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

CHAPTER 4 “THELMA AND LOUISE”

Fifteen years to the day after President Clinton’s human genome celebration in the White House in June 2000, Jennifer Doudna posed in her laboratory at Berkeley for a New York Times photographer. The background is full of typical lab paraphernalia—cubicle freezers tucked under the bench, pipettes hanging on the wall, old yellow radioactive hazard tape. Doudna looks off to the side, wearing a pinstripe double-breasted jacket while holding a pristine white lab coat (creases still visible).

The photo reminded me of a classic Time cover featuring Craig Venter during the height of the genome wars, a lab coat over half of his dark business suit, creating a distinctly Jekyll-and-Hyde appearance. Like Venter, Doudna’s brand had evolved in short order from a dedicated academic researcher working in an obscure branch of biochemistry to an international scientific celebrity credited with spearheading a transformative new field of genome science. Venter was the poster boy for fueling a biotech revolution in genome sequencing. Doudna is the face of the CRISPR revolution, developing the ubiquitous utility tool of molecular biology enabling scientists around the world to edit DNA, from classroom to clinic, and farm to pharma. Doudna’s contemplative gaze might well have signified the ethical controversies hanging in the air, weighing heavily on her shoulders. The ensuing Times story was entitled “The CRISPR Quandary.”¹

I first met Doudna in 1998, her star already ascending years before she or anyone else had heard of CRISPR. Just thirty-five years old at the time, she was making her first visit to the headquarters of the Howard Hughes Medical Institute (HHMI)^I—twenty manicured acres nestled in Chevy Chase, Maryland, just outside Washington, DC. On the faculty at Yale University, Doudna was a newly minted investigator of the nonprofit institute, selected after a rigorous nationwide competition as one of the most talented young scientists in the country.

Doudna had a stellar scientific pedigree, having trained with not one but two Nobel laureates. With the HHMI’s hefty financial support of about $1 million a year (a blip on what was then a $10 billion endowment), Doudna could indulge her scientific curiosity. I congratulated her on her appointment over a glass of wine. She graciously told me about her research plans. I nodded along enthusiastically, hoping my lack of structural biology expertise wasn’t too obvious.

Born in Washington, DC, Doudna was seven years old when her parents moved to Hilo, on the big island of Hawaii. Her father was an English professor at the University of Hawaii (UH) Hilo, while her mother taught history at the local community college. The Hilo scenery provided abundant plants and animals for Doudna to explore and ponder their evolution. When she was about twelve, Doudna’s father left a book on her bed—a dog-eared paperback copy of The Double Helix, Jim Watson’s riveting personal tale of the discovery of the structure of DNA. She ignored it at first, assuming it was a detective novel—which, in a sense, it was.

The Double Helix remains an astonishing story of naked scientific ambition and fierce rivalries. Watson was widely criticized for the sexist manner in which he portrayed Rosalind Franklin. It was Franklin’s unpublished X-ray image of DNA fibers—photograph 51—that inspired Crick and Watson to construct their classic model. Watson literally pieced together the final pieces of the three-dimensional puzzle, showing how the four DNA bases fit together, adenine (A) always pairs with thymine (T), while cytosine (C) partners with guanine (G). The report was published in Nature, eight hundred words of pure gold, “tight as a sonnet” in the words of Colin Tudge.² Franklin died in 1958 and thus was denied a share of the Nobel Prize, which was awarded to Crick, Watson, and her former colleague, Maurice Wilkins, in 1962.

The Double Helix captured Doudna’s imagination, as it has countless young scientists, revealing how biologists could solve the secrets of life by probing the atomic structure of biomolecules such as DNA. Outside the classroom, Doudna experienced isolation and ostracization; she was a minority, referred to by the locals as a haole (a disparaging term for non-native). She found refuge in the library and the lab, delightedly spooling translucent DNA fibers around a glass rod. A family friend, Don Hemmes, let Doudna spend a summer working in his UH lab, playing with an electron microscope while studying worms and mushrooms. When her high school counselor told her “girls don’t do science,” Doudna’s determination only intensified.

