Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing

CHAPTER 3 WE CAN BE HEROES

Great moments in technology and science can emerge from the most unlikely sources. In 1966 Playtex, the company behind the iconic Cross Your Heart bra, entered a NASA competition to design the spacesuit for the first Apollo moon landing. The suits had to be able to withstand pressure and extreme temperature swings. They also had to be flexible, an attribute that Playtex handsomely demonstrated by filming one of its technicians playing American football for hours while sporting a spacesuit. Thus, it came to pass that four Playtex seamstresses sewed the twenty-one-layer A7L spacesuit that Neil Armstrong fashioned on the lunar runway.¹

From the Sea of Tranquility on the moon to the salterns of Santa Pola off the Mediterranean. I’m visiting Alicante, a popular tourist resort on the Costa Blanca in southeast Spain. It is an unlikely candidate for one of the more extreme habitats of life on planet earth. But drive south about fifteen miles, you reach Las Salinas de Santa Pola. Salterns, or salt flats, are as the name suggests, a network of rectangular lagoons characterized by extreme salt concentrations resulting from intense sun and wind. At the perimeter, where the water meets land, the salt crystallizes out, forming a crusty white band like the rim of a perfect margarita.

This place has ecological, historical, and commercial significance. Flamingos and other wildlife abound. A watchtower dates back to the 16th century, where the lookouts of King Felipe II kept watch against the Moors. It looks like a nature reserve, but this is an industrial salt mine. Each lake is the size of a football field, concentrating the salt in stepwise fashion. Today, Bras del Port extracts on average 4,000 tons of salt daily from the Mediterranean Sea. A veritable mountain of salt sits ready for distribution: about 60 percent will be used for water treatment, the rest is for food.

For Francisco Mojica, a microbiologist at the University of Alicante, the study of the halophilic life that thrives in this peculiar habitat is his passion. Where he once toiled in obscurity, today he struggles to escape the media spotlight. In 2017, the leading Spanish newspaper, El País, speculated whether Mojica would make it from the salterns to the Nobel spa.

Mojica has kindly agreed to drive me in his unflashy Volkswagen Passat to las Salinas. It is a journey he makes quite frequently, usually in the company of photographers or film crews who direct him to survey the pristine pink waters or hold a flask of salt water up to the Spanish sun as if for the very first time, like admiring a glass of rioja. In a delicious irony, Mojica never actually collected samples for his own research because as a young graduate student, there were already samples in the lab, taken by his boss a decade earlier.

Mojica first came to the salterns after he had finished military service in 1989 and was looking for a research position. He was offered a PhD position in the microbiology department at the local university in a lab that studied a microbe called Haloferax. “I didn’t have an interest in particular with these organisms. My boss decided the issue of my thesis work,” he told me as we stroll around the salterns.²

Haloferax is not a bacterium (although confusingly it used to be called Halobacterium) but belongs to a distinct group of single-cell organisms called Archaea. To the naked eye there is little to distinguish the two clades. But that belies an evolutionary chasm of some three billion years. The appreciation that Archaea are not just a superficial off-branch of prokaryotes but an entirely separate “third domain” of life is due to the seminal work of evolutionary biologist Carl Woese. DNA sequencing revealed striking genetic differences between Archaea and bacteria, like comparing the operating systems of a Mac and a PC. Ed Yong put it nicely: “It was as if everyone was staring at a world map, and Woese had politely shown that a full third of it had been folded underneath.”³

The water is pink and the salty crusts of the lagoons are drizzled with pinkish red bands that teem with microscopic life. The source of the reddish hue is the production of carotenoids, part of the microbial defense mechanism against salt and sunlight. “It’s like a sunscreen,” Mojica laughs. The color changes with salinity: red shifts to pink as the salt concentration rises from 10 to 30 percent. The same chemicals give the flamingos their trademark pink plumage as they feed on the tiny brine shrimp that, like the Haloferax, thrive in these salty waters.

The salt-loving Archaea of Alicante are by definition extremophiles—lifeforms that are adapted to live in unusually harsh habitats, whether it be underwater volcanic vents, parched deserts, or frozen tundra. For Haloferax, the level of salt in regular seawater just doesn’t cut it: they require ten times as much salt to thrive. Trying to replicate those conditions in the lab is extremely difficult, thus they remain poorly understood compared to their bacterial distant cousins. The two main Haloferax species here are H. mediterranei and H. volcanii (the latter named not because they’ve mistaken a salt lake for a volcanic vent but after the Israeli scientist who discovered them, Benjamin Volcani). I can also smell the presence of anaerobic bacteria responsible for the strong sulfurous odor that wafts over the water.

