Crazy about CRISPR: an Interview with Francisco Mojica.

The CRISPR JournalVol. 1, No. 1 InterviewFree AccessCrazy About CRISPR: An Interview with Francisco MojicaKevin Davies and Francisco MojicaKevin DaviesSearch for more papers by this author and Francisco MojicaSearch for more papers by this authorPublished Online:1 Feb 2018https://doi.org/10.1089/crispr.2017.28999.intAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail In the summer of 2003, Francisco Mojica, a microbiologist at the University of Alicante in Spain, made a momentous discovery. After years of fruitless inquiry into the identity and function of mysterious short DNA segments appearing at frequent intervals in the so-called CRISPR repeats of prokaryotes, a computer search one hot summer afternoon suddenly struck gold: Mojica found a match to a known viral sequence. Soon, other matches were found, and the biological and evolutionary role of CRISPR began to unfold.Kevin Davies, founding Executive Editor of The CRISPR Journal, sat down with Mojica in Spain to reflect on the discovery that kicked off a genome editing revolution. (Fig. 1) (This is a lightly edited record of that conversation, conducted October 23, 2017.)FIG. 1. Francisco Mojica (left) and Kevin Davies.Davies:The first article describing these weird, curious CRISPR repeats was in 1987. What were you doing at the time?Mojica: I started my PhD thesis work in 1989 on the regulation of gene expression in halophilic Archaea [Haloferax mediterranei]. We were trying to investigate the global regulatory response to changes in salinity. These organisms live in environments with very high salt concentrations. We were sequencing regions of these organisms that were related to adaptations to high salinity.Davies:Why is this important in your hometown of Alicante?Mojica: These are salterns [salt lakes]—pools where the sea water gets concentrated, evaporating because of the sun (Fig. 2). These organisms are able to survive even at saturation of sodium chloride. They are extremophiles—salt lovers! The sea water does not have enough salt for them to survive, they need at least ten times more salt to grow. These are amazing living beings, so we were interested just in that process.FIG. 2. The saltern (salt lake) of Santa Pola in southern Spain (left), habitat of Mojica's favorite halophilic archea, seen as red stripes on salt (right). (Courtesy: F. Mojica.)Davies:You found the CRISPR repeats in the genome of these salt-loving organisms?Mojica: We were sequencing regions we thought were related to this salinity, when we saw these repeats. We searched the literature to see whether any similar repeats had been discovered. This was in 1992. We did not have PubMed at that time, so we looked in the old citation index, searched on “repeats”! We had to check every single article dealing with repeats, and see whether those were equivalent to repeats we had just found. Eventually we found the repeats from Escherichia coli [published by a Japanese group in 1987]; they had a similar arrangement, regularly interspersed, but the sequence was different. E. coli and these halophilic Archaea are very different organisms.At the time, we called them tandem repeats (TREPs). That was my boss! I did not agree with that, because they are not in tandem, they are regularly interspaced.Davies:How or when did the name change to CRISPR?Mojica: The name changed twice. In 2000, we described a new family of repeats, just taking into account similar repeats found in 20 different species of bacteria and Archaea. At that moment we gave them the family name “short regularly spaced repeats” or SRSR. In 2001, I got an email from a group in the Netherlands [Ruud Jansen] that had been working with repeats in Mycobacterium tuberculosis. They had been calling them DR, or direct repeats, and they had found four genes related to CRISPR. So they were present only in genomes with CRISPR. They wanted to give a name to these CRISPR-associated (Cas) genes but did not like SRSR! There was no one else to ask, and we wanted a different name, easy to pronounce. I came up with CRISPR… [Jansen] accepted CRISPR, he said it was a great acronym (Fig. 3).FIG. 3. 2001: A Spacer Odyssey - Ruud Jansen and Mojica agree via email to the snappy “CRISPR” acronym.Davies:So from 1992 on, you were trying to ascertain the significance of the CRISPR repeats. Were you just doing computer searches?Mojica: When we found these repeats, we thought they could be related to DNA structure or topology, or a place for cooperative binding of proteins. We tested those hypotheses for many years, but we did not succeed. That is a shame for not publishing negative results, it was interesting… they were palindromic but we did not get any evidence for these structures. Eventually we became interested in the spacers, because when you look in different species, these spacers change a lot. Where do they come from? What is the reason for these spacer regions? We thought they could be synthesized de novo—that was crazy probably—but we were looking for an explanation. We did not find any matches in the databases for many years.In 1995, the first bacterial genome was released. After that, more and more complete genomes were introduced.Davies:For most people, studying a system for so long with just negative results, you must want to give up. You're a very stubborn man!Mojica: {Laughs} Yes, but I knew it was interesting, they are found in many distantly related prokaryotes. Some Archaea have 2% of their genome made up of these repeats, so that tells me this is really important. I remember my collaborators telling me “you're wasting your time” looking for these spacer matches.Davies:In 2003, you were on holiday in Alicante—what happened next?Dr. Mojica: I used to spend my summer break in Santa Pola, now a tourist town south of Alicante. Salt lakes are there, just a coincidence. I really do not like the hot weather, lying on the beach, I prefer going to the laboratory to relax—no crowds, no sand. My wife was at the beach, I told her I would come back after dinner. We had got some sequences from E. coli strains, we were sequencing the CRISPR loci to evaluate whether they could be used for genetic markers. We got a few of these sequences, and as I often did, I BLASTed these sequences against the database. For the first time, it matched a viral sequence. I thought maybe it is a coincidence, but it was not.We knew that strain