It was August 21, 1992 when a graduate student at the University of Alicante, Spain studying the transcriptional responses of the archaeon Haloferax mediterranei to high salinity noticed an unusual pattern of repeated sequences in a gel. Soon thereafter he noticed that a similar pattern of repeats had been reported in Escherichia coli in 1987 and in Mycobacterium bovis in 1991. He had a feeling that such patterns found in distantly related prokaryotes had to be of great biological importance. Steadfastly, he pursued this feeling, eventually leading him to the discovery of a prokaryotic adaptive immune system. This system, which we now know as CRISPR (and which has been extensively covered in this blog, see here), revolutionized our understanding of microbial evolution and, through its application as a tool for genome modification, has revolutionized all of biology. The graduate student who saw that gel in Alicante in 1992 was Francisco Mojica, now a professor at the same university. Francisco very graciously agreed to be interviewed by STC. I jumped at the opportunity to carry out the interview, asking him to relate from a very personal perspective, the discovery of CRISPR. Here is his story.
Roberto: There are many topics related to CRISPR on which I'd love to hear your perspective. Perhaps starting with the recognition, during your doctorate, of unusual repeated sequences that eventually led you to the discovery of the immune system of bacteria and archaea. I am particularly interested in how you interpreted the data, what challenges you had, the difficulties in publication. That would be truly interesting. And, of course, I'd love to hear your opinion regarding the most recent developments on the application of CRISPR-based technologies and how you see the future.
Francisco: How I started with all this CRISPR story was, as is often the case, a bit by chance because I was looking for other things. I was working with extreme halophilic archaea and my interests were to determine how they adapted to growth at different salinities. As you well know, these archaea can grow in saturated solutions of sodium chloride. We started with a project that involved the first DNA sequencing done at the University of Alicante. You can imagine all the problems that involved. Those terrible sequencers! Horrible! Those glass plates to which the ultra thin gels got stuck, sometimes onto one plate and sometimes to the other, but most often half a gel onto each, creating a major mess. Only very occasionally did the sequencing come out. Among the very few good sequences we obtained – and this was among the first ones we did – we found this remarkable pattern: a sequence that was repeated many times at regular distances. Our immediate interpretation was: "Another artifact of sequencing! For once the gel works out and there has to be a problem and we do not know what the real sequence is." So, we repeated it and verified that it was actually real. There really were repeated, regularly spaced sequences! For us this was a tremendous surprise. In any case we looked at the databases of those days. You know, a far cry from today's PubMed. But we saw that there was something similar in E. coli . I'm talking about 1992, comparing sequences then was not what it is today (see here for GenBank statistics).
Do you have a photo of that first gel?
Yes indeed! Here, this photo was published in FEBS, in a publication from last year. In fact the journal cover is the key part of the gel.
We then asked if a similar pattern also existed in E. coli . Yes it did. The two looked a lot alike in terms of the pattern, not in the sequence itself, but in their general structure and organization. We said: "This is fantastic. This is telling us something that no one has seen before. It must be very common in prokaryotes because it is in E. coli and in a halophilic archaeon." But perhaps our most important conclusions were these. First, this finding had to be very important, and second it could not have to have anything to do with the adaptation to high salt, which was what we were looking at in that one DNA region. Because, after all, E. coli … The poor bacterium, you place it in a one molar solution of sodium chloride and it dies. So that led us to do some additional experiments. The first thing we saw was that the repeats, the region of the CRISPR repeats of the halophile, were transcribed. Then we did some experiments in which we introduced multicopy plasmids that contained cloned regions of these repeats that were also on the chromosome. And we saw the best phenotype that we could have hoped for! We found, of course, that the cells receiving these plasmids were dying! There was some sort of interference that we simply could not interpret at that moment. But, well, at the time what the results were telling us is that these repeats had a fundamental function for the cells. So from there onwards I became entranced by these sequences. I kept working with them hoping to understand them. Wondering what could be the reason for this cell death when we manipulated them. And so, let's say, almost ten years went by. During which I did my post-doc…
You were in Utah and Oxford, right?
Yes, first I was in Utah with Sandy Parkinson and then in Oxford with Chris Higgins doing things that have nothing to do with CRISPR at first sight. But, hey, as you well know, everything is related. As it turns out in Oxford I was working on a protein, H-NS, which we later found out is the repressor of the CRISPR system in E. coli !
