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As the co-inventors of CRISPR/Cas9 gene-editing technology, Jennifer Doudna, Emmanuelle Charpentier and Feng Zhang are typically the first names that spring to mind when CRISPR is being discussed. What you may not know, however, is that the CRISPR mechanism was originally discovered back in the 90s by a particularly humble microbiologist, Francisco Mojica, Professor at the University of Alicante.



Kicking off Technology Networks Explores the CRISPR Revolution, Professor Mojica, or "Francis", takes us on a journey back to the original research that, despite being deemed "crazy" by members of the scientific community at the time, led to the CRISPR revolution that is anticipated to edit evolution forever.



Impossible to anticipate



The greatest finds in scientific discovery are typically unanticipated when a researcher embarks on a study. The discovery of CRISPR is no exception: "It was absolutely impossible to anticipate the huge revolution that we are enjoying nowadays," Mojica tells me as he laughs, still seemingly astounded by it.



In 1992, Mojica was working on his thesis at the University of Alicante. A keen microbiologist, he was studying microorganisms belonging to the Archaea family, a group of prokaryotes that he brands "quite peculiar". These microorganisms are halophiles, meaning they require high-salt conditions to survive. Mojica and colleagues were interested in understanding how Archaea are able to grow in high salinity and, when required, adapt to changes in such salinity.



They opted to sequence their DNA to look for clues in the genome. In a pre-Human Genome Project era, this was quite the task. The scientists didn't have the luxury of sequencing data being at their disposal.



Nevertheless, Mojica and the team's efforts were successful. They discovered that the halophiles' DNA possessed a series of regularly spaced repeats, which they labelled tandem repeats (or TREPS). "We saw that these repeated regions in the halophiles were transcribed, meaning they were active. The cell was reading this information in each of the growing conditions that we tested, and so we knew that they had to be important for the cell," Mojica tells me.



The researchers scoured the literature for evidence of previous work outlining the existence of TREPS. They struck gold in the form of a paper published in 1987 by Ishino and colleagues (a huge feat considering that PubMed was yet to be invented). The Japanese researchers had too stumbled across these TREPS in E. coli.1



A "crazy" hypothesis, and a backlash from the scientific community



Mojica's curiosity was sparked. What function did these repeats serve? They existed in bacteria and archaea – thus, surely their origin was ancestral?



"In 1995 we published a paper where we suggested a hypothesis which, nowadays, may sound crazy. We hypothesized that the repeats were involved in the segregation of the chromosomes in cell division," Mojica tells me.2 "It was completely wrong," he laughs, "but it fit with our data at the time."



Mojica's efforts to explore the functional role played by the repeats were greeted with an initial backlash from the scientific community.



"When we didn't have any idea about the role these systems played, we applied for financial support from the Spanish government for our research," he explains. "The government received complaints about the application and, subsequently, I was unable to get any financial support for many years." It immediately occurs to me that the culprits behind the complaints are probably kicking themselves now.



I pause for a second, slightly bewildered, before asking him why. "Initially they said, "You want to explore the role played by some repeats in very peculiar and strange organisms. Maybe you discover the role, maybe you don't. But, in any case, your findings will only apply to these strange organisms."" They doubted the relevance of the research. "That was the first criticism. Next I was implored by the reviewers of the grant to move to a model organism such as E. coli, which I did." Mojica continues, "It was a huge mistake."



Whilst the repeats are transcribed in halophilic archaea, in E. coli, they are repressed – unless you create mutations. "I spent many years trying to understand the function of these repeats in a model organism in which the system was not working. I could not get any results," he tells me, surprisingly maintaining an upbeat, positive tone to his voice at all times during this recollection. Unphased by his lack of results, Mojica persisted. In 2000, TREPS received a rebranding after he discovered that the repeats existed in many other organisms that were hardly close on the evolutionary tree. Going forward, the repeats were to be known as short regularly spaced repeats, or SRSRs.3



CRISPR enters scientific literature



By 2001, both Mojica and Ruud Jansen, of Utrecht University, were searching for the repeats in various prokaryotic organisms. Jansen reached out to Mojica to inform him that his research team had discovered genes next to the repeats and wanted to agree on common terminology for the repeats.



Several names were proposed before an agreement was made. "I thought about a few alternatives, of which I just remember RISR and CRISPR," Mojica says. "I introduced them to Ruud, explaining their meaning and the pros and cons of each. CRISPR was the one that considered all the features of the repeats. We agreed to use it in our future publications." In 2002, the first mention of clustered regularly interspaced short palindromic repeats, or CRISPR, appeared in the scientific literature.4



A dramatic revelation courtesy of the genomic era



In the early 00s, science entered a "genomics era" in which genome sequencing technologies rapidly advanced, paralleled by increased sequencing data being made available to scientists in public databases.



Such data permitted a revelation for Mojica in his work.



When sequencing one particular strain of E. coli, Mojica discovered that there were sequences between the repeats known as "the spacer regions" of CRISPRs that matched the sequence of a particular virus. Further exploration of sequencing data revealed that this was the case in many other, extremely different organisms. These DNA sequences protected the prokaryotes from being infected by viruses carrying the same sequence in its genome; the virus simply couldn't infect the cell. And so, he realized: "This is an immune system. This is an adaptive immune system!"



As he sits with a wide grin on his face recalling the fine details, it is very clear to me that the sheer thrill of making this discovery remains with Mojica to this day. "It was a very nice surprise," he says.



