The Code Breaker, page 12
tracrRNA
In 2009, the year that Charpentier was uprooting from Vienna and moving to Umeå, the CRISPR crowd had coalesced around Cas9 as being the most interesting of the CRISPR-associated enzymes. Researchers had shown that if you deactivated Cas9 in bacteria, the CRISPR system no longer cut up the invading viruses. They had also established the essential role of another part of the complex: CRISPR RNAs, known as crRNAs. These are the small snippets of RNA that contain some genetic coding from a virus that had attacked the bacteria in the past. This crRNA guides the Cas enzymes to attack that virus when it tries to invade again. These two elements are the core of the CRISPR system: a small snippet of RNA that acts as a guide and an enzyme that acts as scissors.
But there was one additional component of the CRISPR-Cas9 system that played an essential role—or, as it turned out, two roles. It was dubbed a “trans-activating CRISPR RNA,” or tracrRNA, pronounced “tracer-RNA.” Remember this tiny molecule; it will play an outsized role in our tale. That’s because science is most often advanced not by great leaps of discovery but by small steps. And disputes in science are often about who made each one of these steps—and how important each really was. This would turn out to be the case for the discoveries involving tracrRNA.
It turns out that tracrRNA performs two important tasks. First, it facilitates the making of the crRNA, the sequence that carries the memory of a virus that previously attacked the bacteria. Then it serves as a handle to latch on to the invading virus so that the crRNA can target the right spot for the Cas9 enzyme to chop.
The process of uncovering these roles of tracrRNA began in 2010, when Charpentier noticed that the molecule kept appearing in her experiments with bacteria. She couldn’t figure out its role, but she realized that it was located in the vicinity of the CRISPR spacers, so she speculated that they were connected. She was able to test this by deleting the tracrRNA in some bacteria. The result was that the crRNAs didn’t get produced. Researchers had never quite pinned down how the crRNAs were made inside a bacterial cell. Now Charpentier had a hypothesis: it is this tracrRNA that directs the creation of the short crRNAs.
Charpentier was moving to Sweden at the time. When the researchers in her Vienna lab sent her an email saying they had shown that the absence of tracrRNA meant that crRNA wasn’t produced, she spent the night drawing up a long plan of experiments for them to do next. “I became obsessed with this tracrRNA,” she says. “I am stubborn. It was important for me to follow up. I said ‘We have to go for it! I want someone to look at it.’ ”5
The problem was that there was nobody in her Vienna lab who had the time and inclination to pursue the tracrRNA. That’s the drawback of being a wandering professor: you leave your students behind, and they move on to other things.
Charpentier considered doing the experiments herself, even though she was in the midst of a move. But she finally found a volunteer in her Vienna lab: a young student from Bulgaria, studying for a master’s degree, named Elitza Deltcheva. “Elitza was very dynamic, and she believed in me,” Charpentier says. “She understood what was happening, even though she was just a master’s student.” She even convinced one of the graduate students, Krzysztof Chylinski, to work with her.
Charpentier’s little team discovered that the CRISPR-Cas9 system accomplished its viral-defense mission using only three components: tracrRNA, crRNA, and the Cas9 enzyme. The tracrRNA took long strands of RNA and processed them into the small crRNAs that were targeted at specific sequences in an attacking virus. They prepared a paper for Nature, which would be published in March 2011, in which Deltcheva got to be the lead author—and the graduate students who had declined to help were lost to history.6
A remaining mystery
Charpentier presented the findings at a CRISPR conference in October 2010 in the Netherlands. She was having trouble getting her paper through the editorial process at Nature, and it was risky to go public with work before it was published. But she thought that perhaps one of the paper’s reviewers would be in the audience and would be convinced to speed up the process.
She was stressed during her presentation because she had not yet figured out what happened to the tracrRNA after it helped to create a crRNA. Was the work of the tracrRNA done by then? Or did the two little RNAs stick together when it came time to guide the Cas protein to cut up an invading virus? One member of the audience asked her directly, “Do the three elements stay together as a complex?” Charpentier tried to deflect the question. “I tried to laugh and to be very confusing on purpose,” she says.
