The Code Breaker, page 8
Mojica’s paper was rejected by two other publications. Finally he was able to get it published in the Journal of Molecular Evolution, which was not as prestigious but served to get his findings in a peer-reviewed publication. Even at that journal, Mojica had to pester and prod the slow-moving editors. “I reached out and tried to get in touch with the editors almost every week,” he says. “Every week was so terrible, such a nightmare, because I knew we had discovered something really great. And I knew that at some point others would discover it. And I couldn’t get them to see how important it was.”9 The journal received the paper in February 2004, did not make a decision until October, and it was not actually published until February 2005, two years after Mojica had come up with his findings.10
Mojica says he was driven by his love of the beauties of nature. He had the luxury at Alicante of doing basic research without showing how it might translate into something useful, and he never tried to patent his CRISPR discoveries. “When you work as I do on weird organisms that live in unusual environments, like very salty ponds, your only motivation is curiosity,” he says. “It didn’t seem likely that our discovery would apply to more normal organisms. But we were wrong.”
As is often the case in the history of science, discoveries can have unexpected applications. “When you do curiosity-driven research, you never know what it may someday lead to,” Mojica says. “Something that’s basic can later have wide consequences.” His prediction to his wife that his name would someday be in history books proved to be correct.
* * *
Mojica’s paper was the beginning of a wave of articles providing evidence that CRISPR was, indeed, an immune system that bacteria adapted whenever they got attacked by a new type of virus. Within a year, Eugene Koonin, a researcher at the U.S. National Center for Biotechnology Information, extended Mojica’s theory by showing that the role of the CRISPR-associated enzymes was to grab bits of DNA out of the attacking viruses and insert them into the bacteria’s own DNA, sort of like cutting and pasting a mug shot of dangerous viruses.11 But Koonin and his team got one thing wrong. They speculated that the CRISPR defense system worked through RNA interference. In other words, they thought that bacteria used the mug shots to find a way to interfere with the messenger RNAs that carry out the instructions encoded by DNA.
Others thought so as well. That is why Jennifer Doudna, Berkeley’s leading expert on RNA interference, would end up getting a phone call out of the blue from a colleague who was trying to figure out CRISPR.
Jillian Banfield
CHAPTER 10 The Free Speech Movement Café
Jillian Banfield
In early 2006, shortly after she published her first paper on Dicer, Doudna was in her Berkeley office when she got a call from a Berkeley professor she had heard of but didn’t know: Jillian Banfield, a microbiologist who, like Mojica, was interested in tiny organisms found in extreme environments. A gregarious Australian with a wry smile and collaborative nature, Banfield was studying bacteria that her team found in a very salty lake in Australia, a hot geyser in Utah, and the extremely acidic waste draining from a California copper mine into a salt marsh.1
When Banfield sequenced the DNA of her bacteria, she kept finding examples of the clustered repeated sequences known as CRISPRs. She was among those who assumed that the CRISPR system worked by using RNA interference. When she typed “RNAi and UC Berkeley” into Google, Doudna’s name was the top result, so Banfield gave her a call. “I’m looking for someone at Berkeley,” she told Doudna, “who is working on RNA guides, and I did a Google search and your name popped up.” They agreed to meet for tea.
Doudna had never heard of CRISPR. In fact, she thought that Banfield was saying “crisper.” After hanging up, she did a quick online search and found just a few articles about it. When she got to the point in an article where it said CRISPR stood for “clustered regularly interspaced short palindromic repeats,” she decided to wait for Banfield to explain it to her.
They met on a blustery spring day at a stone table in the courtyard of the Free Speech Movement Café, a soup-and-salad hangout at the entrance to Berkeley’s undergraduate library. Banfield had printed out the papers by Mojica and Koonin. She realized that, in order to figure out the function of these CRISPR sequences, it made sense to collaborate with a biochemist such as Doudna, who could analyze each component of a mysterious molecule in a laboratory.