Mojica’s obsession with the salt-loving microbes of Santa Pola is the embodiment of basic research. “It was knowing by knowing, to expand knowledge,” he says.⁴ Buried in the circular genetic code of Haloferax, Mojica reasoned, must lie a clue to explain its love of salt. This was not so straightforward: the first complete microbial genome sequence wasn’t reported until 1995, by Claire Fraser and Craig Venter’s group, which also decoded the first complete Archaea genome two years later. Mojica’s lab was not a flashy genome center with the latest DNA sequencing hardware. In the early 1990s, sequencing for many researchers was still a cumbersome manual process, which involved making a large gel sandwich between two glass plates, then separating radioactively labeled DNA fragments by size in an electric current. From the resulting ladder imaged on an X-ray film, Mojica could spell out the corresponding DNA sequence.

In one of his first sequencing attempts, in August 1992, Mojica saw something so surprising, he assumed he’d messed up the experiment. He saw weird repetitive sequences, each about 30 bases long, which he duly noted in his first paper.⁵ “We were absolutely lucky,” he said, after sequencing less than 1 percent of the Haloferax genome. “It was the first paper where CRISPR was taken seriously!” he says.^I Mojica also showed that the repeats were surprisingly transcribed into RNA, suggesting they had some sort of function.

“When you see something very peculiar, you have no alternative but to research it. I thought this was a nice thing to keep working on,” he says as we continue our stroll along the salterns. He had a hunch the mystery repeats might be tied to salt adaptation, perhaps by changing their conformation and thus gene activity by sensing changes in the cell’s osmotic pressure. “At the time, [DNA] supercoiling was the answer to everything in the regulation of gene expression!” It was a nice hypothesis, but wrong.^II

Working after hours in the university library, Mojica eventually unearthed a Japanese report from 1987. Atsuo Nakata and Yoshizumi Ishino^III at Osaka University described a similar repeating motif in the genome of E. coli. While sequencing a gene of interest, the Japanese group had noticed an unusual nearby sequence with a distinctive repeat pattern, like crop circles carved into the DNA terrain. This region consisted of a series (the cluster) of short repeated stretches of palindromic sequence (reading the same forward and backward); each identical repeat, twenty-nine letters in length, was separated by a thirty-two-base stretch of unique sequence (the interspaced DNA). But because nothing similar had been seen before, and there were no clues as to their biological function, the researchers let it slide. The team dutifully wrote up and published their observations, which attracted scant attention at the time.⁶

Two years later,⁷ Mojica described a short sequence repeated hundreds of times in tandem, spanning more than 1,000 bases. Between each pair of repeats was a unique DNA sequence of unknown function. Mojica’s boss suggested calling these repeats—which were also observed in another extremophile, a volcano-loving Archaea—TREPs, for tandem repeats. Surely there was a reason why prokaryotes devote up to 2 percent of their precious compact DNA to these strange repeats? Microorganisms “cannot allow themselves luxuries,” Mojica thought, “they must have an important function.”⁸

Other scientists also stumbled upon these repeats. German microbiologist Bernd Masepohl puzzled over a stretch of thirteen DNA repeats, found in a cyanobacterium, which he called LTRR, for “long tandemly repeated repetitive.” But in focusing on the repeated DNA elements, Masepohl paid little attention to the unique sequences in between.⁹ Another team also came close to solving the mystery of the DNA repeats. In 2002, Eugene Koonin, a Russian expat computational biologist at the National Center for Biotechnology Information at the NIH, and his colleague Kira Makarova, described a series of bacterial genes they suspected to be part of a DNA repair system.¹⁰ What they didn’t realize was that these genes were sitting adjacent to the CRISPR array and—as we shall soon see—play an essential role in the function of CRISPR and gene editing.

After a few years working in Oxford, Mojica returned to Alicante in 1997 to set up his own group. With little funding, Mojica tried to do some very cheap experiments, “even though I had no idea about bioinformatics.” The nagging question was the origin of the spacer DNA, the sequencers interspersed between the repeats. “The easiest thing is to look at the databases and expect that something comes out, but we didn’t get anything—until 2003.” By now, the DNA databases were bursting with bacterial and archaea genomes, many of which carried versions of these repeats.