Unfortunately, the scientist was once again greeted by criticism when he endeavoured to publish his research findings. "The paper was rejected by four different journals for many different reasons. One journal said to us that it wasn't interesting enough and another said we needed more experimental proof. We almost considered not publishing the paper." One of those papers was the journal Nature. He adds: "I guess it was a very new idea. We presented our findings at a conference in Spain and some of my colleagues came to me and suggested that what we were doing was very pretentious and 'overgrown'." Mojica laughs.



The study was eventually published in the Journal of Molecular Evolution in 2005.5



Genome-editing came as a "wonderful surprise"



Following the publication of their discovery in 2005, Mojica and colleagues anticipated that their research findings would have a large impact on the biotechnology, biopharmaceutical and clinical science sectors.



And so, in 2012, when Doudna and Charpentier demonstrated that they had reprogrammed the CRISPR mechanism to function as a gene-editing tool in vitro, Mojica was "wonderfully surprised, and very, very impressed."



Since the 2012 publication, a wide variety of research groups have further developed and manipulated the CRISPR mechanism for an array of purposes, ranging from agriculture, diagnostics, drug development, cancer research – the list goes on, and will be explored throughout the series.



I am intrigued to know what Mojica deems his favourite application of CRISPR thus far. "My goodness, every single one of them has been astonishing," he continues, "I cannot choose one. I must choose two! And they are the two papers published back to back in Science in 2013." Mojica is referring to a paper published by Feng Zhang that was followed by a second paper in the same journal by George Church.6,7



Both research groups outlined their novel use of the CRISPR tool to edit the genome of mice and human cells, igniting the CRISPR genome-editing "revolution".



Filing a patent? I never thought about it



An ongoing patent dispute lurks behind the excitement and flurry surrounding CRISPR technology, which will be explored in a later instalment of the series. Interestingly, Mojica is one of few scientists involved in the discovery of the CRISPR mechanism and its applications that has not filed a patent.



"Some people ask me why I didn't file a patent 10 years ago," he pauses. "I have to confess, I never thought about doing that. In my lab, we aim to understand biology. Filing patents probably should be one of the goals," he laughs before adding, "But it is not."



He then goes on to express his anxieties regarding the impact the patent dispute may have on the progress of CRISPR research and applications. "I'm pretty sure the patent dispute could be slowing down the transfer of experiments and research from the lab to the clinic. I'm not absolutely sure, but I fear that could be a problem, and that isn't fair." He adds "It's quite difficult for me to understand why there is such a long-lasting dispute on getting money from research."



Mojica strikes me as a passionate scientist who truly thrives on the quest for novel discovery and is modest in doing so. When asked in a previous interview how he would react to being awarded a Nobel Prize for his work in the CRISPR field, he admitted, "I will disappear from the planet. I need to rest and relax, and I need time to get back to what motivates me and return to the lab."



Looking to the future of CRISPR



Mojica's work in this the field of CRISPR is certainly far from finished. He tells me, "We are still interested in understanding how the CRISPR mechanisms work in nature; particularly how these systems develop the memory of past infections. There is a huge diversity of CRISPR Cas mechanisms, and different systems work differently in a variety of organisms." "We are using metagenomics high-throughput sequencing to identify more CRISPR Cas systems and variants that are different to those we know of currently. We hope that either our group, or other groups across the globe can look to identify further applications for these systems or improve the current CRISPR tools we have now."



I ask Mojica what he envisions the CRISPR research field to look like in 10 years' time.



"I am a microbiologist, I'm not a specialist in genome editing… but I do read a lot!" he laughs. " It's risky to predict any situation, but I wish that in 10 years' time CRISPR will already be in the clinic, and some patients will have been cured from diseases that currently have limited treatment options. Who knows exactly how many diseases could be tackled by CRISPR."



"A reality right now is CRISPR's application in agriculture. I do anticipate that in some countries, we will soon be eating food that is derived from CRISPR-edited crops. But it's risky to predict any situation."



As our interview comes to a close, I express my sheer gratitude to Mojica for lending his time to me and for sharing his CRISPR story. He replies, humble as ever, "The pleasure is all mine."



Professor Francisco Mojica was speaking with Molly Campbell, Science Writer, Technology Networks.



References:

1. Ishino, Shinagawa, Makino, Amemura, and Nakata. 1987. Nucleotide Sequence of the iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia coli, and Identification of the Gene Product. Journal of Bacteriology. DOI: 10.1128/jb.169.12.5429-5433.1987.



2. Mojica, Ferrer, Juez and Rodríguez-Valera. 1995. Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Molecular Microbiology. DOI: 10.1111/j.1365-2958.1995.mmi_17010085.x



3. Mojica, Dı́ez-Villaseñor, Soria and Juez. 2000. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Molecular Microbiology. DOI: 10.1046/j.1365-2958.2000.01838.x



4. Jansen, Embden, Gaastra and Schouls. 2002. Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology. DOI: 10.1046/j.1365-2958.2002.02839.x



5. Mojica, Díez-Villaseñor, García-Martínez and Soria. 2005. Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements. Journal of Molecular Evolution. https://doi.org/10.1007/s00239-004-0046-3.



6. Cong et al. 2012. Multiplex genome engineering using CRISPR/Cas systems. Science. DOI: doi: 10.1126/science.1231143.



7. Mali et al. 2012. RNA-Guided Human Genome Engineering via Cas9. Science. DOI: 10.1126/science.1232033.