Can genetic engineering cure autism

Gene scissors Crispr / Cas9: How the Nobel Prize winners achieved their breakthrough

Emmanuelle Charpentier and Jennifer Doudna have taken what is probably the most important step in genetic research in recent years. They unraveled a mechanism with which the genome can be changed more specifically than ever before, and developed a highly effective gene scissors from it. For this, the two researchers have now been awarded the Nobel Prize in Chemistry. How does Crispr / Cas9 work? And what options does the process offer?

Discovery, that sounds too random. So fateful, so unintentional. Not what research like hers really is: trying it out, working it off - a tough, methodical hunt with an uncertain outcome, on the trail of a suspicion. An approach that pushes the boundaries of knowledge step by step. So discovery: Emmanuelle Charpentier believes that the term doesn't really fit. "You don't look for something and suddenly find it," she explains, "you decipher a mechanism over months and years."

The French microbiologist has spoken a lot about the mechanism she has deciphered in recent months at conferences in Chicago, Rome and Heidelberg. She calls the result of her research a "game changer".

Game changer: the turning point in the game, the big, all-changing throw. A breakthrough that Emmanuelle Charpentier is sure will bring the Nobel Prize sooner or later.

It is thanks to their research that scientists around the world are able to intervene specifically in the blueprint of life like never before, to manipulate the genetic material, to rewrite it - with a process that is comparatively inexpensive and usually amazing works quickly.

The 49-year-old French woman, director of the Max Planck Institute for Infection Biology in Berlin since 2015, has risen to become a world star in science in less than five years.

In August 2012, when she was still doing research at the university in Umeå, Sweden, she published an article in a specialist journal, only a little over five pages long: “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity”. Together with the American researcher Jennifer Doudna and other colleagues, she describes how the bacterium Streptococcus pyogenes protects itself from viruses. This pathogen can cause scarlet fever or purulent tonsillitis. But Streptococcus pyogenes itself is also vulnerable: by viruses.

The system with which the bacterium fends off these infections consists, as it were, of two areas: archive and cutting tool. In its own genetic material, the unicellular organism stores samples of almost all viruses that it - and its ancestors - have ever come into contact with, as a memory aid. In the event of a renewed infection, the microbe's defense system is activated: It checks the virus on the basis of the archived samples.

The work on Streptococcus pyogenes is the prelude to a revolution

If it detects the intruder, the cutting tool comes into play: A specific enzyme, a kind of molecular tool, cuts the viral DNA. The defense system (which the two researchers call CRISPR-Cas9, according to the archived DNA sequences, CRISPR, and the DNA scissors, Cas9) render the pathogen harmless.

In their essay, however, the scientists not only explain how the mechanism works: They also show how the defense shield can be converted into an all-purpose weapon. Because they have discovered that CRISPR-Cas9 (today mostly just called CRISPR) can be a kind of universal tool for rewriting genetic material. Even more - a technology with which genes can be changed in a targeted manner in bacteria, plants, animals, people.

In short: the work over Streptococcus pyogenes is the prelude to a revolution.

A little more than ten years ago, nobody in science knew anything about the role of this system in bacteria. Today, new studies appear almost daily in which researchers present the results of their experiments with CRISPR. In which they report about attempts to produce peanuts without allergens - or mosquitoes that cannot transmit malaria. Work is already underway to bring extinct animals back to life. Or to cure diseases for which there was previously little hope.

“You start with an idea and think it will take 30 years before something useful emerges from it,” says Charpentier. And then, suddenly, everything goes quickly.

Researchers are already using CRISPR to modify the human genome

There is probably no other development that has advanced genetic engineering so far in such a short time. In simplified terms, one can imagine the targeted modification of genetic material sequences with CRISPR like editing a text file on the computer: The software finds any combination of letters in the document, on a page, in a line. Once the relevant position has been found, typing errors can be corrected, letters or entire words can be deleted, exchanged or inserted.

In the CRISPR process, the letter combination you are looking for is made up of the DNA molecules adenine (A), guanine (G), cytosine (C) and thymine (T) - and in such a way that their sequence corresponds to the section in the DNA sequence that is to be changed.

The enzyme, the actual gene scissors in the CRISPR system, is prepared with the appropriate combination of letters - in a way, it resembles the search mask in the word processing program. As a rule, it now heads for precisely that part of the genome that it is supposed to find. It then binds to the selected section, causing a break in the two strands of the DNA double helix.