In 2000, Mojica renamed his obsession SRSRs (short regularly spaced repeats). That didn’t last long. Later he exchanged emails with Ruud Jansen in the Netherlands, who was studying a family of genes adjacent to the mystery repeats. Jansen felt a new name was needed, so Mojica suggested “CRISPR.” On November 21, 2001, Jansen emailed his enthusiastic approval:

Dear Francis,

What a great acronym is CRISPR. I feel that every letter that was removed in the alternatives made it less crispy so I prefer the snappy CRISPR over SRSR and SPIDR.¹¹

CRISPR finally had a name, as did a group of unusual genes that seemed to piggyback with the CRISPR elements. Jansen reasonably, if unimaginatively, dubbed these “CRISPR-associated” genes, or Cas. For now, Mojica was still focused on the curious CRISPR spacers.

The breakthrough finally came one picture-postcard afternoon in August 2003. Mojica was vacationing with his wife close to home, near the salterns. Feeling the heat, Mojica made an excuse to pop back to his air-conditioned lab, where he could run a few more computer searches. This was routine, almost like playing a video game: Mojica would copy one of the mystery spacer sequences and paste it into a computer program called BLAST that would search for matches in the massive DNA database, GenBank. Mojica had run this program hundreds of times to no avail. His colleagues called his quest a waste of time. But on this day, to his astonishment, the computer flagged a match. This particular spacer from E. coli matched a stretch of viral DNA called P1 that crucially infects the same bacterium. Over the next few weeks, Mojica catalogued dozens of other examples of matches between CRISPR spacers and various viruses.

In October, Mojica submitted the biggest research paper of his life to the top journal—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.” And the result? “It wasn’t even reviewed!”¹²

Perhaps something was lost in translation, but Nature’s editors didn’t consider this to be a conceptual advance “of sufficient general interest” that merited publication in its prestigious pages. Mojica appealed, arguing this was the first description of a bacterial immune system with a memory function. Nature indicated it was willing to reconsider if he could describe the mechanism underlying this immunity. But Mojica’s group couldn’t generate any experimental proof for the hypothesis, just the smoking gun of the sequences. One reason, it turned out, is that CRISPR is repressed in the most popular lab workhorse, E. coli.¹³ It was as if Mojica had incriminating physical evidence but no security camera footage.

Mojica licked his wounds and resubmitted to another journal… and another. Three more journals, including the Proceedings of the National Academy of Sciences (PNAS), all passed on CRISPR. Each delay increased the chances he might get scooped. Finally, in October 2004, Mojica submitted his manuscript to a lesser known journal specializing in evolution. It took a full six months before he finally heard some encouragement from the editor. Three months later, the paper was accepted. “I remember those two years like a nightmare,” he told me. “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?”¹⁴

Most scientific papers are team efforts, the fruit of months if not years of planning, reviewing, and repeating experiments, a continual exchange of ideas between student and mentor. If one member receives the spotlight, other members, rightly or wrongly, can feel aggrieved.

The second of four authors on Mojica’s groundbreaking report was graduate student César Díez-Villaseñor. He watched the accolades showered on Mojica with a mixture of pride and envy. In the early days of CRISPR, “it seemed likely that spacers didn’t have any function at all,”¹⁵ he recalled, because each spacer had a unique sequence, minimizing the likelihood of any sort of common function. But Díez-Villaseñor was puzzled: “If the sequence of the spacers is not really relevant, why bother?” Perhaps their uniqueness meant they were somehow toxic to similar sequences. “The proposition was immediately dismissed, although it didn’t leave my mind.” He says wistfully, “it was hinting the right direction.” Another possibility was that the spacers were being generated by some peculiarly sloppy form of DNA replication. But then adjacent spacers ought to be more similar to each other than distant ones, which was not the case. Díez-Villaseñor charted the CRISPR spacers from E. coli to show his boss, refuting the idea of mutation incorporation. He remembers it was the day before Mojica’s eureka moment.

