The Code Breaker, page 13
So far Doudna’s collaboration with Charpentier had produced two significant advances. The first was the discovery that the tracrRNA played an essential role not just in creating the crRNA guide but, more important, holding it together with the Cas9 enzyme and binding it all to the target DNA for the cutting process. The second was the invention of a way to fuse these two RNAs into a single-guide RNA. By studying a phenomenon that evolution had taken a billion or so years to perfect in bacteria, they turned nature’s miracle into a tool for humans.
* * *
On the day that she and Jinek brainstormed how to engineer a single-guide RNA, Doudna explained the idea to her husband over dinner. Realizing that it would have implications for a possible patent on gene-editing technology, he told her that she needed to have it written up fully in the lab notebook and witnessed. So Jinek went back to the lab that night and wrote a detailed description of their concept. It was close to 9 p.m., but Sam Sternberg and Rachel Haurwitz were still there. Lab notebooks have witness signature lines at the bottom of each page in order to document important advances, and Jinek asked both of them to sign. Sternberg had never been asked to do that before, so he realized that it was a historic evening.5
CHAPTER 18 Science, 2012
When it came time to write a scholarly paper describing CRISPR-Cas9, Doudna and her teammates used the same round-the-clock collaborative methods they had employed in their experiments. The manuscript was shared in Dropbox, with each of their changes tracked in real time. Jinek and Doudna worked during the day in California, handed things off with a late-night Skype call as dawn was breaking in Europe, and then Charpentier and Chylinski would take the lead for the next twelve hours. Because the sun never set in Umeå during the spring, Charpentier announced she could work any hour of the day. “You can’t really sleep much when it’s light all the time,” she says, “and you’re never really tired in those months, so I was on duty at any time.”1
On June 8, 2012, Doudna hit the Send button on her computer to submit the manuscript to the editors of the journal Science. It listed six authors: Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer Doudna, and Emmanuelle Charpentier. An asterisk next to the names of Jinek and Chylinski noted that they had contributed equally. Doudna and Charpentier were listed last because they were the principal investigators leading the labs.2
The 3,500-word paper went into great detail on how the crRNA and the tracrRNA worked to bind the Cas9 protein onto the target DNA. It also showed how the structure of two Cas9 domains determined how each cut one of the DNA strands at a specific location. Finally, it described how they were able to fuse the crRNA and tracrRNA to engineer a single-guide RNA. This system, the authors noted, could be used to edit genes.
* * *
When the editors of Science received the paper, they were excited. Although many of the activities of CRISPR-Cas9 in living cells had been described before, it was the first time researchers had isolated the essential components of the system and discovered their biochemical mechanisms. In addition, the paper contained a potentially useful invention: the single-guide RNA.
At Doudna’s urging, the editors fast-tracked the review process. She knew that other papers on CRISPR-Cas9, including one from a Lithuanian researcher (more on him in a moment), were already circulating, and she wanted to make sure that her team was the first to publish. The editors at Science had their own competitive motivation: they didn’t want to be scooped by a rival journal. They asked CRISPR pioneer Erik Sontheimer to be one of the reviewers and told him he would have to get his comments back in two days, an unusually fast turnaround. He declined the assignment because he was doing his own work on the topic, but the journal’s editors were able to find others to review the paper quickly.
The reviewer comments contained only a few requests for clarification. There was one significant issue that they did not raise. The experiments looked at the CRISPR-Cas9 system of Streptococcus pyogenes, a common bacteria that can cause strep throat. Like all bacteria, it is a single-cell organism without a nucleus. But the paper suggested that the CRISPR-Cas9 system could be useful for gene editing in humans. Charpentier thought that would prompt some questions. “I was thinking that the reviewers would ask if there was any evidence that it worked in human cells,” she recalls. “But they never raised that, even after the conclusion I wrote saying that it would be an alternative to existing gene-editing methods.”3
* * *
The Science editors approved the revisions and formally accepted the paper on Wednesday, June 20, 2012, just as participants were gathering in Berkeley for the annual CRISPR conference. Charpentier had arrived from Umeå and Chylinski from Vienna a few days early so they could be together for the final proofreading and edits. “Krzysztof arrived jet-lagged,” Charpentier recalled, “but that was not the case for me because I had been in Umeå where it had been light all the time and I hadn’t been sleeping in a rhythm.”4
They gathered in Doudna’s seventh-floor office and watched on her computer as the final PDF files and graphics were uploaded into the journal’s online system. “The four of us were sitting in the office watching the status indicators for the uploads,” Jinek recalls, “and there was a lot of excitement when the last one reached one hundred percent.”
