CRISPR – beyond genetics

© iStockPhotos

© iStockPhotos

CRISPR-Cas9 is a tool that enables the genome to be edited, like word processing, enabling us to cut or cut-and-paste genes. Easy to use, highly efficient and quick, CRIPSR has revolutionized genetics. But by modifying human embryos, a Chinese scientist has opened Pandora’s box. In the following article, published in the March 2019 issue of EPFL Magazine, EPFL researchers explain.

On 26 November 2018, Chinese researcher He Jiankui announced that he had created the first genetically modified babies, twin girls born a few weeks earlier. Following in the steps of in vitro fertilization, the scientist from Southern University of Science and Technology in Shenzhen – who has since been fired – disabled a gene to endow the children with immunity to the AIDS virus. The scientific community has reacted with shock, indignation, and condemnation. By interfering with the human genome, He Jiankui violated scientific integrity and crossed a line.

At the same time, the event made the broader public aware of the tool hidden behind the six letters of CRISPR, which stand for Clustered Regularly Interspaced Short Palindromic Repeats. Used for a few years now, CRISPR is already considered one of the most important discoveries in the history of biotechnology, alongside restriction enzymes that enabled DNA to be cut and manipulated, green fluorescent protein that reveals gene expression, and the polymerase chain reaction (PCR) that enables a DNA or RNA sequence to be amplified. In summary, CRISPR took geneticists from the typewriter to word processing.

Discovery out of the blue

Scientists already knew how to modify the genome, but CRISPR made things much simpler, faster, and efficient by giving scientists genetic scissors to cut or cut-and-paste genes wherever they want in the genome. Appearing somewhat out-of-the-blue, CRISPR actually hails from microbiology: it is one of the techniques used by bacteria to combat phages, the viruses that try to colonize their genomes. When bacteria detect the presence of viral DNA, they produce RNA corresponding to that of the invading virus. This RNA then recruits a protein called Cas9 and guides it to the section of the genome corresponding to the viral DNA, which the Cas9 protein will then cut and remove from the bacterium’s genome.

The process is similar to editing a plant, insect, or human eukaryotic cell. The guide RNA, paired with a Cas9 protein, seeks its target sequence in the cell’s DNA, which its partner cuts like molecular scissors. The cut simply deactivates the gene, but another piece of DNA can also be added to Cas9 to replace the part that was cut, e.g. to repair a mutation.

Multiple applications

CRISPR-Cas9 is used mainly in three ways. First, to create mutants for basic research, from worms to monkeys, fruit flies, or mice. “It’s the most effective approach; it works really well,” confirms Bruno Lemaitre, a specialist in the immune system, which he studies using fruit flies (see below). “CRISPR enables us to generate and study a large number of mutations that affect immunity genes.”

Second, CRISPR-Cas9 offers great hope for treating genetic diseases, such as gene therapy for congenital immune disorders – the famous “bubble children” whose hematopoietic (“blood-making”) stem cells in the bone marrow, are genetically modified to rebuild a functional immune system. “Gene therapy with viral vectors is now successfully applied to treat some of these conditions, but we can imagine that in a few years we will use CRISPR for genetic diseases where viral vectors have not been satisfactory,” suggests Didier Trono, head of the Laboratory of Virology and Genetics. The same applies to brain cells, e.g. in diseases like Parkinson’s disease.

We can also modify the genome directly on the first cells of the embryo, which means that the mutation can be passed on to the next generation. This is what He Jiankui did for the first time on humans.

Finally, there are applications in agriculture and the environment. CRISPR-Cas9 facilitates genetic modification of plants that resist predators or can produce materials. For example, researchers at Stanford have managed to make yeast produce opioids. On the environmental front, researchers have created lab mosquitoes that are resistant to malaria, paving the way to potential control the parasite through the insect that carries it. This is especially possible because CRISPR-Cas9 enables gene drive (genetic forcing): a system that ensures 99.5% transmission of a gene to the next generation through sexual reproduction. This could enable the elimination of invasive species, e.g. certain mammals in Australia or New Zealand – or mosquitoes again.

For life and beyond

Of course, the technology has its limits. “One of these is the uncertainty about the tool’s precision,” says Trono. “It is easy to tell whether we have targeted the right area, but harder to know whether we have targeted others too.” This is the so-called ‘off-target effect’. “If we cut the genome in the wrong place, the cell may develop unexpected properties, such as cancerous degeneration. This poses a problem in a therapeutic context.”