“I said with a bit of frustration that spacers had to be taken from previous sequences but, obviously, they had to be present inside the cell at that time.” As he said that aloud, he realized it made perfect sense. “Of course—it must be an immune system!” There were known examples of RNA interference being used as an immune system. “Suddenly, everything that had looked so strange made perfect sense.” Díez-Villaseñor asked Mojica if he was running sequence searches with the spacers. Mojica responded briskly: “Doing that is my job! Don’t do anything.”

To find a microbial immune system that could recognize invading phage was a very big discovery. The next day, doubts started to be dispelled. “Francis came to the lab elated and directly talked to me. He told me he had found the first homology of an E. coli spacer in phage P1.” Later he told another professor that CRISPRs were like “memorabilia from past hosted genetic elements.” Díez-Villaseñor says: “That was probably the most satisfying professional moment in my entire life.”

Mere weeks after Mojica began his publication odyssey,^{16, 17} Gilles Vergnaud in Paris submitted his own CRISPR story, and experienced similar frustrations. With concerns growing about Saddam Hussein’s use of biological weapons, Vergnaud, working for the French Ministry of Defense, was tasked with improving microbial detection methods. At the end of 2002, Vergnaud obtained access to DNAs from dozens of strains of Yersinia pestis isolated during a plague outbreak in Vietnam in the mid-1960s. Using the best genetic methods available at the time, he found the strains were identical except for one region, which they named minisatellite number 6 (MS06). When a graduate student, Gregory Salvignol, sequenced MS06 in more detail, it turned out to be a CRISPR. Moreover, the French team found that it acquired new spacers from viral DNA. They too proposed that these structures were part of a bacterial immune system. The first draft of the manuscript, written in July 2003, included the notion of a “defense mechanism”—a record of “past genetic aggressions.”

But like Mojica, Vergnaud endured his own depressing runaround. He submitted his paper—the first to include “CRISPR” in the title—just behind Mojica, but in November 2003, PNAS dismissed it without review. It was the same story at the Journal of Bacteriology (twice), Nucleic Acids Research, and Genome Research. In July 2004, he submitted to Microbiology, which eventually published his paper in 2005.¹⁸

The early history of CRISPR would look very different if any one of those journals had said yes. As it was, Vergnaud applied for grants from the French National Research Agency over three consecutive years without success. A third report on CRISPR came out from Alexander Bolotin, a Russian microbiologist working for the French ministry of agriculture. Bolotin noted a correlation between the number of spacers and phage sensitivity, deducing that “spacer elements are the traces of past invasions by extrachromosomal elements.”¹⁹

We now know that some 90 percent of Archaea contain CRISPR elements, but only 40 percent of bacteria. Mojica explains that bacteria have a larger repertoire of defense systems at their disposal. CRISPR serves as a genetic barrier of sorts to microbial evolution because it discourages horizontal gene transfer. “Do you prefer to have a barrier to genetic transfer or defend against viruses?” says Mojica.

Spain has only celebrated two Nobel Prize winners in science—Santiago Ramón y Cajal in 1906 and Severo Ochoa in 1959. That puts a lot of pressure on Mojica, even as he laughs off such idle speculation. “It’s good to know that some people think I deserve it, I really appreciate that, but thinking about the possibility of getting the Nobel Prize is—how you say—crazy! There’s no way one could expect to get the Nobel Prize.”

Before heading back to Alicante, Mojica and I stop for a beer before lunch. He is about to embark on a three-week lecture tour of Australia, the sort of career success that most academics covet. I’m curious about how the microbiologist who has been nicknamed el padrino—the godfather—is handling his newfound celebrity. Mojica pauses, reaching for another olive. The restaurant is deserted, but in almost a whisper, he says, “I hate it… I hate it.” This was not the reaction I was expecting. “I just want a quiet life,” he says, shaking his head. “I want to do my research and go home to my wife.”

Anyone taking bets on where the next pivotal step in the CRISPR story would occur could have found extremely long odds on a Danish yogurt company. But for scientists at Danisco, ensuring bacteria used in starter cultures can ward off the constant threat of phage infection is a commercial priority. The next CRISPR breakthrough came in two parts of the world where cheese making is revered—France and Wisconsin.

Philippe Horvath was born about fifty miles south of Strasbourg, close to the German border. As we walk to a restaurant in Vilnius, Lithuania, he stops to catch the score of a World Cup game displayed on a giant outdoor screen. Croatia are winning en route to their surprise appearance in the final of the 2018 tournament. “Did you know my name means ‘from Croatia’ in Hungarian?” Horvath asks.²⁰ (I mean, why would I know that?!)

