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 In early 1953, Perutz shared a Medical Research Council report containing Rosalind Franklin’s unpublished DNA crystallography data with Crick and Watson.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.
CHAPTER 5 DNA SURGERY
In March 2011, Doudna and Charpentier met for the first time at a small conference at the InterContinental San Juan in Puerto Rico, hosted by the American Society for Microbiology. The theme was “Regulating with RNA in Bacteria.” After both women had given their respective talks, Charpentier suggested they take a stroll through Old San Juan. In some respects, they were a study in opposites: one tall with fair hair, the other a shorter brunette. Charpentier was the junior scientist in several respects—five years younger, with a relatively modest publication record, working at a remote Swedish university. Doudna, by contrast, had trained under two Nobel laureates, was a full professor and approaching her fifteenth year as an HHMI investigator. She had also been an author on almost 20 research articles in the top three science journals, compared to just a couple for Charpentier.
As they ambled down cobblestone streets, Charpentier shared results from her upcoming Nature paper—her first as a group leader in the journal. The lab work was led by a Master’s student, Elitza Deltcheva, and a Polish grad student, Krzysztof Chyliński.1 After years of perseverance, she had identified the critical role of tracrRNA in the weaponizing of the CRISPR antivirus defense mechanism. Now she needed help in deducing the role of Cas9 in S. pyogenes and offered a collaboration. Charpentier had identified several different Cas9 proteins from different bacteria, but they always worked with a duplex of RNAs—the crRNA, corresponding to the spacer sequence, and tracrRNA. Sorting out the enzyme’s 3D structure was crucial because “if one wanted to reduce the system into practice, then the structural biology might bring clues to shorten the proteins and do some protein engineering.” There was no doubt this was Doudna’s domain.
The two scientists hit it off. “I really liked Emmanuelle,” Doudna said. “I liked her intensity. I can get that way, too, when I’m really focused on a problem. It made me feel that she was a like-minded person.”2 And Doudna had published her first papers on CRISPR by this time.
Back in Berkeley, Doudna persuaded Martin Jínek to work on the Cas9 project with Charpentier’s lab. Jínek began skyping with Chyliński, the grad student, who had stayed in Vienna after Charpentier, who didn’t have tenure, had taken a group leader position in Sweden. Although they could converse in something approximating Polish—Jínek learned the language watching Polish television as a child—the two scientists mostly communicated in English.
The Charpentier lab had tried to purify Cas9 “but it wasn’t working out,” said Jínek. After the Puerto Rico accord, the collaboration was sealed when Charpentier attended the 2011 CRISPR meeting at Berkeley. The team captured the union with a casual group photo on the steps of Stanley Hall on the eastern edge of the Berkeley campus, where Doudna’s lab was located. Jínek stood in the center of the group, all clad in jeans, with Doudna and Charpentier on his right, Chyliński and Ines Fonfara (a Charpentier postdoc) to his left. They could have been a country music quintet posing for the cover of their soon-to-be-platinum debut album.3
The first task was to purify enough Cas9 protein to obtain crystals to analyze the structure using X-ray crystallography. Jínek’s interest wasn’t so much genome editing at this stage but understanding the molecular mechanism of how Cas9 works. “It’s a system that uses guide RNAs but most likely targets DNA,” he said. “There are parallels with RNA interference, but it’s not RNA that’s being targeted.” This just added to Jínek’s interest in demonstrating that Cas9 was acting as an RNA-guided DNA cutter. Perhaps this wasn’t headline news but it should provide a stimulating swansong for his postdoctoral fellowship before heading back to Europe.
Working with a summer student, Jínek purified Cas9 from S. pyogenes samples shipped from Europe. Wilson remembers Jínek being quite secretive. He gave the precious Cas9 plasmid DNA an inscrutable lab name, MJ923. “This is how he catalogued things until it was safe to talk about,” said Wilson. Jínek’s first experiments using crRNA alone to target DNA failed. That all changed when Jínek added the tracrRNA to the mix.
Jínek’s breakthrough—although he would be loath to use such dramatic language—was fusing the crRNA and tracrRNA, both required for gene targeting, into a single chimeric RNA. This was actually a fairly standard procedure, Jínek reminds me. As a biochemist, he was always looking for the minimal requirements in any reaction or system, attempting to break down the components in a very reductionist fashion. If both RNAs were part of a duplex, then presumably the two ends must be in close proximity to each other. “Then you can stitch them together with a loop,” Jínek said. Those sorts of permutations were not uncommon in the Doudna lab. For example, adding or removing a base from the end of an RNA molecule can dramatically alter the ability to form crystals. Jínek found he could trim the crRNA from one end and likewise truncate the tracrRNA. But he had to maintain a degree of base pairing between the two.
Jínek modestly describes the eureka moment thus: “We had a brainstorming session with Jennifer,” but then clarifies that by “we,” he really meant him. As he sketched the CRISPR parts list on the whiteboard in Doudna’s office, they sensed that by synthetically fusing the two essential RNA molecules, forming a single guide RNA (sgRNA) molecule, they would in principle have the ability to preprogram any guide sequence. In other words, they could specify and target any gene of interest, not just naturally occurring viral sequences. All that was required was to design a custom RNA sequence of some twenty bases that matched the desired target sequence. Doudna remembers it being a “transformative” moment, although when she initially mentioned it to a few Berkeley colleagues, they couldn’t see what all the fuss was about.4 “Krzysztof and Emmanuelle were brought on board shortly after that,” Jínek recalls.
It took Jínek a few weeks to make the chimeric RNAs in-house but he quickly demonstrated that the sgRNA system was able to cut DNA at a matching sequence. Suddenly CRISPR had the makings of another gene-editing technology, in the same toolbox as TALENs and ZFNs. Things moved quickly after that. Jínek presented his results at an internal lab meeting, a weekly gathering in which Doudna’s students and postdocs took turns to present their latest results. Wilson doesn’t recall too many fireworks when Jínek presented his sgRNA results, but he does remember asking if they could use this method for RNA interference. Doudna replied, “We could use this for something better, like genome editing.”