Now - as in a word processing program - a passage can be deleted or a new word (a foreign gene) can be inserted.

For more than 20 years, molecular biologists have in principle been able to use enzymes to eliminate or replace certain genes. But the procedures were expensive and lengthy. Like CRISPR, these early gene scissors work with special cutting proteins that bind to a selected point in the DNA sequence and cut the double strand of DNA there.

But these proteins had to be laboriously changed for each use. It took a long series of trial and error before the protein actually found the desired location. Each success often cost hundreds of failed attempts. Sometimes the cutting tool landed a chance hit, and DNA sections were often eliminated in the wrong place. It was only with CRISPR that the word processing program - in order to stay in the picture - received a more precise search mask: it can usually go to the desired point in the book with great precision.

With CRISPR, the technology is no longer a highly specialized application, but largely a standard process. What used to take months or even years to work now usually only takes days. Almost every small laboratory can use the method.

CRISPR has made genetic engineering, genetic surgery, faster, cheaper and more efficient than ever before. A biology student with standard laboratory equipment would be able to use this system to turn off a gene, says Charpentier. A few days of work, material costs of barely a few hundred euros.

Image description: In order to exchange faulty genes or create organisms with new properties, researchers use the CRISPR technique to insert foreign genetic material (green) into a section of DNA (blue). In order for the foreign gene to get to the correct position in the DNA strand, it is marked with a recognition molecule. This consists of the precise genetic sequence of letters of the location you are looking for and attaches itself accordingly. In the next step, an enzyme, a kind of molecular tool (oval structure), cuts through the DNA strand at the very point where the recognition molecule is attached. This creates a gap in the DNA. The body's own repair mechanisms ensure that the strand break closes again. The foreign gene is prepared in such a way that it is automatically inserted into the gap. The foreign gene is ultimately in the original DNA and can take up its function there.

US companies are already selling CRISPR construction kits that even laypeople can use to try out the technology. The Californian company “The Odin”, for example, offers sets that can be used to exchange a gene in certain bacteria so that they can then grow on special nutrient media.

The start-up even wants to enable people to intervene in their own genetic material. At a biotechnology conference in San Francisco in October 2017, Odin's founder Josiah Zayner, who holds a PhD in biophysics, put a syringe in his forearm. The contents, announced Zayner, target its myostatin gene, which inhibits muscle growth in the body. He will be the first person to try to change his own genome in this way. CRISPR in self-experiment: bigger muscles with gene surgery.

The US Food and Drug Administration (FDA) is already warning that the sale of gene therapy products is against the law and that there are concerns about the associated safety risks.

Many major medical problems appear to be solvable with CRISPR

But CRISPR has also electrified established science. Research institutions, universities and companies are promoting the use of the method. With CRISPR, conditions and needs can already be adapted very effectively to farm animals and crops, pigs with a lower percentage of body fat can be bred or cassava plants and wheat that are immune to common viruses and fungi.

Among other things, medical professionals want to use CRISPR to create better animal models for human suffering. Even complex mental disorders such as autism and schizophrenia, in which a large number of gene mutations are involved in different combinations, could become more understandable and treatment effects could be simulated more easily.

Many major problems in medicine suddenly seem solvable with CRISPR. For example, antibiotic resistance could be eliminated by modifying viruses in such a way that they kill bacteria: the unicellular immune system turns against itself.

Or the lack of donor organs for transplants: maybe hearts, lungs and kidneys can soon be bred in pigs thanks to genetic scissors. So far, many pathogens that are passed on from parent animals to their offspring can be found in their genetic material. With the new technology, US researchers succeeded for the first time in rendering these viruses harmless.

The first CRISPR studies on cancer therapy are already underway in China. At the end of 2016, researchers at Sichuan University in Chengdu injected CRISPR gene-surgically processed immune cells into a patient suffering from aggressive lung cancer for the first time.

Another Chinese team was able to use this technique to remove a complete chromosome for the first time: the third, surplus copy of the 21st chromosome from cells grown in the laboratory from a person with trisomy 21, i.e. Down's syndrome. Would it be possible in the future to heal embryos in which this defect was discovered in the womb using genetic scissors? Much seems possible.