During his PhD at the University of Strasbourg, Horvath studied the genome of Lactobacillus plantarum, which is traditionally used in food fermentation including sourdough, kimchi, pickles, and sauerkraut. I was skeptical this could sustain an entire PhD thesis, but Horvath shoots me a look. “This is not a trivial bacterium like E. coli,” he says sternly. Sauerkraut is serious business in Alsace, where it forms the base of the famous choucroute garnie.

Horvath saw an ideal job advertisement for a molecular biologist in industry and sent off the only application letter he has ever written. He was hired in December 2000 by Rhodia Food (formerly Rhone-Poulenc, a famous French chemical company).^IV Horvath’s expertise in bacterial genetics helped improve the quality of starter cultures—the seed bacteria used to ferment milk into yogurt and cheese, which Rhodia sold to food giants like General Mills, Danone, and Nestlé. Phages that prey on the bacteria used in fermentation are found naturally in milk. “When you have a tank containing 10,000 liters of milk and you add a starter culture that is sensitive to a phage that is present, it’s a disaster!” says Horvath. “The milk remains milk.”

A typical starter culture consists of a high density of three to eight strains of bacteria—about 1 trillion bacteria per gram. Common examples include Lactobacillus acidophilus, Lactococcus lactis, and Streptococcus thermophilus. Horvath explains a freeze-dried starter culture pouch or brick is added to some 2,000 liters of milk. The goal is to minimize the number of rounds of cell division the bacteria need to produce enough lactic acid to lower the pH of the milk. Acidification must occur quickly to protect the milk from spoilage bacteria such as Salmonella and Listeria. “The higher the number of bacteria, the fewer generations you’ll need and the less risk you’ll take in terms of phages,” says Horvath. Humans have fermented milk in this fashion for millennia without knowing the molecular minutiae.

While starter culture customers prioritize acidification and phage resistance, they also value other qualities including texture and aroma. “You have to acidify quickly but you must also produce texture. You’ve experienced liquid yogurt?” Horvath asks me as we are about to tuck into dinner. “This is due to phages that have killed the texturing strain.” Horvath’s group developed starter cultures for a variety of fermentations including more than 1,000 different French cheeses. The starter culture for pizza cheese is far different to those used to produce Camembert. And because phages can manifest at any time, Horvath’s group has to formulate different starter cultures for the same product with unrelated phage sensitivities.

Much of Horvath’s work involves selecting daughter strains that are immune to the phages that attacked the parent. The process is survival-of-the-fittest straightforward: add a phage to a sensitive strain in the lab, wait patiently and search for survivors—naturally occurring mutants called bacteriophage insensitive mutants (BIM)—perhaps because the phage is unable to attach to the bacterial cell surface.

In September 2002, while attending a symposium in the Netherlands on lactic acid bacteria, Horvath came across a poster presented by Alexander Bolotin. The poster mentioned a repetitive DNA motif called “SPIDR” (spaced interspersed direct repeats), which would later be renamed CRISPR. “We have identified a region with repeats that is very useful for strain identification,” Bolotin stated. Horvath was so intrigued that he snuck a photograph.

Back in the lab, Horvath compared the sequence from one of his own group’s Streptococcus strains (LMD-9) with the strain that Bolotin had studied and other strains. To Horvath’s delight, there was a huge diversity of spacers across the SPIDR repeat regions. Every strain was different, resembling a DNA fingerprint. Horvath noticed something else: some of the sequences alternating with the SPIDR repeats matched the DNA of viruses, suggesting a link between spacers and phages. “By comparing spacer sequences with known [viral] sequences, we saw identities with phage sequences. Yes, in 2003!” At the time, only Mojica and a handful of other investigators were remotely interested in the CRISPR repeats. Horvath tried in vain to get his supervisors interested in a project he dubbed “CRISPy-SPIDRs,” but researching obscure virus biology in the food division of a chemical company wasn’t likely to win many converts. “We were told to stop working on that,” Horvath said. He continued his CRISPR research on the side, running computer searches just like Mojica.