Doudna graduated in chemistry from Pomona College in California after briefly flirting with the idea of switching to French, supported by her mentor and undergraduate advisor, Sharon Panasenko.³ She moved to Boston in 1985 for her PhD at Harvard Medical School, working with Jack Szostak, a brilliant RNA biochemist who went on to win the Nobel Prize. She published a major paper with him in 1989 revealing a surprising enzymatic function for certain RNA molecules. Doudna became smitten with RNA, not to be fashionable but to learn more about the versatility and functionality of ribonucleic acid.

In the central dogma of molecular biology first articulated by Francis Crick—DNA >>> RNA >>> protein—RNA was sometimes overlooked. It was considered by some a disposable copy of the genome, lacking the majestic symmetry of the double helix or the exquisite three-dimensional complexity and diversity of proteins, which carry out the essential functions in life. In 1960, Crick and Sydney Brenner postulated the existence of messenger RNA (mRNA), a facsimile of DNA relaying the freshly copied instructions in the book of life beyond the cell nucleus to the protein-manufacturing factories, the ribosomes.

While Doudna was focusing on RNA, she was acutely aware of colleagues who were breaking new ground in other branches of genetics. One was Jim Gusella, who wanted to help patients with the incurable Huntington’s disease. Like a darts player throwing a lucky bullseye, Gusella had fortuitously pinpointed the location of the Huntington’s gene on chromosome 4 in one of his first experiments in 1983. Fatefully, it would take a decade of toil before his team identified the gene itself.

Doudna’s next move was to join another Nobel laureate, spending three years in Colorado working on RNA enzymes with Tom Cech, who won the Nobel Prize in 1989. “I think Jennifer had something to do with both of those [Nobel Prizes],” says her colleague Barbara Meyer, although that would require Doudna mastering in time travel. She opened her own lab at Yale University, and immediately made an impression. Vic Myer, a leading gene editor, recalls, “she was bright, energetic, insightful, and asked very good questions. Fundamentally she is driven by the science, excited by the science, and a great person.”⁴

In 2002, Doudna moved her lab across the country from Yale to Berkeley, California, to be closer to home, family, and a major synchrotron source for her structural biology studies. Her husband, Jamie Cate, whom she met in the Cech lab, set up his own lab next door. The big question scientifically at the time centered on the RNA World hypothesis, the idea that life began on earth in the form of RNA molecules.

Jill Banfield, a microbiologist at Berkeley, characterizes new species of bacteria and Archaea, expanding our understanding of the evolutionary tree of life. Like an archeologist searching for rare living microbes, her research takes her around the world to a range of environments, some more exotic than others: salt lakes in her native Australia, mine shafts in Colorado, geysers in Yellowstone National Park, as well as simple groundwater wells. Banfield’s team has characterized literally hundreds of new microbial species from these extreme locales, the winners in a billion-year-old game of Survivor.

In 2006, Banfield was stumped. Samples of the same species collected from the same location should have identical sequences, she reasoned, but to her surprise no two DNA traces had the same sequence. “It was shocking,” she recalls. She had stumbled on the fast-evolving CRISPR region. The latest news on that front had come in a paper from Kira Makarova and Eugene Koonin at the NIH that included a rare sighting in science: a confession. The duo had previously suggested that the function of the CRISPR-associated genes was in DNA repair. They were wrong. Abandoning that hypothesis, they now proposed that CRISPR was a genetic defense system that targeted viruses via a mechanism called RNA interference (RNAi), which would earn its discoverers, Stanford’s Andy Fire and Craig Mello at the University of Massachusetts, a Nobel Prize.⁵

Banfield typed in “RNAi” and “UC Berkeley” into her search engine. The first name that popped up was Doudna’s. She decided to give her colleague a call. “You’re doing the type of research that I think could be very interesting for something that I’ve stumbled across in my own work,” she said.⁶ And she introduced Doudna to a new piece of scientific jargon: CRISPR. Now it was Doudna’s turn to google Banfield. A few days later, the geomicrobiologist and the RNA biochemist met at the Free Speech Movement Café, a popular central meeting spot in the heart of the Berkeley campus. Seated at an outdoor table, Banfield excitedly told Doudna about her work sequencing bacterial genomes and the clusters of strange palindromic repeat sequences contained in some of her newly discovered organisms.