When I sat down with the two of them to hear about that meeting, they displayed the same excitement they described feeling back then. They both talked rapidly, especially Banfield, and they finished each other’s sentences amid quick laughs. “We are sitting there and drinking tea, and you had a big pile of pages that had all this data of the sequences you had found,” Doudna recalled. Banfield, who usually works on her computer and rarely prints out anything, agreed. “I kept showing you the sequences,” she recalled. Doudna chimed in, “You were so passionate, and you were talking so fast. You had a lot of data. And I’m thinking, ‘She’s really, really excited about this.’ ”2
At the café table, Banfield drew a string of diamonds and squares that represented segments of the DNA she had found in her bacteria. The diamonds, she said, all had identical sequences, but the interspersed squares each had unique sequences. “It’s like they are diversifying so fast in response to something,” she told Doudna. “I mean, what was causing these strange clusters of DNA sequences? How did they actually work?”
Until then, CRISPRs had largely been the purview of microbiologists, such as Mojica and Banfield, who studied living organisms. They had come up with elegant theories about CRISPR, some of them correct, but they had not done controlled experiments in test tubes. “At the time, nobody had actually isolated the molecular components of the CRISPR system, tested them in a lab, and figured out their structures,” Doudna said. “So the time was right for biochemists and structural biologists like me to jump in.”3
CHAPTER 11 Jumping In
Blake Wiedenheft
When Banfield asked her to collaborate on CRISPR, Doudna was initially stymied. She had nobody in her lab to work on it.
Then an unusual candidate walked into her office to interview for a postdoctoral position. Blake Wiedenheft, a charismatic and bear-cub-loveable Montanan with an enthusiasm for the outdoors, had spent most of his academic career, when he wasn’t taking time off to pursue wilderness adventures, collecting microorganisms from extreme environments, from Kamchatka in Russia to Yellowstone National Park in his backyard, just like Banfield and Mojica. His letters of reference were not stellar, but he was earnest and passionate about switching his interest from the biology of small organisms to the biology of molecules, and when Doudna asked him what he wanted to work on, he said the magic words: “Have you ever heard of CRISPR?”1
* * *
Wiedenheft was born in Fort Peck, Montana, population 233, an outpost eighty-one miles from the Canadian border and near nothing else. The son of a fisheries biologist for Montana’s Wildlife Department, he ran track, skied, wrestled, and played football in high school.
As an undergraduate at Montana State, he majored in biology, but he spent little time in the lab. Instead, he enjoyed going into nearby Yellowstone and collecting microorganisms that can survive in the boiling acid springs there. “It made a huge impression on me,” he says, “to scoop up sample organisms from an acid hot spring, bring them back in a thermos, grow them in these artificial hot springs we rigged up in the lab, and then take those samples to the microscope and peer through the lens and see something that has never been seen before. That changed how I imagined life.”
Blake Wiedenheft in Kamchatka, Russia
Montana State was a perfect university for him, because it allowed him to indulge his love of adventure. “I’m always looking for what’s over the next peak,” he says.2 When he graduated, he had no plans to become a research scientist. Instead, like his father, he was interested in fish biology, and he signed up to work on a crabbing vessel in the Bering Sea off Alaska, collecting data for government agencies. He then spent a summer teaching science to young students in Ghana, followed by a stint as a ski patroller in Montana. “I was addicted to adventure.”
But during his travels, he would find himself rereading his old biology textbooks at night. His college mentor Mark Young was studying the viruses that attacked the bacteria in Yellowstone’s boiling acid springs. “Mark’s excitement for understanding how these biological machines work was, literally, infectious.”3 After three years of wandering, Wiedenheft decided there were adventures to be found not only outdoors but in labs. He returned to Montana State as a PhD student under Young, and together they studied how these viruses invade bacteria.4
Although Wiedenheft was able to sequence the DNA of the viruses, he found himself wanting more. “Once I started peering at the DNA sequences, I realized they were uninformative,” he says. “We had to determine structures, because structures, the folds and shapes, are conserved over a longer evolutionary period than the nucleic acid sequences.” In other words, the sequence of letters in the DNA did not reveal how it worked; what was important was how it folded and twisted, which would reveal how it interacted with other molecules.5
He decided that he needed to learn structural biology, and there was no place better for that than Doudna’s lab at Berkeley.