CRISPR also raises the question of modifications to the germ line, which could be transmitted to future generations. “A modification might be positive today, but will it still be in 500 years?” asks Trono. For Denis Duboule, head of the Laboratory of Developmental Genomics, this is not about tools: “Instead of operating with a scalpel, CRISPR means we can operate with a genetic scissors in the egg. That’s all. And all the better if our descendants can avoid a gene like BRCA1 that causes breast cancer in 35-year-old women! First we treat, and worry about consequences later. Still, that does not mean we should just produce anything.”

“These techniques that seem like science fiction today have enormous potential,” Duboule adds. The mutations are stable, it’s as if you are reprinting a book. The problem is not changing a word in the text, but changing the right words. Today, we can’t yet say that this is the right word, but in twenty years we’ll be there.”


Gene drive is worrying too. “We can exert control over wild species, for example to fight against pests and avoid using pesticides,” says Lemaitre. “But this human dominance causes concern even among researchers.” “The danger is even greater because CRISPR’s ease of use increases its reach and experiments could even be done by first-year students,” says Kenneth Oye, Director of the Program on Emerging Technologies at MIT who was invited to EPFL this year. “The result: biosecurity managers should be concerned about possible harmful use of this technique, not just by at a handful of institutions but by many ‘amateur’ biologists. The regulatory mechanisms need to be reviewed.”

As for “transhumanist delusions”, Duboule believes these have nothing to do with CRISPR. “This would be a question about synthesizing human chromosomes. At that point, we could really produce a transhuman genome. But for now, modifying the genome is just like changing the sauce for a pasta dish. It’s still pasta.” And, for the moment, Oye points out, “there is consensus among scientists that the therapeutic applications are appropriate, but human-enhancement modifications are not.”

“We would need a specific form of regulation,” concludes Marie-Valentine Florin, Head of the International Risk Governance Center at EPFL. “But have no illusions: if it goes against the will of scientists or the public, it won’t be followed. Surveys show that people are broadly in favor of gene editing to prevent the birth of children with serious diseases. And there’s a huge business behind this. The main risk comes from clinics that are prepared to do this.”

Brilliant or foolish, He Jiankui crossed a line

Will there be before-and-after “CRISPR babies” – the way the media described the Chinese twins whose genome was modified? The scientific community as a whole disapproved of the He's move, believing he made a mistake by crossing the line. But is it possible to stop a genetic, societal, and economic revolution that is already underway?

“He Jiankui made a serious error of judgment, according to all institutional standards,” says Oye. “The parents probably did not give their informed consent, the potentially serious side effects were not considered, and there are other methods to obtain the desired result. The case is clear, he did everything wrong. But there will come other cases, perhaps more sensitive, loaded with ethical, medical, environmental, and biosecurity issues.”

“Deplorable but irrefutable”

“It was not just criminal; the modification he made also does not make any sense,” deplores Trono. “It protects against HIV, but these children were not particularly exposed. However, inactivating the CCR5 gene can cause other problems: people carrying this mutation – around 3% of Caucasians – are more vulnerable to other viral infections.”

A matter of risk?” This story shows that there are value systems where some risks are acceptable,” says Florin. “We clearly see that, in China, risk tolerance is higher than ours. There is still strong discrimination against HIV-positive people in this country. Parents are therefore ready to take a risk to have a child that they are told will be healthy.”

Furthermore, Duboule sees the Chinese scientific move as a deliberate one that will lead to a geographical shift: “It sounds the death knell of the Anglo-Saxon empire in terms of basic research. Of course, He Jiankui is wrong. But he did this using a state-of-the-art method from a technical perspective, and in a highly intelligent way: he used a gene that was normal in the two chromosomes and broke it. If he had interfered with a mutation like BRCA1, he would not have been able to prove that the mutation was present in the genome he had modified. But in the case in question, we know that what he did is solely through CRISPR. It’s deplorable but irrefutable. He will go down in history as the first to have done this.”