The World Health Organization estimates the number of hereditary diseases caused by a single faulty gene at more than 10,000. Conditions such as Huntington's disease, a nervous disease in which the brain becomes progressively sick and causes movement disorders, for example. Or Duchenne muscular dystrophy, which paralyzes the body. Or sickle cell anemia, which damages the heart, eyes and kidneys, for example. These monogenic hereditary diseases are the obvious target for the use of CRISPR in the near future.

However, some researchers want more than stopping these diseases: They want to turn them off long before they can break out - already in the germ line, i.e. on the way from the fertilized egg cell to the germ cells of the new organism.

The genetic surgical manipulation of egg and sperm cells is supposed to free embryos from hereditary diseases. These interventions would also have an effect on the genetic makeup of future generations: If genes are manipulated at this earliest stage of human development, these changes are later passed on - an intervention in human evolution.

Ethically, these interventions are highly controversial. But they can hardly be prevented in the long term - especially since there is no global consensus on the use of genetic engineering and its limits.

For example, a US research group has already experimented with the use of the technology on human embryos at an early stage of development. Using the gene scissors, the team corrected a mutation in the MYBPC3 gene that causes the heart muscle to thicken - hypertrophic cardiomyopathy, a major cause of sudden death in young athletes.

Emmanuelle Charpentier says she is strictly against such interventions: Nobody should touch the germ line.

Jennifer Doudna, her colleague at CRISPR decryption, is not so sure about this question. If there was a way to help people affected by hereditary diseases, would it be permissible not to do so? Even if it cannot be ruled out that the technology and the wishes will at some point go far beyond medical necessities? That parents begin to shape their unborn children according to their ideas in terms of size, eye color and intelligence?

That is still a distant possibility. In addition to ethical considerations, there are still a number of possible risks in the way of using gene scissors on humans. There are doubts as to how secure the technology is. And concerns about the consequences of their use.

The transport route is currently one of the technical difficulties: In order to bring the cutting enzyme to its place of use, the biologists need so-called gene ferries - harmless or artificially inactivated viruses that transport CRISPR molecules through the bloodstream into the cell interior. But these viral gene ferries are quickly reaching their ballast limits. An alternative might be liposomes: tiny fat particles that attach to cells and release their charge inside the cell.

The problem of possible collateral damage of the CRISPR method is also serious - for example if the gene scissors miss their target and cut the DNA in another section. The consequences of these unplanned DNA breaks cannot be foreseen. In the worst case, the changes could trigger cancer.

At a glance

The template
Bacteria use the CRISPR mechanism as a defense to cut up the genetic material of viruses.

The technology
The researchers Emmanuelle Charpentier and Jennifer Doudna have used this to develop gene scissors for science.

The application
Thanks to their invention, it is now easier than ever to create genetically modified organisms or treat hereditary diseases.

US doctors who had corrected the genetic defect in mice with congenital blindness with the help of CRISPR found a good 100 major changes and more than 1000 point mutations in the genetic make-up of the now sighted mice, in which only a single base pair of the DNA was changed.

Whether the mutations were a result of the CRISPR treatment, as the scientists feared, or could have been found in the genome of the mice before the experiment, is controversial: The study did not compare it with control mice.

Even so, shortly afterwards, a group of scientists warned in a study that CRISPR tools had to be carefully adapted to the genome of the respective patient in order to ensure that the technology would not be misdirected by variations in the patient's genome.

Researchers have long been looking for ways in which these risks can be avoided. A team from the Salk Institute in San Diego, for example, has created a new variant of the CRISPR technology that avoids breaks in the DNA. A new, more controllable process has also been developed at Harvard University. The first clinical studies on gene therapy with CRISPR have already started.

The company "CRISPR Therapeutics", in which Emmanuelle Charpentier has a stake, wants to try to cure the hereditary diseases beta thalassemia and sickle cell anemia.

Both conditions are mostly caused by mutations in a gene that is involved in the production of hemoglobin: a complex of proteins that carries oxygen in the blood. Treatment is designed to reactivate a form of hemoglobin found in infants. Approval has been applied for.

The fact that the first clinical studies have already started is record-breaking fast by the standards of medical research - not even six years after the first talk of a novel, uncomplicated and inexpensive gene scissors procedure.

The gene therapy revolution has only just begun.

This article first appeared in March 2018 in "GEO compact No. 54 - Our heritage, our genes".