Once the final revisions were submitted, Doudna and Charpentier sat together, just the two of them, in Doudna’s office. It had been only fourteen months since they had first met in Puerto Rico. As Charpentier admired the view of the late afternoon sun setting over San Francisco Bay, Doudna spoke of how pleasant it had been collaborating with her. “It was a glorious moment when we finally got to share in person the joy of discovery and also some personal confidences,” Doudna recalled. “We got to take a breath and talk about how hard we’d worked together across thousands of miles.”
When the talk turned to the future, Charpentier indicated she was interested in returning to a focus on the basic science of microbes rather than making tools for gene editing, and she confided that she was ready to move labs again, probably to the Max Planck Institute in Berlin. Doudna asked, somewhat teasingly, whether she would ever want to settle down, get married, have children. “She said she didn’t want that,” Doudna recalled. “She said she enjoyed being alone and treasured her private time and was not looking for that kind of companionship.”
That evening, Doudna organized a celebratory dinner at Chez Panisse, the Berkeley restaurant where chef Alice Waters pioneered farm-to-table cuisine. Not yet a celebrity outside the rarefied realms of science, Doudna was unable to get a reservation at the fancier downstairs dining room, but she got a long table at the more casual upstairs café. They ordered champagne and toasted what they knew would be a new era in biology. “We felt like we were at the beginning of this intense time when the science was all coming to fruition, and we were thinking about what the implications were,” Doudna recalls. Jinek and Chylinski left before dessert. They had to work that night on the slides for the presentation they would make at the conference the next day. On their walk back to the lab, in the last glow of twilight, Chylinski indulged in a cigarette.
Virginijus Šikšnys
Krzysztof Chylinski
Martin Jinek
CHAPTER 19 Dueling Presentations
Virginijus Šikšnys
Virginijus Šikšnys of Vilnius University in Lithuania is a mild-mannered biochemist with wire-rimmed glasses and a shy smile. He studied organic chemistry at Vilnius, got his doctorate at Moscow State University, then returned to his native Lithuania. He became intrigued by CRISPR when he read the 2007 paper by the Danisco yogurt researchers Rodolphe Barrangou and Philippe Horvath showing that CRISPR was a weapon that bacteria acquired in their struggle to fight off viruses.
By February 2012, he had produced a paper, with Barrangou and Horvath as secondary authors, that described how, in a CRISPR system, a Cas9 enzyme was guided by a crRNA to cut up an invading virus. He sent it off to the journal Cell, which summarily rejected it. In fact, the journal did not deem the paper interesting enough to send out for peer review. “Even more frustrating, we sent it to Cell Reports, which is kind of a sister journal to Cell,” Šikšnys says. “They rejected it too.”1
So his next attempt was to send it to PNAS, the publication of the U.S. National Academy of Sciences. One expedited path to be accepted by PNAS is for a research paper to be approved by a member of that academy. On May 21, 2012, Barrangou decided to send an abstract of the article to the member who was most familiar with the field: Jennifer Doudna.