* * *
Wiedenheft is too earnest to be insecure, and that came through when he interviewed with Doudna. “I was coming from a small lab in Montana, and I had enough hubris not to be completely intimidated, though I should have been,” he recalls. He had a few subject areas he planned to pitch, but when Doudna showed interest in CRISPR, which was his first passion, he became energized. “I just started yammering and tried to sell myself best I could.” He went to the whiteboard and mapped out the CRISPR projects being pursued by other researchers, including John van der Oost and Stan Brouns, a team from the Netherlands he had worked with when they came to Yellowstone to collect microorganisms from the hot springs.
He and Doudna brainstormed about opportunities that her lab might pursue, most notably figuring out the functions of the CRISPR-associated (Cas) enzymes. Doudna was struck by his energy and infectious enthusiasm. For his part, Wiedenheft was impressed that Doudna shared his enthusiasm for CRISPR. “She has a knack for seeing around corners to know what the next big thing is,” he says.6
Wiedenheft threw himself into his work in Doudna’s lab with the joyful passion he displayed as an outdoorsman. He was willing to charge headlong into techniques he had never used before. At lunchtime he would go on a hard-core bike ride, then work through the afternoon and evening still wearing his cycling gear, wandering around the lab in his helmet. He once spent forty-eight hours straight on one experiment, sleeping next to it.
Martin Jinek
Wiedenheft’s desire to learn structural biology caused him to latch on to, both intellectually and socially, a postdoc who was the Doudna Lab’s expert in crystallography. Martin Jinek (YEE-nik) was born in the Silesian town of Třinec in what was then Czechoslovakia. He studied organic chemistry at Cambridge University and did his doctoral work under the Italian biochemist Elena Conti in Heidelberg. This produced, in addition to an agile scientific outlook, a hybrid accent that featured very precisely pronounced phrases repeatedly interspaced with the interjection “basically.”7
In Conti’s lab, Jinek developed a passion for the star molecule of this book, RNA. “It’s such a versatile molecule—it can do catalysis, it can fold into 3D structures,” he later told Kevin Davies of the CRISPR Journal. “At the same time, it’s a carrier of information. It’s an all-rounder in the world of biomolecules!”8 His goal was to work in a lab where he could figure out the structure of complexes that combined RNA and enzymes.9
Jinek was good at charting his own path. “He was somebody who could work independently, which has always been important in my lab because I’m not a close hands-on advisor,” she says. “I like to hire people who have their own creative ideas and want to work under my guidance and as part of my team, but not with daily direction.” She arranged to meet Jinek when she went to Heidelberg for a conference, then enticed him to come to Berkeley and sit down with the members of her lab. She felt it was important that people on her team were comfortable with each new hire.
Jinek’s initial work in Doudna’s lab focused on how RNA interference works. Researchers had described the process in living cells, but Jinek knew that a full explanation required re-creating the process in a test tube. The in vitro experiments allowed him to isolate the enzymes that are essential to interfering with the expression of a gene. He also was able to determine the crystal structure of one particular enzyme, thus showing how it is able to cut up the messenger RNA.10
Jinek and Wiedenheft, with their very different backgrounds and personalities, became complementary particles. Jinek was a crystallographer who wanted more experience working with living cells, and Wiedenheft was a microbiologist who wanted to learn crystallography. They took an instant liking to each other. Wiedenheft had a much more playful sense of humor than Jinek, but it was so contagious that Jinek soon acquired it. On one trip they took with other lab members to the Argonne National Laboratory near Chicago, they were working in the huge circular building that houses the Advanced Photon Source, a powerful X-ray machine. It is so large that there are tricycles for researchers to use to get around. At 4 a.m., after working all night, Wiedenheft organized a tricycle race around the entire circuit of the building, which he of course won.11
Doudna decided that her lab’s goal would be to dissect the CRISPR system into its chemical components and study how each worked. She and Wiedenheft decided to focus first on the CRISPR-associated enzymes.