Banfield took her notepad and sketched a circular bacterial genome and then, magnifying a section of the DNA, drew a series of symbols:

Array of Light: In her first meeting with Doudna, Jill Banfield sketched the CRISPR array showing the alternating pattern of repeats (diamonds) and spacers (squares) derived from viral DNA.

This was the CRISPR array, a series of identical motifs about thirty bases in length interspersed with other sequences—Banfield drew squares, giving each a number—that apparently had nothing in common with each other. Banfield knew the spacers were derived from viruses and that they evolved at a faster clip than other parts of the bacterial genome. And now there was the suggestion that CRISPR was the blueprint for an antiviral defense system involving RNAi. In Doudna, one of the world’s experts on RNA structure and function, she’d found the perfect ally.

Banfield’s expertise in cataloguing the vast diversity of microbial life—“the weight of evolutionary history” as she calls it—promises to unearth many new tools for the CRISPR toolbox. In 2013, Kim Seed and colleagues at Tufts University made a remarkable discovery: a phage that has pilfered CRISPR repeats from bacteria—like a hostage grabbing an assailant’s weapon—and turned it on the host cell.⁷ Banfield’s team has also discovered so-called jumbo phages—phages with giant genomes larger than some bacteria, blurring the boundary between life and death—lurking in the gut microbiome of people in Bangladesh with a non-Western diet. The CRISPR machinery not only helps the viruses evade the bacterial defense mechanisms but may also thwart competing viruses.⁸

It would actually be several years before the two women, both leading large groups with different research interests and areas of expertise, managed to collaborate on a research paper, but Banfield had piqued Doudna’s interest. Looking back at her pivotal role in the CRISPR drama, Banfield, one of the most accomplished microbiologists in the world, can only laugh. “One thing they’ll write on my tombstone is: ‘Told Jennifer Doudna about CRISPR-Cas.’ Like, that will be the sum of my life!”⁹

In the first few months of 2007, two new postdocs joined the Doudna lab. Blake Wiedenheft introduced CRISPR to the laboratory; Martin Jínek ensured the lab’s legacy.

Jínek hails from Třinec, a city on the border between the Czech Republic and Poland. One hundred miles to the west is Brno, the birthplace of genetics. Surprisingly, Jínek didn’t pay a visit to Gregor Mendel’s monastery until a few years ago, when he was invited to give a lecture. At sixteen, Jínek won a scholarship to a private boarding school in England. He then spent four years at Cambridge University studying chemistry, but always had an inclination towards biology, especially RNA, thanks in no small part of Doudna’s string of successes with Szostak and Cech. “It’s such a versatile molecule,” he told me. “It can do catalysis, fold into 3D structures, and it’s a carrier of information. It’s an all-rounder”—a term, I surmise, he picked up watching cricket at school.

After completing his PhD in Germany, Jínek wanted to study RNAi. The Doudna lab had just published the structure of an important RNA processing enzyme called dicer. Doudna likened it to a “molecular ruler” that measures RNA sequences prior to cutting them into precise lengths. RNAi was attracting huge biotech interest, underpinning new companies such as Alnylam Pharmaceuticals and Moderna. Jínek arrived in Berkeley just before publication of the Barrangou et al. Science paper, which elevated CRISPR to prime time. Doudna’s group discussed that paper in a journal club meeting. “Everybody was quite excited about it,” Jínek recalls. “We decided that this was going to be an RNA-guided mechanism, like RNA interference. There was going to be some kind of connection.”

