The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race

Charpentier and Doudna’s collaboration worked like a model United Nations, with Hawaii-raised Doudna and Czech-born Jinek working from Berkeley, Parisian Charpentier from Sweden, and Polish-born Chylinski from Vienna. Tasked with making the Cas9 enzyme guide crRNA to chop up a virus in vitro, Jinek and Chylinski met initial failure. They seemed to be missing a crucial piece in their two-component experiment. But when Chylinski threw tracrRNA into the test-tube mix, the three-component mix immediately sliced the viral DNA. Realizing their team was on the verge of a major discovery, Doudna and Charpentier intensified their collaboration. Soon, the foursome had worked out the precise mechanisms of each of the three components of the CRISPR-Cas9 system.

The crRNA contained a 20-letter sequence that acted as a sort of GPS to guide the complex to DNA with a similar sequence. The tracrRNA, which helped form the crRNA, now acted as a scaffold to keep the other components in place as they zeroed in on the target DNA. Finally, the Cas9 enzyme chopped up the DNA. So, the tracrRNA had not one but two functions in the CRISPR system. Further, the system could be made to cut any chosen DNA sequence by adding a different guiding crRNA, making it widely applicable.

Could CRISPR be made even cheaper and easier to use? Jinek and Doudna brainstormed over the minimum amount of crRNA and tracrRNA required to make the system work. They discovered that each of the RNAs could be truncated a bit and still function. They also discovered that the RNAs could even be linked in a way that kept the combined molecule functional, by strategically fusing the tail of one to the head of another. This engineered single RNA molecule would have the guide information (crRNA function) at one end and the clamping handle (tracrRNA function) on the other. The night she and Jinek conceptualized the single-guide RNA, Doudna wrote up the detailed concept in her lab notebook in the presence of Sternberg and Haurwitz. Meanwhile, with Chylinski’s help, Jinek immediately began working on fusing the two RNAs. Within three weeks, they had engineered a working single-guide RNA.

Doudna and her team worked around the clock to submit their findings to the journal Science. The paper was submitted on June 8, 2012, and listed six authors: Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer Doudna, and Emmanuelle Charpentier. Per convention, Doudna and Charpentier’s names were listed last because they were the principal investigators of their respective labs. The editors of Science were excited by the paper’s findings; it was the first time researchers had isolated the basic components of the CRISPR-Cas9 system and described the biochemical mechanism of each. It was also the first time a single-guide RNA had been described.

At Doudna’s request, the paper was fast-tracked. Science was in a hurry to publish the paper so that Doudna’s findings weren’t poached by a rival journal. Though Charpentier expected the editors to question how the single-guide RNA, so far tested only in bacteria, could work in human cells, the query was never raised. As Doudna and Charpentier celebrated their success, Charpentier indicated that she wanted to return to basic microbiology rather than pursue gene-editing tools. Doudna also recalls Charpentier saying she wanted to switch labs to the Max Planck Institute in Berlin.

Doudna and Charpentier were not the only scientists studying CRISPR. Virginijus Šikšnys of Vilnius University in Lithuania became interested in CRISPR after reading the 2007 paper of Danish yogurt scientists Barrangou and Horvath. By 2012, Šikšnys (with Barrangou and Horvath as secondary authors) had submitted a paper to Cell describing how a Cas9 enzyme was guided by crRNA to chop up an attacking virus. When his paper was rejected by Cell and other journals, Šikšnys decided to get an endorsement from Jennifer Doudna, the authority in the field. Since Doudna had competing interests in CRISPR, she only read the abstract of Šikšnys’s paper before recusing herself. Yet the abstract was enough to show Doudna that Šikšnys too had discovered many mechanisms of the Cas9-CRISPR system. Doudna received Šikšnys’s abstract on May 21; her team applied for a patent on CRISPR technology on May 25 and sent their paper to Science on June 8. The seeming hurry caused some scientists, like Eric Lander of the Broad Institute of MIT, to think Doudna wanted to scoop Šikšnys. However, Doudna’s patent and paper had been in the works long before she heard from Šikšnys. Moreover, competition over releasing discoveries first is common in scientific circles.

Both Doudna and Šikšnys presented their findings at a June 2012 CRISPR riven between warring camps. Barrangou, one of the organizers, decided to let Šikšnys present first, since he felt Šikšnys had been piped to the post by Doudna’s publication. When Šikšnys’s presentation made no mention of the latching role of tracrRNA, Doudna, who was in the audience, questioned it openly. And when Jinek and Chylinski presented on behalf of Doudna and Charpentier, they too were grilled over an important point: Eric Sontheimer, one of the first scientists to sense CRISPR’s potential, wanted to know how the single-guide RNA technology would work in human cells.