was resistant to infection by phage (bacteria-infecting virus) P1. At that moment, it was just amazing. I looked for CRISPR in every genome available; we had a special bioinformatic program to look for regularly spaced repeats. We found many CRISPR repeats, suddenly a spacer from Streptococcus matched a sequence from a phage, and the phage infected Streptococcus… Then we matched another. Oh my goodness!Davies:So you wrote the article describing these results?Mojica: We found this discovery in August [2003]. By the end of September or October, we submitted to Nature. I remember the title of the submission was “Prokaryotic repeats are involved in an immunity system.” To convince the editor and referees, we wrote that the existence of an acquired immune system in prokaryotes will have tremendous repercussions in biology and clinical sciences. It was not even reviewed—they said this has already been described. We said, “No, this is the first time! We're talking about an acquired immune system with memory.” I tried to explain that this was not the same as the previously described defense systems, but they asked whether we were able to describe the mechanism in this immunity… We did not have experimental proof. We knew the spacers matched sequences of foreign DNA, and no article had described efficient infection of sequences matching spacers. We had many observations relating to that, and we performed some experiments, but nothing worked. We learned five years later that the E. coli CRISPR system is repressed, silenced under laboratory conditions.Davies:That was not the only rejection, was it? What happened to your article after that?Mojica: It was rejected by three more [including PNAS and Nucleic Acids Research]. Finally it was published in 2005 in the Journal of Molecular Evolution (Fig. 4). One of the referees suggested that our observations would better fit a journal related to evolution. At that point, it did not matter where [we published]. It took something like six months to get the first answer. I was trying all the time to get in touch with the editor, we wanted to publish as quickly as possible. Eventually after six months, they told us they were interested in the article. Three months later, we heard it had been accepted. I remember those two years like a nightmare! I promise, when you have something so big in your hands, and you send it to very good journals, and all of them agreed it was not interesting enough to be published, you think, is it me who is crazy or something else?FIG. 4. Two years and five journals later, the landmark study by Mojica and colleagues is finally published in the Journal of Molecular Evolution. (Published with permission of Springer.)Davies:Was it an anticlimax once the article finally came out?Mojica: Just one month after we published, another article appeared saying that the spacers of a large set of Yersinia pestis strains also derived from phages. Then a third article appeared with almost the same conclusions. These three articles telling more or less the same [story] caught the attention of people. In 2007, an article [Barrangou, R. et al. Science 2007] experimentally demonstrated that we were dealing with an adaptive immune system, and that the Cas proteins were functionally related to CRISPR for the first time. There was no proof of that connection (previously). They demonstrated that Cas proteins were necessary to achieve this immunity, and when Streptococcus thermophilus is challenged with a phage, we get a few survivors, and they have acquired new spacers. This demonstration also helped to trigger the field.Davies:It has been a little more than ten years since your landmark article was published. How do you view the transformation CRISPR has had on life sciences?Mojica: I was very excited when [CRISPR researchers] met for the first time in 2008, after the article from Barrangou et al., scientists of ten research groups met in Berkeley. I realized we had succeeded in catching the attention of the scientific community—at least the microbiologists. That was huge. After 2012, when we saw that some tools could be developed from this prokaryotic immune system, and in 2013 when it was demonstrated to be a very good tool for genome engineering, and now we see 10–15 articles published every single day using CRISPR, you say, what have we done?! This is amazing, it was unforeseen, it was difficult to know for sure these repeats were important.So I am very happy—prokaryotes have many consequences for life on earth, and now we have the best molecular biology tools ever with such huge potential. So no scientist could be happier than one that realized that this small boy, that no one else cares about, became an important person.Davies:You have seen CRISPR push into so many areas. Are there any aspects that give you concern?Mojica: We think about situations in the past when huge developments were made, when there was rejection by the public. But now these promises are seen as normal. I think we will need some time to get used to the possibility of doing something that was not possible a few years ago.I do not like the idea of changing the germline—not at all—but sometimes you think it is not ethical not to do that when it is possible to cure someone. I do not have kids, but I can understand anyone who has a little boy with a [serious] genetic disease, if there is no alternative. But there are alternatives to CRISPR—if you can avoid doing manipulation in the germline, I think you should.Davies:Are you back in the laboratory still studying your salt-loving organisms?Mojica: No, I moved to E. coli. These salt lovers are hard to work with. I am still working on CRISPR, I am interested like everyone in the world in this technology, but I am still interested in basic research and contributing to knowledge. The output is this wonderful technology and many other applications being developed from CRISPR.** To hear the entire podcast conversation, visit: www.crisprjournal.comFiguresReferencesRelatedDetailsCited byEvolution and molecular mechanism of CRISPR/Cas9 systems Volume 1Issue 1Feb 2018 InformationCopyright 2018, Mary Ann Liebert, Inc.To cite this article:Kevin Davies and Francisco Mojica.Crazy About CRISPR: An Interview with Francisco Mojica.The CRISPR Journal.Feb 2018.29-33.http://doi.org/10.1089/crispr.2017.28999.intPublished in Volume: 1 Issue 1: February 1, 2018Online Ahead of Print:January 8, 2018PDF download