That issue—and what Charpentier knew about it—might seem arcane. But it led to a set of disputes that illuminates how CRISPR researchers—and Doudna in particular—can be very competitive about who deserves credit for each small advance. The fact that the tracrRNA did in fact stick around and play an important role in cleavage would later be among the discoveries published in the seminal 2012 paper that Charpentier would write with Doudna. But to Doudna’s annoyance, Charpentier would sometimes imply, years later, that she already knew this fact in 2011.
When I press her, Charpentier admits that her 2011 Nature paper did not, in fact, describe the full role of the tracrRNA: “It seemed clear to me that the tracrRNA needed to continue to be associated with the crRNA, but there were some details we didn’t fully understand, so we didn’t put this in the paper.” Instead, she made the decision to save writing about the full tracrRNA function until she could find a convincing way to prove it experimentally.
She had studied the CRISPR system in living cells. To get to the next step would require biochemists who could isolate each chemical component in a test tube and figure out precisely how each one works. That is why she wanted to meet Doudna, who was scheduled to speak at the March 2011 conference of the American Society for Microbiology in Puerto Rico. “I knew we were both going to attend,” she says, “and I put in my mind that I would find a chance to talk to her.”
Puerto Rico, March 2011
When Jennifer Doudna walked into the coffee shop of the hotel in Puerto Rico on the second afternoon of the conference, Emmanuelle Charpentier was at a table in the corner sitting by herself, as she often liked to do, looking far more elegant than the other patrons. Doudna was with her friend John van der Oost, the Dutch CRISPR researcher, who pointed Charpentier out and offered to introduce her. “That would be great,” replied Doudna. “I’ve read her paper.”7
Doudna found Charpentier to be charming: just a hint of shyness, or feigned shyness, along with an engaging sense of humor and very stylish aura. “I was instantly struck by her intensity but also her sly humor,” Doudna says. “I immediately liked her.” They chatted for a few minutes and then Charpentier suggested they get together for a more serious discussion. “I’ve been thinking of contacting you about a collaboration,” she said.
The next day they had lunch, followed by a stroll along the cobblestone streets of old San Juan. When the discussion turned to Cas9, Charpentier became excited. “We have to figure out exactly how it works,” she urged Doudna. “What’s the exact mechanism it uses to cut DNA?”
Charpentier was taken by Doudna’s seriousness and attention to detail. “I think it’s going to be fun to work with you,” she told her. Doudna was similarly moved by Charpentier’s intensity. “Somehow, just the way she said that it would be fun to work with me made a chill run down my back,” she recalls. The other enticement was that it was just the sort of detective tale that gave Doudna a sense of purpose: the hunt for the key to one of life’s basic mysteries.
* * *
Right before Doudna left for Puerto Rico, she had a career-counseling conversation with Martin Jinek, the postdoc in her lab who had been working on the structures of Cas1 and Cas6. He was having doubts, which turned out to be unwarranted, about whether he would be successful as an academic researcher, and had thought about becoming an editor at a medical journal instead. But he decided against it. “I think I’m going to be in your lab about one more year,” he told her. “What would you like me to work on?” He was especially interested in finding a CRISPR project of his own, he said.
So when Doudna heard Charpentier’s pitch, she thought it would be a perfect project for Jinek. “I’ve got a wonderful biochemist who’s also a structural biologist,” she told Charpentier.8 They agreed that they would connect Jinek with the postdoc in Charpentier’s lab who had worked on her earlier Cas9 paper, Krzysztof Chylinski, a Polish-born molecular biologist who had stayed in Vienna when she moved to Umeå. Together this foursome would make one of the most important advances in modern science.