A short time later, Wiedenheft, a swarthy scientist from Montana, visited for an interview with Doudna and talked about studying CRISPR. “We were all primed for that by the journal club—Jennifer included,” Jínek said. The man from Montana got the job and performed the first CRISPR experiments in Doudna’s lab. Wiedenheft was interested in the pathways by which phages infect bacteria and conversely, how bacteria ward off phage infection. Like Banfield, he had collected microbial samples in Yellowstone and other extreme locations. “Without Blake being in the trenches, I don’t know if the Doudna lab would’ve had a meaningful thrust in that direction,” said Ross Wilson, who joined the lab two years later.¹⁰

Jínek wasn’t working on CRISPR himself but he’d regularly talk science with Wiedenheft. The Czech’s first involvement was helping his friend learn about protein crystal structures for a first look at a DNA-cutting enzyme called Cas1, published in 2009.¹¹ Some afternoons, the two men would take a break to go biking in the Berkeley hills. Wilson shakes his head recalling the sweaty duo returning to the lab in their spandex shorts.

Just as Doudna’s interest in CRISPR was getting off the ground, her head was turned by an irresistible offer in 2009 from one of the most famous names in biotech. She admits she was undergoing a mini midlife crisis, looking for a new scientific challenge. “Am I going to get to the end of my career and feel like I did some cool stuff, had some fun, published some papers we’re proud of, but did I really solve any problems?” she told journalist Lisa Jarvis.¹²

The offer came from Richard Scheller, Doudna’s former colleague at HHMI before he became head of R&D at Genentech, one of the most successful and coolest biotech companies in the world.¹³ Doudna would have the chance to apply her RNA expertise in the search for novel drugs and therapies. Her appointment as Genentech’s new vice president of Discovery Research was announced in a press release, and she listed her change of address in an article with Jínek in Nature.¹⁴ She hoped that most of her group would join her in South San Francisco, but industry wasn’t appealing to Jínek. Plan B was to move down the hall and finish his postdoc in the lab of Doudna’s husband.

However, Doudna soon had a change of heart. “I realized what I’m good at doing and what I really like. It all boiled down to creative, untethered science,” she said. After a somewhat painful two months, she resigned and returned to Berkeley, where she reclaimed her HHMI professorship. Having given up most of her administrative obligations, she was free to pursue “crazy, creative projects” that might not be clinically relevant but she considered cool science. And the craziest project in the lab was CRISPR. “Had I not made the foray to Genentech and then back to Berkeley, I might not have done any of the CRISPR work,” she acknowledged.¹⁵

In a 2010 lab photo, Doudna is pictured with her troop of CRISPR devotees—Wiedenheft, Jínek, graduate student Rachel Haurwitz, and longtime lab manager Kaihong Zhou. There is an air of innocence in the group, blissfully unaware of how their lives were about to change. Jínek had finally run out of independent funding after four years but Doudna was happy to keep him around. In 2011, he began searching for faculty positions back in Europe, but wanted to enjoy a last hurrah to end his Californian adventure. Jínek fancied taking a closer look at the little-known type II CRISPR system. Doudna soon offered him a gilt-edged opportunity.

It wasn’t just the Doudna lab that was struck by the Danisco CRISPR story. On May 23, 2007, just two days after the Science paper came out in print, Virginijus Šikšnys emailed Horvath to offer a collaboration. In the Lithuanian’s world, the discovery of a new antiviral defense system called CRISPR was almost as momentous as the collapse of the Soviet Union. For years, Šikšnys had been trying to understand how bacteria defend themselves against viruses. Suddenly, scientists from a yogurt company had described an entirely new bacterial immune system. It was the start of an important partnership that would have a major impact on the development of genome editing.