But, well, those things happen... and there was absolutely no chance that at that time I could have imagined that H-NS had anything to do with those regularly spaced sequence repeats. When I returned to Alicante I started my own group and dedicated myself exclusively to the study of these repeats. This took place in 2003. Meanwhile, in 2000 I had confirmed that indeed, as we suspected, these repeats are a very common feature in prokaryotes. You know that by 1995 sequencing had greatly improved and complete genomes of prokaryotes began to appear. We analyzed them all and we saw that the repeats were indeed present in archaea and in bacteria. Everywhere we looked we found them. We then defined the repeating family as SRSR (short regularly spaced repeats). But then we renamed them CRISPR. A group in the Netherlands asked that we rename them. They had discovered the cas genes at that time, the genes associated with CRISPR. They wanted a new name that related the sequences to their proteins and they did not like SRSR. So, I proposed CRISPR, it seemed good and that name stuck!
In 2000 we published the description of the family of repeated sequences. We continued investigating what they could be doing. In 2003, we discovered a striking sequence identity in the spacers of strains of E. coli that we were sequencing. We saw that one of the spacer sequences was identical to a sequence present in phage P1, which infects E. coli . But, critically, phage P1 did not infect the particular strain that contained the spacer with P1 sequence identity! That result set off our alarms. We performed exhaustive comparative analyses of the spacers of all the CRISPR clusters that we were able to identify in databases. We found that a small but significant percentage of spacers corresponded to phage and plasmid sequences! But not from any phage or plasmid. Rather, phages or plasmids that infected the strains that belonged to the taxonomic group that contained those spacers. Then, of course, to us this showed... Well, it was all very clear... At least from our point of view this was, without a doubt, an immune system. Because we also saw that an effective infection of these phages, of these plasmids, had never been described in these specific strains that had spacers whose sequence was identical to sequences in those phages and plasmids. Because to us it was so very clear that it was an immune system, we decided to send our manuscript to Nature. But, much to our surprise, Nature told us that this had already been described…
Wow! Did they cite a particular study or did they just say "this has already been described?" It seems to me that Nature's editors simply did not understand.
Our manuscript was rejected by the simple statement: "It is not new because it has already been described." We wrote a response letter saying: "No! It has not been described. This is a unique discovery and it will undoubtedly have a tremendous impact on biology, on its clinical applications and on biotechnology." And all they said in their subsequent response was that even so, they were not interested.
So, well… We sent it to other journals and the sad and depressing fact is that all of them gave us pretty much the same response. They were not interested. Or they needed an experimental demonstration of what was simply a proposal. And it has to be acknowledged, ours was indeed a proposal. We had not designed nor performed experiments that would have proven that this was an immune system. That was missing. It was not because of lack of trying. We tried but nothing worked at the time because we were working with E. coli , trying to prove it in E. coli ! But as I told you before, in E. coli, the system is repressed! We still do not know, in fact, what the natural conditions are, in nature...
Of course, because it must be that in certain conditions the system in E. coli gets derepressed. It must be so!
It must be so, it must be so... But hey, you know that situations define the things you do. For us, there was no alternative but to keep trying, and trying. But it wouldn't work, it wouldn't work. Eventually, we published this article with just the proposals but without the experimental demonstration that it was an immune system. We also proposed the mechanism by which RNAs were the key to finding the target of the immune system simply by pairing bases with their complementary sequences. Many aspects of this were demonstrated two years later and the rest three years later. Basically, this was the story of the discovery of the acquired immunity system of prokaryotes.
Phenomenal. There we have it in a nutshell. Very good. And then, from the beginning, I imagine, when you told Nature when you initially sent them your manuscript, you sensed that this discovery would have enormous impact not only in microbiology, but in biology in general and eventually also in biotechnology. You already must have had some idea that there were important applications. Today we see that, indeed, that has been the case.
But I did not imagine the magnitude of the current level of application.
No, not that level.
I did not imagine an impact beyond microbiology. I thought of biotechnology but within the areas of applied microbiology. Biotechnological applications, such as generating microorganisms resistant to viruses, strains that interfered with the transfer of plasmids. Perhaps utility in increasing the genetic stability of microorganisms that are used in biotechnological processes. I could imagine many possible applications but they were constrained to the field of microbiology.
Let me interrupt you there. Within microbiology, how do you see the role of CRISPRs in the origin of species in bacteria? If there are species in bacteria… I say this because we just had a post in this blog regarding the concept of species in bacteria. But CRISPRs' role in bacterial evolution... For me their discovery had a basic, fundamental impact in our understanding of bacterial evolution. You saw it from the beginning too, I imagine. Today, how do you see the role of CRISPRs in evolution?
