Charpentier, PhD
French microbiologist Emmanuelle Charpentier, PhD, has played a critical role in launching the field of CRISPR gene editing while moving between laboratories and countries in her search for a stable and independent research environment. While working at Umeå University, she collaborated with another CRISPR pioneer, Jennifer Doudna, PhD, a professor at the University of California, Berkeley, to show that an RNA-guided CRISPR enzyme, Cas9, could introduce double-stranded breaks in targeted DNA sequences. This work culminated in a classic paper that appeared in Science in 2012 and listed Charpentier and Doudna as co-corresponding authors.
Since then, Charpentier’s contributions have been heralded by the scientific community and celebrated with dozens of prestigious awards. At present, she occupies a lofty position at the Max Planck Institute for Infection Biology, namely, director of the Department of Regulation in Infection Biology.
During a recent visit to New York City, Charpentier agreed to a rendezvous—appropriately in a French bistro—with GEN’s Kevin Davies, PhD, who asked the peripatetic scientist to describe her training, her role in the CRISPR revolution, and her future plans. Much of the discussion highlighted the scientific gains Charpentier achieved after she finished her postdoc stints in the United States and returned to Europe. This part of the discussion is presented here, adapted with permission from The CRISPR Journal, which originally published the whole conversation between Charpentier and Davies.
Davies: You’ve been an independent investigator in Vienna, Sweden, and now Germany. Was this something you had to do to keep finding resources to continue your research?
Charpentier: Yes. In 27 years, I’ve worked in five countries, seven cities, 10 institutions, 14 different offices, and 13 different departments. It’s a very big turnover!
It was not that it was planned, but I realized during my postdoc that it was going relatively fast. After a short time in a lab, I had seen what I wanted to see, I had done my time, I was successful, I needed to move on. I could feel very quickly when it was the right time to move on and avoid getting stuck in a position where maybe I would be unable to evolve the way I wanted to evolve. Each time I went somewhere, there was always an incentive or a better position.
Davies: As you look back over the previous 5–10 years, what are one or two of the major insights or turning points in the story?
Charpentier: This was clearly the experiment in 2009 that found a link between tracrRNA—the second RNA of the CRISPR-Cas9 system—and the CRISPR-Cas9 system. It was a very simple experiment.
The idea I had was that tracrRNA was involved in the activity of the CRISPR-Cas9 system. We had a knockout of tracrRNA because we were studying tracrRNA. (We were going in the wrong direction.) We also had a knockout of CRISPR RNA because we were interested in understanding the CRISPR-Cas9 mechanism. It became a simple experiment when we realized that when we were knocking out tracrRNA, maturation of CRISPR RNA no longer occurred, and when we were knocking out CRISPR RNA, maturation of tracrRNA no longer occurred. We realized right away that Cas9 was important in the stability of those two RNAs.
Very quickly it came to our minds that Cas9 forms a complex with this RNA duplex, and that tracrRNA can form a duplex with CRISPR RNA. I remember that it was the next day, after we considered other organisms, when we figured out that tracrRNA was a unique molecule. We realized it was not defined by sequence identity among other tracrRNA molecules in bacteria; instead, it was a molecule with an extremely variable nucleotide sequence. We compared tracrRNA molecules with one another and saw that they have a common characteristic: an anti-repeat sequence that base-pairs to the cognate repeat of CRISPR RNA.
It was also clear that tracrRNA would not be the end of the story. If you were looking at the other CRISPR-Cas systems—type 1 and type 3 systems, described by the groups of [Luciano] Marraffini with [Erik] Sontheimer for type 3, also Michael Terns, and for the type 1 system, Stan Brouns and John van der Ost—it was clear that those systems work with a complex of proteins guided by CRISPR RNA.
Davies: It was 2011 when you met Jennifer Doudna?
Charpentier: Yes. The CRISPR field then was fascinating because it was bringing together a very interesting crowd of diverse scientists. You had the bacterial geneticists, the molecular biologists, the biochemists, and the structural biologists. There were different papers starting to describe the structures of those proteins, and Jennifer Doudna had already done substantial work on the structure of those proteins.
The structure work was important because we had already identified these diverse Cas9 proteins of different sizes with different sequences, always with a duplex of RNAs but of different structures. My idea was that it would be important to understand what is or is not conserved in terms of structure on those proteins that have different sizes and sequences. It would be important to gain insight into these mechanisms because if one wanted to reduce the system to practice, then the structural biology might provide clues for shortening the proteins [for genetics (especially in higher eukaryotic organisms but also in microbes)] and doing some protein engineering.
When I went to the meeting [in Puerto Rico in 2011], I knew I would meet Jennifer Doudna. I was looking forward to meeting her because I didn’t know her. [This was around the time] I published in Nature work that I had presented [a year earlier] at the first official CRISPR meeting, which had been organized by John van der Oost and held in Wageningen, the Netherlands. I figured out that the CRISPR scientists knew one another because they had attended this secretive meeting that took place in Berkeley prior to 2010, where I had not been invited because no one had any idea that I was working on CRISPR.
When I presented my work at the Wageningen meeting, Jennifer was not present. But Jennifer and her colleagues discovered that I was working on CRISPR, and they discovered the famous story of tracrRNA. It was very nice because this was, I think, the highlight of everything. The pioneers all came up to me and shook my hand and said, “I think you got the story!” So, yes, I had in mind approaching Jennifer and asking her whether she would be interested in deciphering the structure of Cas9.
Davies: Your collaboration clicked straightaway?
