CRISPR-Cas9 has taken the world by storm in just a few years with the promise of making genome editing much easier and faster than ever before. But how does this gene editing tool actually work? How can it benefit biology research? What will happen when we start using it to edit human DNA? And what’s the fight between its developers all about?
CRISPR-Cas9 has been called the ‘biggest biotech discovery’ of the century. This gene editing tool has already revolutionized biology research in the lab, making it easier to study disease and faster to discover drugs. The technology will also significantly impact industrial production based on microorganisms and the development of crops and food.
But the one application that has made it famous is the modification of the human genome, which brings the promise of using CRISPR to cure disease. The first clinical trials testing CRISPR-Cas9 in people are underway in China and about to start in Europe and the US. So while scientists start venturing into tweaking our own DNA, it is worth to take the time to fully understand what CRISPR is, whether the expectations put in it are realistic, and what the actual benefits and risks of using the technology are.
First of all, what is CRISPR-Cas9?
CRISPR is short for ‘clustered regularly interspaced short palindromic repeats.’ The term makes reference to a series of repetitive patterns in the DNA of bacteria and archaea that were discovered and extensively researched by Spanish scientist Francis Mojica in the 90s. These patterns are the basis of a sort of primitive immune system that bacteria use to ‘remember’ the DNA of viral invaders. Cas9 is a protein that can recognize the sequence stored within CRISPR patterns and cut all DNA molecules with a matching sequence.
But it wasn’t until 2012 that Jennifer Doudna and Emmanuelle Charpentier, at the University of California, Berkeley, took the discovery a step further. They published a scientific paper showing what happened when the CRISPR-Cas9 system was taken out of bacteria and introduced in eukaryotic cells — the ones that make up more complex organisms like plants or animals.
“When you cut the DNA of a bacteria, you kill it. But in eukaryotes, when you cut DNA you activate a repair mechanism that opens the possibility to rewrite DNA,” Mojica told me. “Jennifer and Emmanuelle did it in vitro and it worked wonderfully.”
Another two papers published just a few months later by Feng Zhang and George Church from the Broad Institute also reported some early uses of CRISPR as a gene editing tool, which has led to a patent dispute between Zhang and the Doudna-Charpentier team. But we’ll talk about that later.
It is important to note that CRISPR is by far not the first system that allows us to edit DNA in all sorts of organisms. Other technologies used extensively before are TALEN and zinc-finger nucleases (ZFNs). And in fact, some experts point out that these tools, which have been in use enough time to become quite refined, are more accurate than CRISPR-Cas9.
But CRISPR brings an important advantage over these other techniques, which is that they are much easier and faster to use. Most previous technologies required creating a molecule from scratch designed to make changes in very specific DNA sequences. With CRISPR, the same Cas9 molecule can be directed to any sequence just by providing it with a guide RNA molecule. This is much easier to synthesize, and companies like Synthego in the US have spotted a good business opportunity producing them for researchers.
What can CRISPR do?
In theory, CRISPR could be applied to modify the DNA of virtually any living being for all sorts of different applications. In biotech and pharma companies, CRISPR is becoming the go-to tool for drug discovery. In academic research labs, the gene editing tool has already become quite popular and is being used by many to modify the genome of organisms ranging from bacteria and worms to mice, pigs, and even monkeys. This is very valuable to understand the function of any gene of interest, whether it is one that causes disease or one that makes a crop produce better yields or survive harsh conditions.
In agriculture, CRISPR could be used to produce crops that grow faster or that resist drought much faster than with traditional breeding techniques. It can also be used to add new features, such as making tomatoes spicy, or remove others — for example making gluten-free wheat or decaf coffee beans. However, regulations can limit the use of these technologies. While the US has already seen the launch of CRISPR-modified crops, the European Union decided to set strict GMO regulations that scientists believe are hindering the potential of the technology.
But right now, most of the money seems to be in using CRISPR-Cas9 to engineer human DNA. With over 10,000 diseases caused by mutations in a single human gene, CRISPR offers hope to cure all of them by repairing whatever genetic error the patient has.
There are two main approaches to using CRISPR as a human therapeutic. The first is called ex vivo gene editing, and involves extracting human cells, engineering them in the lab, and reinjecting them into the patient. This method is similar to that used for most gene therapies already in the market, and it allows more control over the process. However, it can become quite expensive given each patient requires an individual manufacturing process for their therapy.
The second method is called in vivo gene editing and involves delivering CRISPR-Cas9 into the patient’s body to edit the DNA directly from within the cells. CRISPR could be delivered inside nanoparticles or encoded into DNA and be cleared out of the body once it has completed its mission.
Wait, is editing human DNA with CRISPR safe? Or ethical?
Those are the big questions right now. Especially after CRISPR gene editing was used to create the world’s first gene edited babies. These ‘CRISPR babies’ carry a mutation that protects them against HIV infection. The experiment resulted in a strong pushback as scientists around the world questioned the ethics of altering human DNA without fully understanding the possible consequences. Indeed, there is now proof that people carrying these mutations might be at risk of catching certain infections and dying younger.
Despite all the talk about CRISPR and the money invested in it, there are still no clinical results from human trials, though there will be soon. A recent study pointed out that since the Cas9 protein is naturally found in bacteria that infect humans, the immune system of many of us is already primed to attack it.
