Although the first article about gene editing scissors CRISPR-Cas9 was published by Prof. Virginijus Šikšnys and his team just 11 years ago, the benefits of this technology for society can already be felt. “[...] It was recently announced that the UK has registered the first CRISPR-Cas9 based therapy for the treatment of inherited diseases,” the researcher says.
We spoke with Prof. Šikšnys at his office at the Life Sciences Center of Vilnius University (VU LSC).
Q: Tell us, please, how the CRISPR-Cas9 technology developed from the beginning?
A: Understanding how technologies develop is an important and interesting question. People might think that researchers come to the lab, develop technology, and the next day it’s already implemented. In fact, it is a long process that usually starts by looking for answers to very general biological questions aimed at understanding the world and environment around us. CRISPR-Cas9 technology is a nice illustration of this process since it emerged trying to understand how viruses interact with bacteria.
We all know how viruses affect people, as the COVID-19 pandemic has obviously shown. However, viruses also interact with bacteria in the invisible world with the same conflicts as our own.
Viruses attack bacteria in order to reproduce and often kill bacteria when viral replication is completed. In other words, if there were no bacteria, there would be no viruses. To cope with the viral threat bacteria developed multiple antiviral defense barriers, but since viruses need to get into bacteria to reproduce, they are constantly trying to overcome the defenses. I sometimes call it an “arms race” where each side is looking for ways to defend against or defeat the enemy, which forces the enemy to find new weapons, and this happens endlessly… This is how bacteria and viruses co-evolve.
This struggle is also important in our everyday life. When we eat yogurt, we may not often think (unless it’s written on the packaging) that live bacteria were used to make it. During fermentation, the milk is infected with bacteria that can turn it into yogurt. If viruses enter this process, the bacteria are often killed, and the fermentation fails as the milk is simply spoiled. This issue is important for the entire dairy industry.
The people who first noticed that CRISPR-Cas9 systems work as bacteria’s defenses against viruses worked for companies that produce starter cultures for the dairy industry. By examining the DNA of bacterial cultures, they saw that bits of viral DNA appeared in the bacteria genome. Researchers then began delving into how it was possible that bacteria have segments of viral DNA. Finally, it was found that there are certain genes close to the areas where the DNA fragments settle and these genes were important both for the incorporation of viral DNA and antiviral defense. This was the first evidence that the CRISPR system protects bacteria from a virus.
Q: And how did you get interested in CRISPR-Cas9?
A: We got involved in it in 2007, when the first article describing CRISPR-Cas systems was published in the journal Science. I got very interested after reading that article. Its authors suggested that bacteria have a novel antiviral defense system but no one understood how it worked. I was curious to try figuring it out. Then we asked the authors of the article to send us cultures of those bacteria, we purified the proteins and began to study them using biochemical methods.
We found that one of the proteins of the CRISPR-Cas systems, called Cas9, acts as a key element of the defense system. It can recognize and destroy the viral DNA. The protein recognizes the virus by using a short RNA molecule that looks like a fingerprint of the invaders' DNA. After recognition, the viral DNA is cut into pieces. We realized that if we changed the fragment of RNA that recognizes the virus, we could direct the Cas9 protein to any location on the DNA. Thus, the idea was born that it could be a programmable tool for cutting DNA. We described this in our first article.
Due to the easy re-programmability of Cas9, researchers immediately started using it as a tool that enables the targeted modification of genomes including mammalian cells.
I’d like to end where I started - CRISPR-Cas9 technology came about because we and other researchers were looking for answers to common biological questions: in this case, we wanted to understand how bacteria defend themselves against viruses. It seems to me that this is how most of the technologies are initiated. It all starts with basic research, followed by applied research and further development of technological developments. If we eliminated at least one link in this chain, nothing good would happen.
Q: And you, as one of the pioneers of the technology, which CRISPR-Cas9 achievements please you the most?
A: I’ve just returned from the conference we organized – “CRISPR-Cas9: from biology to therapeutic applications”.
We published the first article about CRISPR-Cas9 in 2012. Now, just 11 years later, the technology is already in the clinic. Most recently, it was announced that the first CRISPR-based therapy for the treatment of inherited diseases just has been registered in the UK.
Of course, 11 years may seem like a very long time, but in reality, it is a very short period of time. In just 10 years, we have gone from understanding the biological process to clinical therapies.
CRISPR-Cas9 is primarily applied to inherited genetic diseases that result due to changes in the DNA molecule. The therapy I’ve mentioned, which has already been approved in the UK, is intended for the treatment of sickle-cell anemia and beta-thalassemia. Until now, these were incurable and life-threatening diseases, and the patients are often helped only by blood transfusions.
