarchical structures, which I think that people subconsciously like to see. But scientists, by close association with biotech, also risk a backlash that people make disengage with them, and begin to see credible facts as merely framing one more business venture. Importantly, we trust that what scientists say is probably true, but there is no guarantee of this trust or belief. In fact, trust is jeopardy as scientists connect their work to modes of technology as a means to personal power, half-million dollar cancer drugs, a billion-dollar CRISPR patent battle, and the like.

Science does not provide a positive script—but information to help build that script. For instance, a hypothesis is a proposition or belief that can be tested; but as Karl Popper once suggested, a hypothesis cannot be proven, only disproven (one black swan proves not all swans are white, but more white swans do not) since a given can never be completely proved—there is always the chance of a challenge by new data. Science offers no starting points, and there are questions of whether science is, in fact, leading us to any complete view of nature, which will be unchallenged, or, in some way, enlightened.

Increasingly, some scientists deny a Theory of Everything. Physical systems may be in a state of competition; in other words—there is no logic at the basis of reality. Therefore, while science is a useful tool, we have to at least entertain the prospect that it only leads to an abyss of time—an ongoing building and rebuilding of human histories. I suspect it will fail as a singular means to guide us to any conflict-free reality, and that we are far from done struggling with the consequences of the modernist break.

First U.S. Human Embryo Gene Editing Experiment Successfully “Corrects” a Heart Condition


IN BRIEF

A study published today in the journal Nature confirms earlier reports of the first-ever successful gene-editing of embryos in the U.S. Though controversial, the treatment could one day be used to address any of the 10,000 disorders linked to just a single genetic error.

CORRECTING MUTANT GENES

Last week, reports circulated  that doctors had successfully edited a gene in a human embryo — the first time such a thing had been done in the United States. The remarkable achievement confirmed the powerful potential of CRISPR, the world’s most efficient and effective gene-editing tool. Now, details of the research have been published in Nature.

The procedure involved “correcting” the DNA of one-cell embryos using CRISPR to remove the MYBPC3 gene. That gene is known to cause hypertrophic cardiomyopathy (HCM), a heart disease that affects 1 out of 500 people. HCM has no known cure or treatment as its symptoms don’t manifest until the disease causes sudden death through cardiac arrest.

How CRISPR Works: The Future of Genetic Engineering and Designer Humans
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The researchers started with human embryos created from 12 healthy female donors and sperm from a male volunteer who carried the MYBOC3 gene. The defective gene was cut out using CRISPR around the time the sperm was injected into the eggs.

 As a result, as the embryos divided and grew, many repaired themselves using the non-edited genes from the genetic materials of the female donors, and in total, 72 percent of the cells that formed appeared to be corrected. The researchers didn’t notice any “off-target” effects on the DNA, either.

The researchers told The Washington Post that their work was fairly basic. “Really, we didn’t edit anything, neither did we modify anything,” explained Shoukhrat Mitalipov, lead author and a researcher at the Oregon Health and Science University. “Our program is toward correcting mutant genes.”

A [CONTROVERSIAL] NEW ERA?

Basic or not, the development is remarkable.“By using this technique, it’s possible to reduce the burden of this heritable disease on the family and eventually the human population,” Mitalipov said in an OHSU press release.

However, gene editing is a controversial area of study, and the researchers’ work included changes to the germ line, meaning the changes could be passed down to future generations. To be clear, though, the embryos were allowed to grow for only a few days and none were implanted into a womb (nor was that ever the researchers’ intention).

In fact, current legislation in the U.S. prohibits the implantation of edited embryos. The work conducted by these researchers was well within the guidelines set by the National Academies of Sciences, Engineering, and Medicine on the use of CRISPR to edit human genes.

University of Wisconsin-Madison bioethicist Alta Charo thinks that the benefits of this potential treatment outweigh all concerns. “What this represents is a fascinating, important, and rather impressive incremental step toward learning how to edit embryos safely and precisely,” she told The Washington Post. “[N]o matter what anybody says, this is not the dawn of the era of the designer baby.”

Before the technique could be truly beneficial, regulations must be developed that provide clearer guidelines, according to Mitalipov. If not, “this technology will be shifted to unregulated areas, which shouldn’t be happening,” he explained.

More than 10,000 disorders have been linked to just a single genetic error, and as the researchers continue with their work, their next target is BRCA, a gene associated with breast cancer growth.

Mitalipov hopes that their technique could one day be used to treat a wide-range of genetic diseases and save the lives of millions of people. After all, treating a single gene at the embryonic stage is far more efficient that changing a host of them in adults.

CRISPR, Life Altering Genetic Innovation


The scientific community is in the midst of a gold rush in new technological applications all made possible by the CRISPR/Cas9 system. CRISPR, short for “clustered regularly interspaced palindromic repeats” is quite possibly the biggest innovation in biological science since PCR was developed over three decades ago. This is literally life altering genetic innovation.

CRISPR_technology.png

Scientists have been modifying genomes for years, so what’s the big deal behind this new technology?

In the past, prior to 2010, in order to modify the genome of a mouse, researchers would transfer embryonic stem cells into a mouse embryo containing the genetic mutation of choice. It would then take three generations to see the desired mutation and actually start utilizing the mutation for research purposes. This resulted in large amounts of time and money spent on breeding two unnecessary generations of mice without the guarantee of success. If a researcher wished to modify five genes of interest, this process would be repeated, you guessed it, five more times.

Taking this into account, CRISPR only needs one generation. This system is precise, efficient, and flexible, allowing for multiple mutations to be made all at once. Its efficacy has been proven time and time again in mice, monkeys, and recently in non-viable human embryos by a group of researchers in China, which proves its potential to treat ANY genetic disease.

As applications of this system are developed, many billion-dollar opportunities will arise. With this much money at stake along with world changing potential, rights to the invention are sure to create a heated patent battle at the USPTO, begging the question; who owns the technology anyways?

Professor Jennifer Doudna of UC Berkeley and Emmanuelle Charpentier from Umea University in Sweden filed on March 15, 2013 – one day before the first-to-file rule took effect – and claimed a priority date of May 25, 2012. On the other hand, Feng Zhang of the broad institute of MIT and Harvard in Cambridge, Massachusetts filed on October 15, 2013 under the accelerated examination program. The Broad Institute received patent No. 8,697,359 in April of 2014 claiming priority to a provisional application filed in December 2012.

As the Broad Institute continued to file applications for the technology, Doudna filed a Suggestion of Interference claiming that the Broad Institute Patents interfered with Doudna’s previous application. Pre AIA gives right to who created the invention first, unlike the first-to-file rules of today. An interference procedure is underway with oral arguments set for November 2016.

At stake are the rights to exclusively make, use, license, and sell the invention. The CRISPR/Cas9 system has the ability to completely alter how we treat genetic diseases, and may lead to the actualization of ‘designer babies’ – babies born with their traits hand picked by the parents. The discovery of a lifetime is up for grabs and it will be interesting to see who emerges with rights to the technology. Each party has issued liscences to large biotech companies ready to use the technology in grand-scale implication, however these projects have been delayed, pending the USPTO decision of this patent battle.

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