In June 2012, Jínek and Chyliński presented their discovery at the annual CRISPR conference, which had returned to Berkeley. Šikšnys presented his own unpublished results, which also demonstrated that Cas9 was a DNA-cutting enzyme. The overall impact “wasn’t revolutionary,” Wilson says, probably because there was no overt mention of gene editing. But Jínek sensed growing excitement. The CRISPR field was poised to move from an obscure branch of molecular microbiology to cutting-edge biotech.
On June 8, 2012, Doudna submitted her paper to Science. Jínek and Chyliński were given joint top billing, while Doudna and Charpentier’s names completed the author list (the standard convention in the natural sciences, reflecting the funders and directors of the work, like the opening credits of a film). Both were listed as co-corresponding authors—an even division of credit. The Science editors moved quickly, accepting the manuscript in a mere twelve days, and posting the article online twenty days after submission.5
The Science paper showed that the CRISPR-Cas9 system was customizable, able to cleave almost any given DNA sequence in a test tube on demand. The nifty sgRNA would enable any other researcher to adapt the system for their own purposes. Showing ample self-restraint, Doudna and Charpentier closed by stating that CRISPR showed “considerable potential for gene-targeting and genome-editing applications.” It was the modern-day equivalent of Crick and Watson’s teasing understatement in 1953: “It has not escaped our notice that the specific [base] pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”
The authors chose their words carefully because these experiments were limited to bacterial DNA—the team hadn’t shown that CRISPR-Cas9 would work in plant or animal cells, including human. Whether that was a formality—E. coli DNA is the same twisty inert molecule as H. sapiens DNA—or a massive technical challenge given the added biochemical complexity of the cell nucleus of humans and other higher organisms, was the $64,000 Question.
The UC Berkeley press office crafted a press release to tout the importance of Doudna’s report. The release trumpeted “potentially big implications for advanced biofuels and therapeutic drugs, as genetically modified microorganisms, such as bacteria and fungi, are expected to play a key role in the green chemistry production of these and other valuable chemical products.”6 But tellingly there was no mention of any clinical applications in humans, which was far beyond the scope of the paper.
Asked to supply a quote on the invention of “programmable DNA scissors,” Doudna’s comments are revealing for their candor and reluctance to overhype the results. “We’ve discovered the mechanism behind the RNA-guided cleavage of double-stranded DNA that is central to the bacterial acquired immunity system,” she said. “Our results could provide genetic engineers with a new and promising alternative to artificial enzymes for gene targeting and genome editing in bacteria and other cell types.” And she added: “Although we’ve not yet demonstrated genome editing, given the mechanism we describe it is now a very real possibility.”
Despite the media outreach, the paper didn’t catch fire beyond the scientific community. The New York Times didn’t see fit to publish an article on CRISPR until 2014.7 Doudna’s hometown San Francisco Chronicle also passed.8 But for insiders, the colliding worlds of CRISPR and genetic engineering sparked genuine interest. Stan Brouns hailed Cas9 as “the Swiss Army knife of immunity.”9 Fyodor Urnov, who coined the term “genome editing,” has no doubt where the Doudna-Charpentier paper ranks in the annals of scientific literature. “I will never forget reading the last paragraph of the…” he pauses, grasping for the right words like a nuclease embracing a guide RNA. “Immortal is a strong word so I’m going to use it carefully: IMMORTAL Science paper, in which they describe [that] Cas9 can be directed.”10
But the overriding question was: Could a bacterial enzyme, hundreds of millions of years old, make the massive evolutionary leap and find its DNA target in the alien surroundings of a eukaryote cell nucleus? Human DNA might have the same four-letter alphabet as bacterial or viral DNA, but in its natural habitat, the double helix in eukaryotic cells is wrapped, bundled, and looped like a garden hose around protein cores in a material called chromatin. Nobody knew for sure how Cas9 would fare with chromatin.
Two experts weighed in on this very issue. Barrangou said that the potential use of the CRISPR-Cas system for genome editing in human, plant, and other complex cells hinged on whether the molecular scissors could cleave chromatin. “Only the future will tell whether this programmable molecular scalpel can outcompete ZFN and TALEN DNA scissors for precise genomic surgery,” he wrote.11 He told me later: “2012 was not genome editing… It’s the CRISPR-Cas9 technology, the single-guide technology.”12
Dana Carroll, a pioneer of ZFNs, agreed. Unlike CRISPR, the known genome editors were derived from DNA-binding proteins active in eukaryotic cells. “There is no guarantee that Cas9 will work effectively on a chromatin target,” Carroll wrote. “Only attempts to apply the system in eukaryotes will address these concerns.”13 In other words, the proof was in the pudding. Carroll concluded: “Whether the CRISPR system will provide the next-next generation of targetable cleavage reagents remains to be seen, but it is clearly well worth a try. Stay tuned.”
Stay tuned is the kind of stock throwaway line that scientists have written hundreds of times in journal reviews and commentaries. This is how science works, by raising more questions than answers. The distinguished Utah biochemist could have no idea that his words would be dissected and parsed not only by fellow scientists but also by armies of patent attorneys sparring over the inventorship of CRISPR gene editing for years to come.
In science, the race to be first to publish a groundbreaking result means the world: acclaim, funding, promotion, tenure, prizes. Every day, researchers place their trust in the editors of the leading biomedical journals. At three of the leading journals—Nature, Science, and Cell (sometimes dubbed the CNS journals)—the editors are full-time professionals, not part-time academics. As Mojica, Vergnaud and many others can attest, unsympathetic, indecisive, or ill-informed editors and reviewers can make mistakes or cause excruciating delays in publishing decisions, often requiring authors to waste months in search of a suitable home for their findings. In early 2012, the publishing gods struck again.
For five years, Šikšnys, the Lithuanian biochemist, had been collaborating with Horvath and Barrangou. After successfully transferring the CRISPR system from S. thermophilus to the lab-friendly E. coli, he surprisingly found that it could still defend against invading DNA, despite the two bacteria species being very distantly related in evolutionary terms.14 The next step was to sequentially strip away each of the four Cas genes adjacent to the CRISPR array and watch what happened next. Removing three of the four genes had no effect on phage defense, but inactivating Cas9 crippled the defense system, like disabling an alarm system. It was a sure sign that Cas9 was the key ingredient in the interference provided by CRISPR. One of Šikšnys’ students, Giedrius Gasiunas, succeeded in isolating active Cas9 in a tube. “I still remember the excitement when he actually did the first experiment,” Šikšnys recalled, cutting DNA in a programmable fashion using the purified Cas9. He had published dozens of papers in good journals, but this was a big story worthy of a very big journal.