Doudna was just finishing her paper with Charpentier, so she recused herself. She read only the abstract, not the full paper. But reading the abstract was enough for her to learn that Šikšnys had discovered many of the mechanisms of how, as the abstract said, “DNA cleavage is executed by Cas9.” The abstract also declared that this could lead to a method for editing DNA: “These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.”2
The fact that Doudna subsequently hurried to push her own team’s paper into print would cause a small controversy, or at least a few raised eyebrows, among some members of the CRISPR crowd. “You should look at the timing of Jennifer’s patent filing and the submission of her paper to Science,” Barrangou told me. At first glance, it can look suspicious. Doudna got Šikšnys’s abstract on May 21, and she and her colleagues filed a patent application on May 25 and submitted their paper to Science on June 8.
In fact, the Doudna team’s patent application and paper had been in the works well before she got Šikšnys’s abstract. Barrangou emphasizes he is not accusing Doudna of doing anything wrong. “It was not improper or even unusual,” he says. “It’s not like she stole anything. We sent it to her. We can’t blame her. This is how science is accelerated, when you know that it’s a competitive situation. It gives you an impetus to push the process.”3 As it turned out, Doudna remained friendly with both Barrangou and Šikšnys. Their mix of competition and cooperation were part of a process they all understood.
There was, however, one rival who did question Doudna’s haste: Eric Lander, director of the Broad Institute at MIT and Harvard. “She tells the Science editors that they have competition, she races the paper in, and Science rushes the reviewers,” he says. “The whole thing gets done in three weeks, and so she scoops the Lithuanians.”4
I find Lander’s implied criticism of Doudna interesting, even a bit amusing, because he is one of the most cheerfully competitive people I know. The fact that he and Doudna are both very comfortable with being competitive has, I suspect, made their rivalry more intense. But I also think it meant that they understood each other, in the way that the two rivals in C. P. Snow’s novel The Masters were able to understand each other better than any outsider could. Lander told me over dinner one night that he had the emails Doudna sent to the editors of Science that proved she pushed them to hurry her 2012 paper into print after she saw an abstract of Šikšnys’s paper. When I ask Doudna about this, she readily agrees that she told the editors of Science there was a paper being submitted to a competing journal and requested that the reviewers accelerate their process. “So what?” she says. “Ask Eric if he’s ever done that.” So the next time I have dinner with Lander, I tell him that Doudna wanted me to ask him that question. He pauses, laughs, and then merrily concedes, “Of course I have. It’s how science works. This is completely normal behavior.”5
Šikšnys presents
Barrangou was one of the organizers of the June 2012 CRISPR conference in Berkeley, the one that Charpentier and Chylinski had flown over to attend, and he invited Šikšnys to present his work there. This set the stage for a face-off between the two teams that were racing to describe the CRISPR-Cas9 mechanisms.
Both Šikšnys and the Doudna-Charpentier team were scheduled to present their work on the afternoon of Thursday, June 21, the day after Doudna uploaded the final version of the Science article and went with her colleagues to celebrate at Chez Panisse. Barrangou had decided, even though Šikšnys’s work had not yet been accepted for publication, that he should present first, followed immediately by the presentation of the Doudna-Charpentier team.
In the annals of history, the priority had been sealed: the Doudna-Charpentier paper had already been accepted by Science and would be published online June 28, while Šikšnys would not get published until September 4. Nevertheless, Barrangou’s decision to let Šikšnys be the first to present at the Berkeley conference had the potential to give him a small claim to some of the glory—if his research turned out to match or exceed that of the Doudna-Charpentier team. “I was in charge of the order of speakers,” Barrangou says. “I got a request from someone in Jennifer’s lab to move their talk to before Virginijus. I rejected that. Virginijus had sent his paper to me first, back in February when we were trying to get it published in Cell, and I thought it would be fair for Virginijus to present first.”6
* * *
So just after lunch on Thursday, June 21, Virginijus Šikšnys gave a slide presentation, based on his unpublished paper, in the seventy-eight-seat ground-floor auditorium of Berkeley’s new Li Ka Shing Center, where the conference was being held. “We isolated the Cas9-crRNA complex and demonstrated that in vitro it generates a double-strand break at specific sites in target DNA molecules,” he announced. He went on to say that this system could someday become a gene-editing tool.