Yes, in fact, we finally sent our original article to the Journal of Molecular Evolution thinking precisely about the tremendous evolutionary repercussions that CRISPR had. And we've kept thinking about them in this way for many years. We've kept thinking that CRISPRs probably participate in the generation of evolutionary lineages, most probably. What is clear, at least in some bacteria, is that sudden acquisitions of many spacers is aimed against, say, a particular virus. For some reason there are sudden changes, acquisitions of spacers that confer an immunity and that give rise to an evolutionary lineage. That is what is seen when you analyze, in detail, the situation in E. coli , where we do most of our work. You look at the spacers of different evolutionary lines and you see that certain spacers are maintained in a group of evolutionary related bacteria. And those spacers are not on other evolutionary lineages even if they are close. It gives us the impression that acquisition of spacers leads to the generation of new lineages. Experimentally, this is very difficult to prove. That's why we've never been able to get at it. But it gives us the sense that this is the role of CRISPRs in evolution.
It always gave me the impression that the system is like an accordion, constantly expanding and contracting by gaining and losing spacers. Don't you think so? That when a CRISPR region has no spacers, that's when suddenly a lot of other DNA in the form of phages and plasmids, can enter the cell and become established as horizontally acquired genes. That leads to evolution of a new lineage. But then the cell rapidly gains spacers and that lineage is protected from further incoming DNA. Something like that.
Exactly. I am convinced that CRISPRs are key in the generation of new evolutionary lineages. So the evolutionary consequences are tremendous. There is this very strong positive selection, indeed, for spacer containing lineages in that they resist incoming phages. Any cell not having that spacer will die in that environment. That gives rise to new evolutionary lineages.
Evolution is very beautiful.
Tell me a bit more about CRISPR applications to non-microbial genomes.
Yes, of course. I must admit that I did not participate in anything regarding the development of these technologies. People are continually saying to me: "Hey, thanks to you... " Thanks to my nothingness...
Well wait! Indeed, many thanks to you in the very basic sense. For the fact that you made the basic discovery... You should get a lot of the credit.
The success of the technology is… it's, well, truly amazing, isn't it? What is now possible to do with CRISPR-Cas! From the start, I saw that the thing looked good! But I did not imagine that this could reach to the point it has. It's amazing. That you can modify the genome of any living organism of your choosing, with great ease, it's incredible. I have seen several cases very recently. In fact, I used to have little or no connections with scientists that are not microbiologists. Lately, of course, I get invited to give talks at congresses on transgenesis, plants, biomedicine, neurobiology, etc. And the gratitude you see in these people. They are the ones who have suffered for many years from the lack of powerful gene modifying tools. Now they have seen this door opening, allowing them to address any question that comes to mind. Somebody said to me, very recently: "It's just that you cannot imagine it... A few years ago, we said... ah... how could we solve this question? Now the situation is totally different. Almost anything you can think of you can address. Now we can say, let's see what we can dream up, what is a good problem, because we are going to be able to solve it with CRISPR, at least with any question related to some genetic modification." It truly is a wonder. From my point of view, because this technique is letting you know more. It is as if the basic science of microbiology has managed to advance knowledge such that we can continue doing more basic research. It is now possible to study at a functional level any biological system. It also allows you to know diseases and, probably, to cure them. What else can I say?
That must be a very pleasing situation for you. It should give you great satisfaction to have contributed by providing the basic discovery and to see that it has this type of application.
How far it has gone! And how far will it go?
Yes, it seems we have only seen the beginning. Do you want to say a few more words about microbiology in general? Some words of advice for our blog's audience, perhaps in particular for those who are just starting their careers in microbiology. Some words about your experience throughout your career for those who love microbiology.
Well, if they love microbiology, I say they have hit the jackpot. Because from my point of view, with my experience, it was very clear to me from the beginning. I changed my university precisely looking for one within my region, not far away from home because otherwise the cost of living would have been too high, I looked for a university that had fine offerings in microbiology. From the beginning of my studies at the university, I realized the importance, for me at least, of microbiology. The interesting thing was to know those microbes, those bugs, so strange, right? And then over time, I confirmed the tremendous potential that microbes have due to the many more years of evolution than we have had. Real years, and in generations, as they reproduce so much faster than we do. The applications that can be derived from microbiology – as history shows – are many. And the ones that are expected to come are many more. Because so far, we have been quite limited by the "need to culture" bias. But that is changing rapidly. So, we can start to study, to know and to take advantage of all these microbes, which are the vast majority and which are the normal ones, those that have resisted cultivation under the conditions of a laboratory. If we consider just the field of CRISPRs, what we know now is a minimal part of what there is. We are already finding fantastic tools. Imagine all that there is yet to discover in the microbiology of natural environments. Their exploration will yield many more surprises. We've already had so many surprises, restriction enzymes, PCR, everything that has led to the development of molecular biology. All this I think is just the beginning. So, from my perspective, the future of microbiologists seems very bright from my perspective.