Attitudes changed after Rhodia was acquired by Danisco, catapulting the Danish food ingredient company to second in the starter culture market, trailing only another Danish company, Chrysanthum. In 2004, every other loaf of bread and a third of ice creams contained Danisco ingredients such as color, texturants, or emulsifiers. Suddenly flush with money, Horvath felt reborn. That December, he was finally able to buy a DNA sequencing instrument. “What did we do? CRISPR sequencing! The more we sequenced, the more obvious it became!”

Horvath was on the verge of a crucial discovery in the brief history of CRISPR. It came courtesy of a new colleague, a French expat based, appropriately, in Wisconsin—the cheese state.

“I used to be French,”²¹ says Rodolphe Barrangou, professor of food science at North Carolina State University (NCSU). We’re speaking in the dramatic setting of the university’s 21st-century Hunt Library, like something out of The Jetsons. Barrangou’s self-confidence borders on a swagger, an impression magnified by his penchant for wearing cowboy boots to alleviate strain on his back following a serious basketball injury. He drives a modest Honda Accord that he has maintained since graduate school, adorned with a personalized CRISPR vanity plate.

Barrangou was born in Paris, but after he moved to North Carolina for his PhD, he fell in love with the Tar Heel state. During his PhD, which was funded by Danisco, Barrangou’s twin interests were developing next-generation probiotics and starter cultures to ferment milk (“Maybe in the spirit of Pasteur” he says, a little cheesily). His first publications were on the bacteria and attendant viruses involved in sauerkraut fermentation, including the SPIDR repeats.

In February 2005, Barrangou and his wife drove their Honda to Madison, Wisconsin, where Barrangou joined Danisco. The reports that year by Mojica and others marked the first signals of a direct connection between CRISPR elements and viruses. Barrangou began characterizing the genomes of starter cultures, checking in with Horvath. He used the CRISPR repeats to fingerprint the S. thermophilus starter cultures (a key ingredient in yogurt manufacturing), using “those peculiar loci to tell which strain is which and where it came from.” The more they sequenced, comparing new strains with older cultures thawed from Horvath’s freezer in Dangé-Saint-Roman, the more it became obvious that the CRISPR repeats could grow and evolve.

A trio of experiments clinched the association. First, Barrangou asked, what happens when a bacterial strain is exposed to a virus? “We saw the [bacterial] immune system activate itself and pick up new pieces of DNA from the viral genome and integrate them into the CRISPR locus in a particular order,” he says. This strongly supported the notion of a link between the spacer content and phage resistance.

Barrangou handled the next experiment, as his lab in Wisconsin was the only lab at Danisco permitted to do this kind of genetic engineering. Horvath was comparing two bacterial strains: DGCC7710 and a daughter (mutant) strain called 7778, which had arisen following a phage challenge performed in 1990. “Suddenly, the black box was opened!” Horvath said. In the resistant daughter strain, Barrangou found two additional spacers in the CRISPR region. “We had the sequence of the phage used in 1990: spacers 1 and 2 were present in the phage. So what did we do? We engineered to prove it was sufficient to provide resistance.” Removing both spacers in the daughter strain resulted in loss of resistance. “Add the two spacers in the parental strain, and without any challenge, it becomes resistant. Bingo!”

Understandably, Barrangou calls this his favorite experiment of all time. “When you swap the two immune systems between two strains, you swap their resistance to some of these sensitivities to viruses. That was essentially the proof that there is a direct link between the CRISPR genotype and the antiviral phenotype.”

The third experiment, Barrangou admits, was a bit lucky. Adjacent to the CRISPR motifs are the CRISPR-associated, or Cas, genes, which encode the nucleases that actually cleave the viral DNA. Inactivating the two biggest Cas genes had a major impact on the CRISPR system: knocking out Cas9 abolished the immune potential, whereas inactivating another gene, Csn2, left the immune potential intact but scrapped the ability of the CRISPR array to acquire new spacers. “This is where sometimes you have to be serendipitous, right?”