A revolution in the daily lives of EPFL researchers

An abundance of mutants

In his lab at the School of Life Sciences, Bruno Lemaitre studies the immune system using fruit flies. “We mutate Drosophila genes to study their role in the immune system. To get to know the function of a gene, the genetic approach involves mutating it to suppress it and seeing what happens.” And with CRISPR, mutating a gene has almost become child’s play. In his lab, he grows a hundred strains of flies generated by CRISPR with a mutation in a given gene, and produces around ten of them each year.

“What was impossible before becomes possible – like mutating a large number of genes or making double or triple mutants,” the professor explains. For example, at the end of February his own lab announced a major advancement in understanding our immune system’s first line of defense, called “innate immunity”. Until then, it was impossible to understand clearly the role of antimicrobial peptides, small proteins produced by animals with antibiotic properties. The multiple genes that encode these antimicrobial peptides hampered their study until now.

So the researchers used CRISPR to destroy no less than 14 different genes encoding antimicrobial peptides in fruit flies. By suppressing one, several or even all of these 14 genes, the scientists managed to determine their role in fighting infections. Surprisingly, some antimicrobial peptides proved to be extremely specific in their action, targeting a particular pathogen.

Manipulating expression of the genome

Didier Trono, Head of the Laboratory of Virology and Genetics, is working at the cell genome level. For him, CRISPR is part of the arsenal of powerful and elegant genomic tools that are available to researchers. “We use it more like a Stabilo Boss highlighter than scissors,” he says. “It enables us to target parts of the genome, to either activate them or suppress them. In other words, we ask the CRISPR system to perch on bits of DNA, and, by combining them with other proteins, we can activate or suppress the underlying area. So, we can manipulate the expression of the genome in a surgical way, without a scalpel, using derivatives of the CRISPR system.”

“Moreover, this is a more subtle approach than cut-and-paste, which some cells don’t like at all. For example, if we try to use human embryonic stem cells, they react to a Cas9 cut with a cascade of events that leads to their death.”

“A real epistemological change”

For over 30 years, Denis Duboule has been working on the mouse genome to understand the fundamental mechanisms of the evolution of mammals. The advent of CRISPR was a revolution for him. His Laboratory of Developmental Genomics works in particular on neighboring mutations, i.e. those that are very close to each other on the same chromosome. In this case, there is a much higher probability of the mutations being transmitted from one generation to the next. “CRISPR is extraordinary here,” says Duboule. We have developed a method based on electroporation, which no longer requires injections. We are also working on in vitro fertilization, so less interbreeding of mice is required. We don’t even need to make mice strains anymore, because the frequency of the mutation is so high – fifty percent.”

However, he adds that “there are very few things that we can now do with CRISPR that we couldn’t do before.” But this small technological advance leads to a major epistemological change, that is, in the rules and principles of research. The fact that there are no more constraints – in time, cost, or efficiency – means that the experimental design has changed. “We now plan experiments based not on feasibility but on scientific interest. Before, we did what we could do; now we ask how we should do it.”

Another shake-up: confidentiality. “Before, we could talk about unpublished results at conferences because we knew it would take colleagues two years to do the same thing. We’re more careful today. Where it took two years to publish results, it now takes three months.”

So, is science progressing more rapidly? “Not really, because even if today we can do in two months what used to take twelve, there's still the question of budgets. Although this new technology is cheaper, it still carries a hefty price tag. This means we have to prepare our experiments well in advance. But this will change: in a few years’ time, it will cost almost nothing.”

A bitter patent war

The first article on the use of CRISPR-Cas9 appeared in Science back in 2012. It was authored by Jennifer Anne Doudna of Berkeley (University of California) and Emmanuelle Charpentier, who holds an honorary PhD at EPFL since 2016. Six months later, Feng Zhang, at the Broad Institute of MIT, published an article on the use of CRISPR-Cas9 in eukaryotic cells.

Both teams applied for patents; in April 2014, Feng Zhang scored first with the United States Patent and Trademark Office, because he used a fast-track procedure. Legal action was taken by the two other researchers. In September 2018, a US federal appeals court upheld the Patent Office’s decision. However, on February 8, the US Office announced that it will grant a patent to the University of California.

The stakes are primarily financial: the scientific community benefits from the free use of the technique, but commercial use means they are required to pay royalties to the patent holders. But it’s also a question of prestige: CRISPR could well be worth a Nobel prize.