Vilnius, the capital of Lithuania, is not exactly the Mecca of molecular biology. When I first visited in the Spring of 2017, the main cultural attraction was the former KGB headquarters.^II The city center is a curious mix of narrow medieval streets in the old town, where I washed down a pungent beaver stew with real mead, adjacent to rows of designer clothing boutiques.

The Vilnius Institute of Biotechnology is a few miles outside the city center. A chemist by training, Šikšnys built a reputation studying the 3D structure and properties of bacterial restriction enzymes (there are four thousand all told).^III CRISPR offered an exciting new research opportunity. But first, he needed to transfer the CRISPR system into a bacterium other than S. thermophilus, for good reasons. “We don’t know how to make cheese and yogurt!” Šikšnys joked.

The obvious choice was E. coli, the lab workhorse. But which CRISPR system? It turns out S. thermophilus has four different CRISPR systems—all have a cluster of spacers but differ in the architecture and adjacent Cas genes. Šikšnys chose the simplest (type II) system with the smallest number of Cas genes, including one called Cas9, known to be required for phage resistance. With his intimate knowledge of DNA-cutting enzymes, Šikšnys noticed a couple of spots in the Cas9 structure that resembled catalytic active sites he’d observed in restriction enzymes. It was a sign that Cas9 might have interesting DNA-cutting properties of its own.

The Lithuanian’s entry into the CRISPR timeline was one of many new dots on the map. But it took five years to connect the dots from yogurt and pizza starter cultures to the brink of a universal gene-editing technology. The next steps were to answer some practical questions: How were the CRISPR spacers captured from the phage invaders and stitched into the bacterial genome? And how were they weaponized to target and destroy incoming phage?

Moineau’s group first described a key recognition sequence,¹⁶ the critical first touchpoint of Cas9 alighting on DNA. As phages mutated or “escaped” CRISPR-mediated destruction, Moineau catalogued a series of single-base mutations in the CRISPR repeats as well as this recognition motif, which Mojica dubbed the proto-spacer adjacent motif, or PAM.¹⁷

Over the next few years, researchers around the world began piecing together the molecular details of the CRISPR immune system. It was like the British children’s party game pass the parcel: at each round the music stopped with a different participant peeling off another layer of wrapping before revealing the gift. After documenting CRISPR in her own microbial samples,¹⁸ Banfield reached out to Barrangou to propose they organize a meeting of CRISPR disciples. That summer in 2008, about thirty scientists gathered at Berkeley’s Stanley Hall. Members of Doudna’s lab, located on the seventh floor of the same building, dropped in. Barrangou put the wine and beer costs on his corporate credit card.

The action next shifted to the Netherlands. Studying CRISPR in E. coli, John van der Oost, a microbiologist at Wageningen University, and Stan Brouns showed that the CRISPR spacers are first transcribed into a long contiguous RNA, which is then sliced into discrete CRISPR RNAs corresponding to individual spacers. This RNA then forms a complex with Cas proteins that targets the corresponding phage.¹⁹ (The E. coli CRISPR is of the type I variety, in which the role of Cas9 is played by a complex of five proteins known as the Cascade complex.) The result was a CRISPR milestone, duly recognized a decade later when van der Oost shared Holland’s most prestigious science award, the Spinoza Prize. “I don’t think John’s work will ever be forgotten,” says Koonin, the unofficial master record-keeper of CRISPR gene evolution.²⁰

In Chicago, Erik Sontheimer and his Argentine postdoc, Luciano Marraffini, designed some clever experiments using Staphylococcus epidermis to settle another big question: does CRISPR-Cas target the viral RNA—mimicking RNAi—or DNA? Marraffini suspected it would be more efficient for bacteria to dispose of viral infections if they cleaved DNA, taking a machete to the viral genome in one fell swoop. He was correct.²¹ Practically speaking, they wrote, “the ability to direct the specific addressable destruction of DNA that contains any given 24-48 [base] target sequence could have considerable functional utility, especially if the system can function outside of its native bacterial context.”