The priority for Danisco wasn’t so much to trumpet the results in a major science journal but to patent the CRISPR discovery. Filing the initial patent application on August 26, 2005, gave the company one year to provide additional examples to illustrate the patent. Horvath’s group had to keep quiet and hope nobody scooped them. The inventors were listed as Horvath, Barrangou, their respective bosses, Christophe Fremaux and Dennis Romero, and Patrick Boyaval (the boss of Fremaux and Romero). With the clock ticking, Horvath and Fremaux reached out to Canadian virologist Sylvain Moineau, who was also an expert on S. thermophilus. Moineau was skeptical at first until he reproduced the Danisco team’s results in his own lab. One of Moineau’s postdocs, Hélène Deveau, came to his office one day: “You’re not going to believe this,” she said. DNA analysis showed the CRISPR array had increased in size as the bacteria she was studying gained resistance to phages. “It was just history from there,” Moineau said.²²

The patent was converted in August 2006, meaning the results were now publicly available. Danisco’s head of innovation, Egan Beck Hansen, had a PhD in phage biology so he understood the significance of the discovery, and agreed with Horvath it was time to publish. Horvath typically favored specialized microbiology journals, but Barrangou boldly suggested they try Science. His colleagues scoffed at the idea but Barrangou persisted. Besides, if the paper was rejected, they would only lose a few weeks and they could always resubmit elsewhere.

Horvath was listed as the senior author but Barrangou drafted most of the manuscript. “He is good at painting the big picture, I am more in the details,” Horvath told me. Barrangou and Horvath submitted their manuscript to Science in October 2006, but it was rejected. CRISPR was not the hot commodity it is today. One of the three reviewers objected that the results were not observed in the classic model bacteria such as E. coli or Bacillus subtilus. However, Caroline Ash, the editor handling the paper, told Horvath he could resubmit if they added some more data. Barrangou and Horvath set about disrupting more Cas genes to show they were involved in phage resistance, and generated more BIMs and spacer sequences. Ash accepted a revised version of the paper, which was published in March 2007—the 20th anniversary of the first report of the mysterious sequences that would become a household word.²³

The Danisco team had shown experimentally that the biological function of CRISPR-Cas systems is to provide adaptive immunity in bacteria against viruses. It was the first appearance of CRISPR in the famous pages of Science magazine. More importantly, the predictions of Mojica, Vernaud, and Bolotin had been proven correct. Yogurt for the win!

Looking back, Horvath said the paper marked “a small revolution in microbiology, a new immune system but not the big revolution that occurred in 2012.” The paper immediately resonated with biochemists from the Baltics to Berkeley. Horvath and Barrangou began presenting their results on the science circuit. Over the next few years, CRISPR became a bigger and bigger deal. “We knew it was cool,” says Barrangou. “You could use it for genotyping and vaccination and cutting viral DNA.” But there were complications. “In the dairy industry, as soon as you speak about DNA, they suspect GMO manipulations and genetic engineering,” says Horvath. “It’s a sensitive topic.”

In May 2011, DuPont bought Danisco for $6.3 billion. Today, 100 percent of all commercial cultures at DuPont (and other companies) are enhanced using CRISPR screening. “Whether you have yogurt, a bite of cheese, whether you put that on your nachos or pizza or cheeseburger, in Beijing or Paris or London or New York or Buenos Aires, you are consuming a fermented dairy product that was manufactured using a CRISPR-enhanced starter culture,” says Barrangou. Today, the legacy of Barrangou and Horvath’s discovery can be found in products such as CHOOZIT SWIFT 600, one of Dupont’s most successful starter cultures, especially designed for pizza cheese. DuPont sells hundreds of starter culture packs developed from studies using Horvath’s library of some 7,000 phages.

One year after his team’s landmark paper, Horvath was asked why the Science paper had become so frequently referenced. He predicted CRISPR would have a major impact on improving the quality of functional food ingredients and a detrimental impact on the population of bacterial viruses.²⁴ Asked whether their work had any broader ramifications, Horvath replied: “Our research does not have any social nor political implication.”

The yogurt maker extraordinaire would soon eat his words.

I. The term “CRISPR” wasn’t coined until 2001. Although the true significance of Mojica’s 1993 discovery was unknown at the time, twenty-five years later, the date would be immortalized on IMAX movie screens around the world as the opening credit in Rampage.

II. The CRISPR repeats have nothing to do with adaptation to salinity. “It is still a mystery!” Mojica says. He has yet to receive funding to study the question further.

III. Ishino is now a biochemistry professor at Kyushu University. He studies DNA repair in thermophilic archaea, and is searching for new CRISPRs.

IV. In 1997, Rhone-Poulenc split its chemical and pharma businesses to form Rhodia and, two years later, Aventis. The food company remained with chemical division.