CHAPTER 17 CRISPR-Cas9
Success
When Doudna returned to Berkeley, she and Jinek began a series of Skype calls with Charpentier in Umeå and Chylinski in Vienna to plot a strategy for figuring out the mechanisms of CRISPR-Cas9. The collaboration was like a model United Nations: a Berkeley professor from Hawaii, her postdoc from the Czech Republic, a Parisian professor working in Sweden, and her Polish-born postdoc working in Vienna.
“It became a twenty-four-hour operation,” Jinek recalls. “I would do an experiment at the end of my day, I would send an email to Vienna, and Krzysztof would read it as soon as he got up in the morning.” Then there would be a Skype call, and they would decide what the next step should be. “Krzysztof would execute that experiment during the day and send me the results while I was asleep, so that when I woke up and opened my inbox there would be an update.”1
At first, Charpentier and Doudna would join the Skype calls only once or twice a month. But the pace picked up in July 2011, when Charpentier and Chylinski flew to Berkeley for the fast-growing annual CRISPR conference. Even though they had bonded over Skype, it was the first time that Jinek had personally met Chylinski, a lanky researcher with an affable personality and an eagerness to be involved in turning basic research into a tool.2
Emmanuelle Charpentier, Jennifer Doudna, Martin Jinek, and Krzysztof Chylinski at Berkeley in 2012
In-person meetings can produce ideas in ways that conference calls and Zoom meetings can’t. That had happened in Puerto Rico, and it did so again when the four researchers got together for the first time in Berkeley. There they were able to brainstorm a strategy for figuring out exactly what molecules were necessary for a CRISPR system to cut DNA. Physical meetings are especially useful when a project is in an early phase. “There’s nothing like sitting in a room with people and seeing their reactions to things and having a chance to bat around ideas face to face,” Doudna says. “That’s been a cornerstone to every collaboration that we’ve had, even those where we are conducting a lot of the work by electronic communication.”
* * *
Jinek and Chylinski were initially unable to make CRISPR-Cas9 chop up the DNA of a virus in a test tube. They had been trying to make it work with just two components: the Cas9 enzyme and the crRNA. In theory the crRNA would guide the Cas9 enzyme to the virus target, which would then get chopped up. But it didn’t work. Something was missing. “It was extremely puzzling to us,” Jinek recalls.
This is when the tracrRNA reenters our tale. In her 2011 paper Charpentier showed that tracrRNA was required for producing the crRNA guide. She later said that she suspected it played an even larger, ongoing role, though that possibility had not been part of their initial round of experiments. When those experiments failed, Chylinski decided to throw tracrRNA into his test-tube mix.
It worked: the three-component complex reliably chomped up the target DNA. Jinek immediately told Doudna the news: “Without the tracrRNA, the crRNA guide does not bind to the Cas9 enzyme.” After that breakthrough, Doudna and Charpentier became more involved in the daily work. Clearly they were heading to an important discovery: determining the essential components of a CRISPR gene-cutting system.
Night after night, Chylinski and Jinek would ping-pong results back and forth, each adding a tiny bit of the puzzle, with Charpentier and Doudna joining the increasingly frequent strategy calls. They were able to discover the precise mechanisms of each of the three essential components of the CRISPR-Cas9 complex. The crRNA contained a twenty-letter sequence that acted as a set of coordinates to guide the complex to a piece of DNA with a similar sequence. The tracrRNA, which had helped create this crRNA, now had the additional role of acting like a scaffold that held the other components in just the right place when they glommed on to the target DNA. Then the Cas9 enzyme began slicing away.