Sontheimer and Marraffini saw a glimmer of clinical relevance—the possibility “to impede the ever-worsening spread of antibiotic resistance genes and virulence factors in staphylococci and other bacterial pathogens.” It was one small step on the road to precise CRISPR genome editing, albeit one that exclusively used the delete button. “We were the first to recognize and explicitly articulate the possibility that CRISPR could be repurposed for genome engineering,” said Sontheimer.²² But the celebration was short-lived: Sontheimer’s patent application was denied for lack of experimental evidence, as was an ambitious grant application that was years ahead of its time. (As we shall see in chapter 12, it was the beginning of a contentious legal battle for inventorship.)

As interest in CRISPR grew, additional types of CRISPR systems were discovered. At the University of Georgia, Michael Terns showed that type III CRISPR systems target RNA rather than DNA. Meanwhile, Doudna published her first papers on CRISPR with Wiedenheft and Haurwitz.

In 2010, Moineau, who has been fascinated by phages since he first saw them under an electron microscope, took center stage. “If you go to the ocean and take water in the palm of your hands, you have more viruses in your hands than there are humans on this planet,” he says.²³ His forte was the phages that infect S. thermophilus—the key ingredient for yogurt and cheese. Moineau says we eat more than one sextillion (10²¹) S. thermophilus cells per year.

Moineau’s interest is the ongoing arms race between phage and bacteria. Still collaborating with Horvath, Barrangou, and the Danisco team, Moineau’s lab demonstrated that CRISPR RNAs cleaved their DNA targets directly, literally cutting a circular DNA molecule (a plasmid inside a bacterium) in one spot into a linear fragment, producing clean blunt ends in the vicinity of the PAM sequence.²⁴ Moineau published the story in Nature, one of the highlights of his career.

Meanwhile, researchers continued to expand the family of Cas genes by combing through the expanse of microbial life on earth, much of the credit belonging to Koonin and Makarova. The number of different CRISPR classes and subtypes has grown increasingly complex, with six classes known by 2020. Fortuitously, the Strep bacteria studied by Horvath and Barrangou have a type II system, the most rudimentary of CRISPR systems. And that was huge.

The first time I emailed Emmanuelle Charpentier, in early 2017, I received an immediate response. It was an “out of office” message that said:

Due to my full schedule associated with attendance of prize ceremonies, I will not be able to reply to your email…

Well, that was a first. She wasn’t kidding. Charpentier has won dozens of prestigious awards since 2012. Asked about the distraction of being a semi-permanent fixture on the awards circuit, she told Le Figaro: “It’s very exotic! Let’s say that’s not the reason I did research. As a researcher, I like being isolated in my laboratory with my team.”²⁵ Her PhD supervisor said she was so resourceful, she could start a lab on a desert island.²⁶ It didn’t quite come to that, but after two decades of nomadic existence—moving from the United States to Austria to Sweden—she has found a home in Berlin. Time will tell if she can match the success she earned in 2012.

Charpentier may have a lower public profile in the United States than Doudna or Zhang, but it is a different story in Europe. A 2015 profile in Le Monde dubbed her “the charming little monster” of genetic engineering, with “the air of a mockingbird, perched in the forest of the best-rooted authorities of science in France, Europe, and America.”²⁷ She is called “pugnacious and courageous,” inspired by three muses—curiosity, daring, and freedom. The following year, the same newspaper, in an article titled “The new icons of biology,” hailed Charpentier and Doudna as the “Thelma and Louise” of biomedical research, acquiring scientific honors and prizes like a squirrel harvests hazelnuts.²⁸

In September 2018, just a few weeks after receiving the Kavli Prize in Oslo, Charpentier flew to New York to deliver a memorial lecture at Columbia University.²⁹ As usual, her appearance attracted a packed house, as hundreds of students and faculty crammed into the auditorium including the gangly figure of Nobel laureate Richard Axel. Arriving late, the eminent neuroscientist folded himself into a front row seat, as intrigued as everyone else to hear the petite Frenchwoman on course to follow his journey to Stockholm. After an interminable introduction, Charpentier politely chided her host for exaggerating her resume. “I did not publish that many papers!” she said. “I’ve always been more on the perfectionism side—I focus more on the quality than the quantity.”