Sickle-cell anemia is a blood disease that develops from a mutation in the hemoglobin gene. By using CRISPR-Cas9 technology, a pathway is activated in the body that produces normal hemoglobin. Clinical trials have shown excellent results, many patients have been transfusion-free for a year. Just imagine how their quality of life has improved.
Q: Can you explain in more detail how CRISPR-Cas9 therapy works?
A: The stem cells, responsible for the production of the red blood cells, are in our bone marrow. If these cells carry a mutated hemoglobin gene then the red blood cells that originate from these cells will produce a defective hemoglobin molecule that will cause the disease. Now researchers can take bone marrow cells, change them in the lab, and transplant them back into the human bone marrow. The replaced cells start producing blood cells containing the correct hemoglobin version. Such therapy is long and complicated, but the result is excellent. Otherwise, a person would not be able to recover from that disease.
Q: And what about genetic syndromes, for example, the Down syndrome? Is it possible to cure with CRISPR-Cas9 such complex diseases that affect not only the physical but also the intellectual abilities of a person?
A: The reason researchers focused on blood diseases is that some of them, like sickle-cell anemia, are caused by a single mutation. After the correction of that single mutation, the person will become healthy. Other diseases, such as Down syndrome, have a genetic basis, but these are complex changes that occur in large numbers in the human genome. It is complicated to change all of them at once. Of course, researchers are thinking about how to do it, but these are questions for the future.
Another reason is that, in the treatment of some blood diseases, cells that are responsible for producing red blood cells, for example, can be removed from the bone marrow, “repaired” in the lab and returned to the human body. If we wanted to apply the same therapy without removing those cells, we would need other tools.
For example, if we want to use CRISPR-Cas technology to correct DNA errors in other tissues or organs, we need to find ways to “target” that tool to a specific organ. If the bone marrow cells can be removed, you can’t remove the brain or the heart. So researchers are looking for ways to deliver these tools directly into the body.
Q: What researchers are looking for is how to target the tool to specific organs?
A: Yes. One approach that is being actively explored is based on iRNA vaccine delivery technology, which many of us have already tried. Vaccines contain lipid nanoparticles, inside which an iRNA molecule is inserted encoding a certain protein. The iRNA serves as the information to synthesize the protein that triggers the reaction, which then gives us protection against the real virus.
Researchers have an idea to insert iRNA of genetic scissors into lipid particles and inject them into the body. They are currently looking for ways to target them to certain organs. Lipid particles, like most of the fat molecules, often end their journey in the liver. It is not surprising that CRISPR tools packaged in lipid nanoparticles will soon be used to treat liver diseases.
Q: But there is still the question of how to take CRISPR-Cas9 into the brain or other cells?
A: Various delivery vehicles are explored for this purpose. One of the alternatives is to use viruses as delivery vehicles. There are viruses, that are not dangerous to us because we live with them, but they can be reprogrammed so that they will carry DNA that encodes the gene scissors. These engineered viruses can be specific to certain tissues, for example, the heart, the brain, etc. and injected directly into the blood or a specific organ. In short, the next step is to do the gene editing in the body circumventing the need to remove cells for the therapy.
Q: Let’s talk about your research, what are you studying now?
A: As I’ve said, gene scissors can be inserted into a virus to deliver it to the right tissue or organ. But viruses have their own limitations… Some of them are rather small and have a limited cargo capacity, therefore you won’t put a large DNA molecule inside. It just doesn’t fit! And the CRISPR-Cas9 tool is pretty big.
Now we are looking for tools to fit the virus. One of the tools we described in the articles published in the journal Nature (2021 and 2023) is very compact. It is not part of the CRISPR systems, but comes from other DNA elements, which are called “mobile genetic elements”.
In fact, they are the ancestors of CRISPR systems. We have identified its molecular function and solved the structure. We hope that this compact gene editing tool will help us to circumvent the packaging limit of viruses. And we continue to look for new tools with unique features.
Q: What you are talking about is very interesting and important for society. Do you think public awareness of genetic engineering topics is important? Could it help get more funding for research?
A: Yes, of course, Lithuania already has a very ambitious goal – to reach 5% of GDP for the life sciences industry in 2030. This field is increasingly better funded. We hope that it will work and Lithuania will become a high-tech country leveraging the academic and business potential.
Of course, it is very important to have direct contact with society. Researchers may not all be very good communicators, they sometimes speak too complexly for most people to understand. What you do is very important… In the old days, when many people could not read, there was a category of people, who interpreted the Bible. They read the Bible and explained it in a language people could understand. I think that’s what you and other journalists are doing: you’re trying to explain to the public what researchers are doing and why it’s important to all of us. After all, the open dialogue with society determines whether the technologies developed by researchers will be acceptable to society or not.
Q: Thank you for the talk.
A: Thank you!
Interview by Goda Raibytė-Aleksa
Photo credits: Justinas Auškelis and V. Šikšnys personal archive