Cas1
Let’s pause for a quick refresher course.
Enzymes are a type of protein. Their main function is to act as a catalyst that sparks chemical reactions in the cells of living organisms, from bacteria to humans. There are more than five thousand biochemical reactions that are catalyzed by enzymes. These include breaking down starches and proteins in the digestive system, causing muscles to contract, sending signals between cells, regulating metabolism, and (most important for this discussion) cutting and splicing DNA and RNA.
By 2008, scientists had discovered a handful of enzymes produced by genes that are adjacent to the CRISPR sequences in a bacteria’s DNA. These CRISPR-associated (Cas) enzymes enable the system to cut and paste new memories of viruses that attack the bacteria. They also create short segments of RNA, known as CRISPR RNA (crRNA), that can guide a scissors-like enzyme to a dangerous virus and cut up its genetic material. Presto! That’s how the wily bacteria create an adaptive immune system!
The notation system for these enzymes was still in flux in 2009, largely because they were being discovered in different labs. Eventually they were standardized into names such as Cas1, Cas9, Cas12, and Cas13.
Doudna and Wiedenheft decided to focus on what became known as Cas1. It’s the only Cas enzyme that appears in all bacteria that have CRISPR systems, which indicates that it performs a fundamental function. Cas1 had another advantage for a lab that was using X-ray crystallography to try to discover how the structure of a molecule determines its functions: it was easy to get it to crystallize.12
Wiedenheft was able to isolate the Cas1 gene from bacteria and then clone it. Using a vapor diffusion, he was then able to crystallize it. But he was stymied when he tried to figure out the exact crystal structure because he did not have enough experience in using X-ray crystallography.
Doudna drafted Jinek, who had just finished publishing a paper with her on RNA interference,13 to help Wiedenheft with the crystallography. Together they went to the particle accelerator at the nearby Lawrence Berkeley National Laboratory, and Jinek helped analyze the data in order to build an atomic model of the Cas1 protein. “In the process, I got infected by Blake’s enthusiasm,” he recalls. “After that, I decided to stay involved with the CRISPR part of Jennifer’s lab.”14
They discovered that Cas1 has a distinct fold, indicating that it is the mechanism that bacteria use to cleave a snippet of DNA from invading viruses and incorporate it into their CRISPR array, thus being the key to the memory-forming stage of the immune system. In June 2009, they published their discovery in a paper that was the Doudna Lab’s initial contribution to the CRISPR field. It was the first explanation of a CRISPR mechanism based on a structural analysis of one of its components.15
Rodolphe Barrangou
Philippe Horvath
CHAPTER 12 The Yogurt Makers
Basic research and the linear model of innovation
Historians of science and technology, including myself, often write about what is called the “linear model of innovation.” It was propagated by Vannevar Bush, an MIT engineering dean who cofounded Raytheon and during World War II headed the U.S. Office of Scientific Research and Development, which oversaw the invention of radar and the atom bomb. In a 1945 report, “Science, the Endless Frontier,” Bush argued that basic curiosity-driven science is the seed corn that eventually leads to new technologies and innovations. “New products and new processes do not appear full-grown,” he wrote. “They are founded on new principles and new conceptions, which in turn are painstakingly developed by research in the purest realms of science. Basic research is the pacemaker of technological progress.”1 Based on this report, President Harry Truman launched the National Science Foundation, a government agency that provides funding for basic research, mainly at universities.