In her book, Doudna said her first impression of Charpentier was “soft-spoken and retiring.” That was a fair reflection of Charpentier’s lecture, a surprisingly subdued account of her CRISPR journey, framing her work in the context of legendary French molecular biologists like François Jacob and Jacques Monod. She dwells on a quote from Monod in 1970:

Modern molecular genetics offers us no means whatsoever for acting upon the ancestral heritage so as to improve it with new features—to create a genetic “superman”: on the contrary, it reveals the vanity of any such hope: the genome’s microscopic proportions today and probably forever rule out manipulation of this sort.³⁰

Charpentier seldom looks up at the audience and strikes me as guarded, reluctant to let loose and share stories. Axel fidgets in the front row. She wonders if her own mobility “helped me to understand the way bacteria defend themselves against mobile elements.” It’s more a statement of fact than an attempted joke. But if humor is in short supply, humility is not. Charpentier acknowledges a crucial slice of fortune. For years the pet organism in her lab was a bacterium called Streptococcus pyogenes, which can cause life-threatening infections. This was the source of the Cas9 protein. “Cas9 in Strep pyogenes is very efficient,” Charpentier said. “We’ve tested other Cas9 proteins, some are close in efficiency, but if we’d identified mechanisms in another bacterial species, I’d not be here in front of you.”

After her lecture, Charpentier fields questions from the audience: She bemoans a recent European court ruling on GMOs (“a big disappointment for scientists in Europe”) and dismisses concerns about CRISPR’s safety. “Delivery is a bigger bottleneck than the CRISPR mechanism itself,” she says confidently. Students line up to take selfies with her; she is a bona fide scientific celebrity after all. I join the queue to invite Charpentier for an interview before she heads to dinner with some university VIPs.

The next morning, we rendezvous on Manhattan’s Upper West Side. She arrives looking chic in blazer and jeans, two months shy of her fiftieth birthday. By contrast, I am sporting fresh Central Park pigeon excrement down my laundered white shirt. “It is good luck, I think,” she smiles, as we head to a nearby French bistro.

Charpentier was born fifteen miles south of Paris in 1968, six months after the student protests and civil unrest. From an early age, she was intent upon going to college, inspired by her older sister. Her father taught her the Latin names of plants, which might have inspired her to pursue biology. At age twelve, Charpentier came home from school one day and told her mother that she would eventually study at the Pasteur Institute. Ten years later, she made good on her promise. “I got my worst grade for [microbiology] and it became my specialty!” she laughs. She earned her PhD in microbiology in 1995, writing her thesis in a library overlooking the Cathedral of Notre-Dame.

Charpentier recognized that life as a research scientist “would fit the many aspects of my personality—my curiosity, intellectual drive for knowledge, enjoyment of communicating knowledge to others, and working as a team, and my desire to turn complex scientific discoveries into practical applications that would help society.”³¹ She spent the next six years in the United States, beginning at the Rockefeller University in New York. She studied bacteria responsible for skin infections in mice, searching for new biochemical pathways and drug targets. After working with Streptococcus pneumoniae, she turned to S. pyogenes, which became her favorite organism. Her experience in America introduced her to many talented researchers, many with a strong entrepreneurial spirit. That lesson, too, was not lost on her.

There were no openings at the Pasteur when she was ready to return to Europe, but she landed at the University of Vienna in laboratories named after the Nobel laureate Max Perutz, a contemporary of Crick and Watson.^IV “It was important to be independent and to not have anyone around me,” she said. I’m intrigued by Charpentier’s willingness to travel in search of freedom and funding. Without missing a beat, she says: “In twenty-seven years, I’ve worked in five countries, seven cities, ten institutions. Fourteen different offices, thirteen different departments, and fourteen apartments. It’s a very big turnover!” Each move was motivated by an incentive or a better position.