* * *
One evening, right after a key experiment had produced positive results, Doudna was at home cooking spaghetti. The swirls in the boiling water reminded her of the salmon sperm she had studied under a microscope back in high school when learning about DNA, and she started to laugh. Her son, Andy, who was nine, asked her why. “We found this protein, an enzyme called Cas9,” she explained. “It can be programmed to find viruses and cut them up. It’s so incredible.” Andy kept asking how it worked. Over billions of years, she explained, bacteria evolved this totally weird and astonishing way to protect themselves against viruses. And it was adaptable; every time a new virus emerged, it learned how to recognize it and beat it back. He was fascinated. “It was a double joy,” she recalled, “a moment of fundamental discovery of something that is so cool, and being able to share it with my son and explaining it in a manner where he can get it.” Curiosity can be beautiful that way.3
A gene editing tool
This amazing little system, it quickly became clear, had a truly momentous potential application: the crRNA guide could be modified to target any DNA sequence you might wish to cut. It was programmable. It could become an editing tool.
The study of CRISPR would become a vivid example of the call-and-response duet between basic science and translational medicine. At the beginning it was driven by the pure curiosity of microbe-hunters who wanted to explain an oddity they had stumbled upon when sequencing the DNA of offbeat bacteria. Then it was studied in an effort to protect the bacteria in yogurt cultures from attacking viruses. That led to a basic discovery about the fundamental workings of biology. Now a biochemical analysis was pointing the way to the invention of a tool with potential practical uses. “Once we figured out the components of the CRISPR-Cas9 assembly, we realized that we could program it on our own,” Doudna says. “In other words, we could add a different crRNA and get it to cut any different DNA sequence we chose.”
In the history of science, there are few real eureka moments, but this came pretty close. “It wasn’t just some gradual process where it slowly dawned on us,” Doudna says. “It was an oh-my-God moment.” When Jinek showed Doudna his data demonstrating that you could program Cas9 with different guide RNAs to cut DNA wherever you desired, they actually paused and looked at each other. “Oh my God, this could be a powerful tool for gene editing,” she declared. In short, they realized that they had developed a means to rewrite the code of life.4
A single-guide RNA
The next step was to figure out if the CRISPR system could be made even simpler. If so, it might become not just a gene-editing tool but one that would be much easier to program and cheaper than existing methods.
One day, Jinek walked down the hall from the lab into Doudna’s office. He had been experimenting to determine the minimum requirements for the crRNA that served as a guide and the tracrRNA that clamped it to the target DNA. They were standing at a whiteboard propped in front of her desk, and he was sketching out a diagram of the structure of the two small RNAs. Which parts of the crRNA and tracrRNA, he asked, were essential for cutting up DNA in a test tube? “It appeared that the system had some flexibility as to how long the two RNAs had to be,” he says. Each of the little RNAs could be truncated a bit and still function. Doudna had a profound understanding about the structure of RNA and an almost childlike joy in figuring out the ways it worked. As they brainstormed, it became clear to them that they could link the two RNAs together, fusing the tail of one to the head of the other in a way that would keep the combined molecule functional.
Their goal was to engineer a single RNA molecule that would have the guide information on one end and the binding handle on the other. That would create what they ended up calling a “single-guide RNA” (sgRNA). They paused for a moment and looked at each other, then Doudna said, “Wow.” As she recalls, “It was one of those moments in science that just comes to you. I had this chill and these little hairs on my neck standing up. In that moment, the two of us realized that this curiosity-driven, fun project had this powerful implication that could change the direction of the project profoundly.” It’s a fitting scene to imagine: the behavior of a little molecule being able to get the little hairs on Doudna’s neck to stand up.
Doudna urged Jinek to begin work right away on fusing these two RNA molecules to work as a single guide for Cas9, and he hastened back down the hall to place an order with a company for the necessary RNA molecules. He also discussed the idea with Chylinski, and they quickly designed a series of experiments. Once they had figured out what parts of the two RNAs could be deleted and how they could be connected, it took only three weeks to make a single-guide RNA that worked.
It was immediately obvious that this single guide would make CRISPR-Cas9 an even more versatile, easy-to-use, and reprogrammable tool for gene editing. What made the single-guide system particularly significant—from both a scientific and an intellectual property standpoint—was that it was an actual human-made invention, not merely a discovery of a natural phenomenon.