There were, however, some gaps in the Šikšnys paper and presentation. Most notably, he spoke of the “Cas9-crRNA complex” and made no mention of the role of tracrRNA in the gene-cutting process. Although he described the tracrRNA role in creating the crRNA, he did not realize that it was necessary for this molecule to stick around in order to bind crRNA and Cas9 onto the DNA site targeted for destruction.7
For Doudna, this meant that Šikšnys had failed to discover the essential role played by the tracrRNA. “If you don’t know that the tracrRNA is required for DNA cutting,” she later said, “there is no way you could implement it as a technology. You haven’t defined what the components are to get it to work.”
There was competitive tension in the air, and Doudna was intent on making sure that Šikšnys’s lapse involving the role of tracrRNA was highlighted. She was seated in the third row of the auditorium, and as soon as Šikšnys finished she raised her hand. Does your data, she asked, show the role of the tracrRNA in the cleaving process?
At first Šikšnys did not engage on the point directly, so Doudna kept pressing him to clarify. He did not try to refute her. “I remember there was a hint of debate in the discussion that followed Jennifer’s question, and she was very firm in making her voice heard that the tracrRNA was an essential part that was overlooked in the work that Virginijus presented,” says Sam Sternberg. “He did not disagree, but neither was there a full admission that he had missed it.” Charpentier was likewise surprised. After all, she had written about part of the tracrRNA role in 2011. “What I don’t understand is why Šikšnys, after reading my 2011 paper, did not look further into the role of tracrRNA,” she says.8
To be fair, Šikšnys deserves a lot of credit, which I hope I’ve given him, for making many of the biochemical findings at about the same time as Doudna and Charpentier. Perhaps I have put a bit too much focus on the role of the tiny tracrRNA, both because I’m writing the book from Doudna’s vantage point and because she emphasized it in many of our interviews. But I actually do think it’s important. In explaining the amazing mechanisms of life, little things matter. And very little things matter a lot. Showing precisely the essential role of the two snippets of RNA—the tracrRNA and the crRNA—was key to understanding fully how CRISPR-Cas9 could be a gene-editing tool and how the two RNAs could be fused together to create a simple single guide to the right gene target.
Wow
Immediately after Šikšnys finished, it was time for Doudna and Charpentier to deliver what most attendees by then knew was a set of big breakthroughs. The two sat next to each other in the audience, having decided that the presentation would be made by the postdocs who had done most of the hands-on experiments, Jinek and Chylinski.9
When the presentation was about to begin, two Berkeley biology professors walked in with some of their postdocs and students. Doudna had been talking to them about collaborating on getting CRISPR-Cas9 to work in humans, but most of the other participants did not know who they were. Sternberg guessed they were patent lawyers. Their appearance heightened the sense of drama. “I remember people being surprised as a dozen or so unknown people filed in,” Doudna says. “It was sort of a heads-up that something special was about to happen.”
Jinek and Chylinski tried to make their presentation fun. They had prepared the slides so that they could take turns explaining each of the experiments they had done, and they had practiced twice before their appearance. The audience was small, informal, and friendly. Nevertheless, it was very clear they were nervous, especially Jinek. “Martin was very stressed, which made me stressed for him,” Doudna says.
There was no need to be nervous. The presentation was a triumph. Sylvain Moineau, a CRISPR pioneer at the University of Laval in Quebec, stood up and said, “Wow!” Others hurriedly emailed and texted their lab colleagues back home.
Barrangou, the Danisco researcher who had been a collaborator on Šikšnys’s paper, later said that, as soon as he heard the presentation, he knew that Doudna and Charpentier had taken the field to a whole new level. “Jennifer’s paper was clearly so much better than ours,” he admits. “It wasn’t close. It was the tipping point that moved the CRISPR field from an idiosyncratic interesting microbial-world feature to a technology. So Virginijus and I, we had no hard feelings whatsoever.”