Charpentier’s first exposure to CRISPR came in 2006. Two of her students, Maria Eckert and Karine Gonzales, were performing computer searches for DNA sequence matches similar to the studies reported by Mojica one year earlier. This time CRISPR was not the bait but the prize. Eckert and Gonzales were scouring the S. pyogenes genome for traces of small RNAs (encoded by genes that produce RNA molecules rather than proteins). One of the most abundant hits was a novel trans-activating CRISPR RNA (tracrRNA). The gene encoding this tracrRNA sat in the vicinity of the CRISPR array, although the significance of that location wasn’t immediately obvious. Charpentier’s major interest was not CRISPR or bacterial immunity, but her tracrRNA had a certain je ne sais quoi. It would prove a critical piece of the genome editing puzzle.

In 2008, Charpentier decided to leave the history of Vienna for the hinterland of northern Sweden and Umeå University. The weather was bleak but Charpentier warmed to the atmosphere and people at the Laboratory for Molecular Infection Medicine Sweden. She focused on tracrRNA, collaborating with German biochemist Jörg Vogel, planning experiments on the flights back and forth to Sweden.

The next year, her group established a link between the CRISPR-Cas9 system and tracrRNA. “It was a very simple experiment,” she recalls. When the group knocked out tracrRNA, they found that the CRISPR RNA (crRNA) was not made, and vice versa. The logical conclusion was that Cas9 formed a physical complex with these two RNA molecules. When Charpentier’s team compared tracrRNA sequences from a variety of bacteria, they found one thing in common: a sequence that forms a duplex with crRNA. While other groups were describing more complex types of CRISPR systems, the beauty of the type II system in S. pyogenes was that only a single gene, Cas9, was necessary for viral interference, along with crRNA. And then there were three. Charpentier and Vogel had brought the third component—tracrRNA—into the story. “Yes, it is an essential component, because Cas9 is an enzyme guided by two RNAs,” she said.³²

One month after Charpentier submitted her polished account of the discovery of tracrRNA to Nature in September 2010, she traveled to the Netherlands to give a talk at the annual CRISPR conference, which had ballooned to about two hundred CRISPR aficionados. Few members of the CRISPR club knew about the French scientist working in Sweden with colleagues in Austria and Germany. But now, Charpentier recalled with a smile, “they discovered the famous story of tracrRNA.” It was the pinnacle of Charpentier’s career to date. “The pioneers all came up to me and shook my hand and said, ‘I think you got the story!’ ” One notable absentee was Doudna, whose research wasn’t consumed by CRISPR just yet.

With her paper accepted by Nature in early 2011, Charpentier plotted the next step of her story, which she would set in motion at an upcoming conference. She needed an expert in RNA biochemistry and structural biology. “I had in mind to approach Jennifer and to ask her whether she would be interested in deciphering the structure of Cas9.”³³

I. HHMI was set up as a nonprofit medical institute by the businessman-investor-aviator Howard Hughes. It was initially little more than a tax shelter but today HHMI spends a portion of its $20-billion endowment funding hundreds of researchers in the life sciences. Doudna has been an investigator for more than twenty years, and looks set for another twenty. Several other leading CRISPR researchers, including Feng Zhang and Luciano Marraffini, are also HHMI investigators.

II. The Museum of Occupations and Freedom Fights.

III. I was part of the first wave of PhD students in the 1980s who had the luxury of just ordering these enzymes from a catalogue. My predecessors recounted horror stories of spending hours camped in the cold room having to purify them from scratch.

IV. In early 1953, Perutz shared a Medical Research Council report containing Rosalind Franklin’s unpublished DNA crystallography data with Crick and Watson. That sneak peak proved critical in the assembly of the double helix.