Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing

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.”

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.

In 1974, a former Nature editor named Benjamin Lewin launched a journal called Cell, devoted to “the molecular biology of cells and their viruses.” Whereas Nature and Science printed on gossamer-thin paper the consistency of a toilet roll, Cell looked and felt like a fashion magazine for science, with a thick cover, sensual glossy paper, and a heretical Helvetica logo and typeface. Whereas Nature editors would typically squeeze authors down to fewer than 1,000 words for their reports, Lewin gave scientists free rein to lay out and discuss their results without arbitrary page limits. And Lewin, who authored a string of successful textbooks on genetics, knew the science and befriended the scientists. He set up shop in Harvard Square, where top scientists from Harvard and MIT would often hand deliver their latest manuscripts. And although Cell was a biweekly publication, in the ultracompetitive world of molecular biology, Lewin routinely scooped his rivals. In 1990, someone composed a Cell parody¹⁵ and distributed it by fax. It was called Cool.^I Lewin sold his company Cell Press to the Dutch publishing giant Elsevier in 1999 for more than $100 million.

Šikšnys was proud of his Cas9 results, which he concluded “pave the way for the development of unique molecular tools for RNA-directed DNA surgery.” He submitted the paper, with Barrangou and Horvath as co-authors, to Cell in March 2012, but soon regretted the decision. Within a week, the editor had rejected the paper without consulting any outside experts, doubtful that the story was of “sufficient general interest.” Šikšnys was upset. “We thought that it was a big thing because we showed in this paper that in principle you can reprogram this Cas9 to [cut] any sequence.” He resubmitted to Cell’s sister journal, Cell Reports—a notch lower on the prestige pole—but Cell’s song remained the same. The clock was ticking.¹⁶

By now it was May: Šikšnys resubmitted to the Proceedings of the National Academy of Sciences—a famous journal but lacking the wow factor of the CNS glamour journals. He addressed the cover letter to a member of the editorial board whom he felt would have the most expertise to appraise his manuscript: one Jennifer Doudna. But by the time his revised paper was finally published¹⁷ in September 2012, Šikšnys was yesterday’s news. It was an important paper in its own right, but didn’t have the single-guide RNA that Jínek had developed. His results were a day late and a dollar short, and while the experiments were conducted around the same time as (if not before) Jínek, the belated publication date made the study look merely like a confirmation of the Doudna-Charpentier breakthrough.

“These two papers, if you look side by side, look nearly identical,” Šikšnys told me. “The only thing I think that they showed [beyond our paper] is that they can use this single-guide RNA.” Unfortunately, PNAS took more than three months to review and publish the report. The rejection by Cell in itself was not a problem, says Horvath. “The problem appeared when we saw the [Jínek et al.] Science paper!” Would history have been different if his paper had been published in Cell? Horvath shrugged: “Nobody knows,” he said. “Nobody will ever know.”

Over the past few years, the CRISPR narrative has been shaped around the dream team partnership between Charpentier and Doudna. As the profile and fame of the two women grew, Šikšnys was, except for CRISPR insiders, the forgotten man. That began to change following an insightful article in WIRED by Sarah Zhang, who spotlighted the Lithuanian’s lament.¹⁸ Šikšnys earned some belated international recognition when he shared the 2018 Kavli Prize with his two illustrious peers.

After their “immortal” 2012 study, the transatlantic collaboration between Charpentier and Doudna devolved naturally. Jínek was busy juggling his research with job interviews. Charpentier was relocating from Sweden to Berlin. Chyliński was writing up his PhD thesis. Charpentier joined the Berkeley team on a follow-up report in 2014, but all good things come to an end. “We didn’t decide to end the collaboration,” Jínek recalls. “I’d enjoyed it immensely. We made a really good team.”

After almost six years with Doudna, Jínek took a faculty position in Zurich in early 2013, where he continues to study the finer details of Cas9 and other DNA-cutting enzymes. In 2016, he won a prestigious Swiss prize for young biochemists, named after the physician Friedrich Miescher, the father of DNA. In 1868, Miescher set up a lab in Tübingen Castle, where he studied white blood cells in pus isolated from surgical bandages collected from a nearby clinic. Miescher extracted a substance from the cell nuclei that he first described in a letter in February 1869. He dubbed the substance, which “[does] not belong to any known type of protein,” nuclein.¹⁹ Almost 150 years later, Jínek won the Miescher Prize for his seminal role in laying the groundwork for editing nuclein.

And yet, Jínek’s vital contribution to the CRISPR story remains underappreciated. Jínek is humble to a fault, not one to draw attention to himself. When he was asked after a lecture in Germany what he considered the keys to his breakthrough, he offered three reasons. First was the old “we stand on the shoulders of giants” quote.^II Nobody does science in a vacuum, he said. Second was stressing the value of curiosity, the fundamental importance of basic research, and the freedom to pursue the unexpected. Jínek’s goal with Doudna was simply to understand how RNA-targeting enzymes work. “I trained as a structural biologist. I think very deeply about how molecules work,” he said. “I get very excited when I see molecules like this!” he said, gesturing to a cartoon of Cas9 and drawing laughter.