Charpentier: Well, yes. When you meet, sometimes you feel that it is going to work out, sometimes you think it is not going to work out. What is important for any collaboration is a good gut feeling and a good interaction with a colleague. For laboratory scientists who are going to work on a project, there is a need to get along with one another. This need was met in an interesting way by my students, Krzysztof Chylinski and Martin Jinek. Krzysztof is Polish, and Martin is from the Czech Republic. They could understand one another a little bit when they were speaking Polish and Czech.
We held a number of Skype meetings. I was joining Krzysztof and Martin most of the time. The biological questions were very straightforward. What was important was: first, to have the Cas9 purified, and second, to plan my laboratory’s assays, which we wanted to run plus/minus tracrRNA or CRISPR RNA. We wanted to show cleaving activity and any activity other than cleaving; we wanted to look for protospacer adjacent motif, sequence, and domain requirements; and we wanted to show reduction to practice. This became the Science paper. Along the way, Martin was trying to improve the conditions to get the CRISPR structure, which ended up in a publication.
Davies: Was there a eureka moment along the way?
Charpentier: For me, no. But there was a second eureka moment after a Northern blot analysis indicated that there really is an interaction, etc. A follow-up observation the next day told us everything, which is also a study that we ultimately continued in collaboration with the group of Jörg Vogel in Germany.
A second eureka moment in my laboratory was when my student Krzysztof showed that Cas9 does indeed cleave with tracrRNA and CRISPR RNA. This was it. In the test tube, we have those three components. And it is cleaving. The first assay that [we] did is actually the first figure of the Science paper, so all the controls were working right away.
The fact that [CRISPR] would surely be useful as a gene technology, this came earlier—even the idea that it could be useful for the silencing and recombination of genomes in higher organisms, [as week as] for the treatment of human genetic disorders through new ways of developing gene therapies.
But this was also because, prior to starting my master’s studies in microbiology at the Pasteur Institute, I had done a bachelor’s thesis in the early 1990s in a laboratory focusing on human gene therapies. This was one of my early interests. That is why I started with bacterial genetics, and genetics has always been the “red thread.” I developed genetic tools to have better tools to do bacterial genetics when I was a postdoc in the United States. So, it has always been an obsession.
Ultimately, we developed better tools for Streptococcus pyogenes, and these tools were very useful to make sure that we understood the role of tracrRNA, CRISPR, etc. These tools were allowing us to do precise genetics.
Davies: Much was made upon the publication of the Science paper, particularly in retrospect, that the big question remaining was to demonstrate that this gene editing system would work in human cells. Did you feel that was a formality?
Charpentier: For me, it was not a concern because of other systems that have been used to do knockout mice with proteins originating from bacteria. It was working so well in vitro, and then you test it in bacteria, in higher organisms. So, I was very positive [laughs]. Maybe it is my way of approaching science.
Davies: Did you quickly see the potential of this technology and decide to launch a biotech company? Or was it a case of people coming to you?
Charpentier: No. This was 2012 and very much thought through because of my early idea. When I was in the United States (at end of the 1990s), I got very interested in principal investigators who were looking for ways they could become involved in biotech companies. The vector system I developed in the Novick laboratory [at New York University’s Skirball Institute] was ultimately commercialized. I always had in mind that one day it would be nice if my research could lead to anti-infective strategies. Indeed, there is a way to harness CRISPR-Cas9 in this regard, at least as a sort of antibacterial. But the right application for me was human gene therapy.
In 2012, my laboratory was also in the middle of a new move and very busy. I contacted Rodger Novak, who put me in contact with an investor he knew, Shaun Foy. I just told him, “I’m working on CRISPR.” I explained to him, there is a possibility of applying the technology directly in human gene therapy. “Maybe you think I am crazy,” I said. “I do not know timing, etc.” This was the start of CRISPR Therapeutics and ERS Genomics, with first a wish to establish the company in the United Kingdom or in
Switzerland.
In early 2013, I approached Jennifer Doudna and told her that I was more interested in going toward therapeutics. She was more interested in the toolbox, kits, and so on. At that time, I also approached Feng Zhang, who said he would be interested in joining the company. But ultimately, the Broad went in another direction. We
followed our own way.
Davies: We are on the verge of entering the clinic. The literature is full of concerns, scares, off-target effects, p53, immune effects. Do you feel that delivery may be the biggest challenge ahead of meaningful therapeutic benefit using CRISPR gene editing?
Charpentier: Yes, I think so. As much as it is important to work on the specificity of CRISPR-Cas9, the bottleneck remains the delivery systems—to have CRISPR-Cas9 delivered in the maximal number of cells in the most efficient way, in the safest way, with no secondary effects or unwanted reactions.
Davies: You’ve been in Berlin now for three years. Are you happy there?
Charpentier: Yes. I joined the Max Planck Institute for Infection Biology three years ago, and now I have started a new small institute focused on the science of pathogens. We’ve been in negotiation to find space in Berlin near the campus of Humboldt University and the University Hospital La Charité, and we hope to start planning the building soon. We hope that the building will be ready in five to six years, where we’ll have the opportunity to recruit five or six independent group leaders to focus the research of the institute on the science of pathogens.
Davies: Your life has changed almost beyond recognition, I suspect, in the past five years. Do you wish you could go back to relative anonymity—to just being in the laboratory, turning on the radio, and doing your work?
Charpentier: No, I don’t have that luxury anymore. I am conflicted. Let me put it this way: I have so many things to take care of that, in a way, I have to remain pragmatic. I try to organize myself as much as I can to deal with the overwhelming situation on different angles, whether it is the CRISPR-Cas side effects, or also a difficult thing that I encountered lately—finding ways of having a totally independent laboratory in an independent locality. This was the reason I started this new institute. But sure, I miss the time where I could be in the laboratory, feeling a little freer, enjoying more quiet, and focusing on research.