An immune reaction against CRISPR could not only render the therapy useless but could induce severe side effects. Scientists are wary of repeating the same mistakes as when gene therapy was first tested in the 90s, resulting in the death of 18-year old Jesse Gelsinger and years of delay in the development of gene therapy.
Now, even if CRISPR proves to be safe in humans, is it ethical to modify the human genome? The first applications of the technology, aimed at curing genetic diseases, seem quite straightforward. But where should the line be drawn? At what point does a therapy become a tool for eugenics?
Although the point in time when we are able to modify all sorts of human features at will is far ahead in the future, it is never too early to start thinking about how the technology should be regulated and in what cases its use should be allowed or banned. Many scientists, including Doudna, seem to agree that germline editing — that is, DNA modifications that children will inherit, should be left out. At least for now.
Who is developing CRISPR-Cas9 therapies?
Since the first publications showcasing CRISPR-Cas9 as a gene editing tool back in 2012, a number of companies have been set up by the developers of the technology. Based in Switzerland and the US, there is CRISPR Therapeutics, co-founded by Emmanuelle Charpentier. Working in partnership with Vertex Pharmaceuticals, the company has already started a first clinical trial with CRISPR in Europe and the US — after the US FDA lifted a temporary hold — targeting the blood disorders β-thalassemia and sickle cell disease. The company is using an ex vivo approach where the hematopoietic stem cells of the patient are genetically engineered outside the body.
In the US, the first CRISPR clinical trial started earlier this year, run by scientists at the University of Pennsylvania. On top of academic efforts, the US counts also with Intellia Therapeutics, co-founded by Jennifer Doudna, whose first target will be an in vivo treatment for the rare neurological disease transthyretin amyloidosis.
Co-founded by Doudna and Charpentier’s competitor Feng Zhang, there is also Editas Medicine, working in therapies for genetic blindness and cancer among others. Doudna originally co-founded Editas along Zhang, but stopped all involvement with it just a few weeks after Zhang was granted his CRISPR patent and issues concerning intellectual property began to appear.
Who owns the intellectual property of CRISPR tools?
“The intellectual property in this space is pretty complex, to put it nicely,” says Rodger Novak, co-founder and previous CEO of CRISPR Therapeutics. “Everyone knows there are conflicting claims.”
The team of Doudna and Charpentier at UC Berkeley filed a first patent application for CRISPR in May 2012, a few months before their paper was published. Zhang and the Broad Institute filed theirs in December that year, but they paid the US patent office to fast-track the review process. This resulted in Zhang’s patents being issued before there was a decision on his competitors’.
UC Berkeley then initiated a process to invalidate the Broad’s patent on the basis that Doudna and Charpentier had developed the technology and applied for a CRISPR patent earlier. The patent office ended up ruling in favor of the Broad Institute last year, after both parties combined had already spent over $20M (€16M) in legal fees.
“It reminds me of reading about really unhappy rich people,” said George Church about the patent fight. “They have such a big blank check that they just make each other miserable.”
“Everything here is very exaggerated because this is one of those unique cases of a technology that people can really pick up easily, and it’s changing researchers’ lives. Things are happening fast, maybe a bit too fast,” commented Charpentier. “I am very confident that the future will clarify the situation. And I would like to believe the story is going to end up well.”
Indeed, for her the situation is quite favorable on the other side of the Atlantic. While Zhang seems to have ‘won’ in the US for now, in Europe the tables are turned. The European patent office granted Charpentier two patents for the use of CRISPR in a wide range of applications.
What’s next for CRISPR?
With its potential already demonstrated in research, the next big milestone for CRISPR will be demonstrating to be safe and effective as a treatment. But there are many other cool applications underway. One of them is the use of CRISPR to modify the pig genome so that their organs can be transplanted into humans without creating rejection, thus circumventing donor shortage. eGenesis, co-founded by George Church, is already working on it. Another is the use of CRISPR as a diagnostics tool.
But CRISPR might still surprise us as new variants and applications are developed. A version called CRISPR-Cpf1 that might make certain modifications easier is already being extensively researched. “You can imagine that many labs — including our own — are busily looking at other variants and how they work,” Doudna said. “So stay tuned.”
While researchers keep refining the gene editing tool, CRISPR is becoming very popular between DIY scientists and biohackers. Some believe that the relatively simple methods that this technique requires might help democratize science and bring it closer to people outside the lab. However, some recent cases of biohackers injecting themselves with experimental treatments have alarmed the public it remains to be seen how these uses will be regulated.
In any case, the impact of CRISPR in biology is already tangible and will undoubtedly go down in history as a big discovery. The cherry on the cake will come when the technology wins the Nobel prize, which many have been unsuccessfully predicting will go to CRISPR for years.
“It is possible they are waiting for CRISPR to demonstrate all the potential that is expected, but it would be unfair,” CRISPR discoverer Francis Mojica told me. ” What CRISPR has already achieved is much more than what other tools that have received the Nobel have achieved. The prize has gone to tools used to cut and copy DNA in the test tube. CRISPR can be used to edit genomes, change expression levels, visualize DNA, kill bacteria, develop diagnostics, and many more applications, even to store a movie within DNA.”
“I am convinced it will get it. When? I don’t know.”
Images via Soleil Nordic /Shutterstock; The Conversation; Knaw /Flickr CC2.0; Science