Some politicians take pleasure in cherry-picking federal research grants (“sexual preferences in fruit flies,” etc.) as evidence of wasteful government spending. But the CRISPR gene-editing discovery and the scientific, medical, and economic bounty it has delivered, would not have happened but for public funding of unfashionable research conducted by diehard microbiologists, evolutionary biologists, biochemists, and structural biologists studying CRISPR purely for the thrill of discovery, not the lure of money or prizes. Fundamental and applied research are not “two spigots that can be operated independently,” says Stuart Firestein, a professor at Columbia University. “They are one pipeline, and our job is to keep it flowing.”²⁰

Jínek’s third key to success was serendipity—being in the right place at the right time. “I was very privileged to be in a great academic environment, where we were encouraged to think about our projects, to ask the right questions, to think outside the box, where we had the freedom to explore ideas, to work with other people, exchange ideas, and be quite pragmatic about the way we worked.” The rest, he said, was just hard work: 10 percent inspiration, 90 percent perspiration.

In December 2018, Jínek was invited back to his hometown to give a TED-style talk in his native language.²¹ He spoke about the rapid progress in DNA sequencing, the $1,000 genome (a shout-out would have been nice!) and the diagnosis of genetic diseases. Now, thanks in part to his work, we have the ability to rewrite genes and transform medicine. To illustrate gene editing, he used a famous Czech nursery rhyme, Skakal pes pres oves (“the dog was hopping over the wheat”). To explain how bacteria capture the signatures of viruses in the CRISPR array, he showed a photo of an ockovaci prukaz—an international vaccination booklet containing stamps for various immunizations. As for germline editing, regulation had to be a global decision, not nations or researchers alone but the entire human race.

The Doudna-Charpentier Science paper was the impetus for several groups to demonstrate CRISPR-Cas gene editing in living cells. Doudna was at an initial disadvantage. Her specialty was RNA and structural biology, not cell biology and human genetics. Other groups clearly had the edge in expertise and materials. But Jínek was optimistic. After all, researchers had succeeded in using bacterial DNA-cutting enzymes at the heart of the ZFN and TALEN systems. Some engineering would definitely be required—adding a nuclear localization signal to ferry the Cas9 complex into the nucleus, and making some subtle changes to the DNA sequence (a process called codon optimization) to better fit a mammalian cell. But a priori, Jínek reckoned there was “nothing fundamentally different or any impediment to getting it to work in mammalian cells.”

On October 3, Doudna’s inbox chimed with a message that supported Jínek’s belief. The sender was Jin-Soo Kim, a leading molecular biologist in South Korea. His lab had been pursuing CRISPR editing since Doudna and Charpentier’s “seminal paper” and was preparing to submit a new report on “Genome editing in mammalian cells.” Kim generously asked if Doudna (and Charpentier) would be interested in publishing together. “I do not wish to scoop you because your Science paper prompted us to start this project,” Kim wrote. But he wasn’t interested in getting scooped, either.²²

Six weeks later, Church likewise emailed Doudna and Charpentier “a quick note to say how inspiring and helpful” he had found their CRISPR paper. “I’m sure you have received similar appreciative comments from other labs.”²³ Church divulged that he was trying to get CRISPR working in human stem cells, only adding to the stiffening competition. Doudna felt the pressure of growing demands and responsibilities. “My inbox was exploding and journal editors were calling me,” she recalled. “It was just crazy. You could see this tidal wave coming toward you.”²⁴

Jínek worked diligently in his final few months in Doudna’s lab to prove his hunch. On December 15, 2012, Doudna and Jínek sent their latest manuscript to the online journal eLife, funded in part by HHMI. The rhetorical question posed in the opening of their paper—could the bacterial Cas9 system work in eukaryotic cells?—had been answered in the affirmative. Given the evident competition from Church, Kim, and who knows who else, Doudna was hoping for a quick decision. She got it. On January 3, 2013, the eLife editor emailed her to say her paper had been accepted. Both referees, one of whom revealed himself to be Dana Carroll, loved the work. The editor called the manuscript “excellent” and concluded that the ease of programmability meant that CRISPR would probably supplant ZFNs and TALENs for genome editing. Doudna’s results would “likely have a transformative impact in the field of genome engineering for human and many other species with complex genomes.”²⁵

But Doudna’s delight was short-lived. That afternoon she received another email:²⁶

Greetings from Boston and happy new year!

I am an assistant professor at MIT and have been working on developing applications based on the CRISPR system. I met you briefly during my graduate school interview at Berkeley back in 2004 and have been very inspired by your work since then.

Our group in collaboration with Luciano Marraffini at Rockefeller recently completed a set of studies applying the type II CRISPR system to carry out genome editing of mammalian cells. The study was recently accepted by Science and it will be publishing [sic] online tomorrow. I have attached a copy of our paper for your review.

The Cas9 system is very powerful and I would love to talk with you sometime. I am sure we have a lot of synergy and perhaps there are things that would be good collaborate [sic] on in the future!

Very best wishes,

Feng

The Boston bombshell came from Feng Zhang at the Broad Institute. Just like the double helix, the story of CRISPR genome editing has a complementary strand, the twists and turns of which are still being unraveled.

I. Cool’s scope of articles read: “Cool only publishes articles that it deems to be astonishingly cool beyond belief. Any dorky shit submitted will be returned immediately to the authors postage due; there is just too much cool shit submitted (mostly by Cool Dudes) to waste our precious thick glossy sexy stock on it.”

II. Scientists do love to trot out Isaac Newton’s famous quote about “standing on the shoulders of giants” to acknowledge the scientific contributions of their colleagues and predecessors, although there is a school of thought that says Newton was actually trolling his diminutive rival, Robert Hooke.