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

CHAPTER 7 PRIZE FIGHT

In 1994, I attended a science conference in Philadelphia for my journal, Nature Genetics. During a coffee break, a geneticist named Dennis Drayna took me to one side and whispered that his team at Mercator Genetics had made a thrilling discovery: the gene mutated in patients with hemochromatosis, one of the most common genetic disorders in people of European descent. I said we’d be thrilled to review the paper and would handle it expeditiously. I sent copies by courier to four referees to ensure the results were thoroughly vetted. But a week later, as the faxed reviews landed on my desk, my worst fears were realized: a split decision, two reviewers loved the paper but the other two expressed concerns. In desperate need of a tiebreaker, I called the best man for the job: Eric Lander. He read the paper over a weekend, and transmitted his unequivocal endorsement, which we proudly published in 1995.^I

Lander trained as a mathematician and taught economics at Harvard. He’s a Rhodes Scholar and a MacArthur Genius Award recipient. In the late 1980s, he decided to apply his skills to biology—his brother is a leading neuroscientist—taking a fellowship at the Whitehead Institute in Cambridge, a new flagship research center on the fringe of the MIT campus. His stature grew rapidly in the 1990s, as he helped build the theoretical framework for human gene mapping before helping to lead the international genome project consortium’s response to the Celera threat. Lander wrote the lion’s share of the landmark draft human genome paper published in Nature in 2001.

After you’ve helped to orchestrate the successful effort to sequence the human genome, and quietly cofounded several successful biotech companies, what do you do for an encore? Lander set his sights on building a new biomedical institute (literally overshadowing the Whitehead) affiliated with both Harvard and MIT, anchored by a world-class genome center but venturing into cancer biology, neuroscience, cell biology, chemistry, and eventually CRISPR and genome editing. The philanthropists Eli Broad and his wife Edythe have committed $700 million to Lander’s institute, which the billionaire art collector calls his greatest treasure. In 2008, President Obama named Lander to cochair the White House science council, praising Lander’s work on the Human Genome Project as “one of the greatest scientific achievements in history.”¹

Over the years, like the general manager of a dynastic baseball team, Lander has assembled a team of all-star scientists including top Harvard chemists Stuart Schreiber and David Liu, former Harvard provost Steve Hyman, and Merck’s former head of research, Ed Scolnick. He identified Zhang’s potential before CRISPR became the biggest game in science and Lander could take some personal pride in having this rising phenom under his roof at the Broad.

As the competition for credit and awards heated up, one McGovern Institute executive, Charles Jennings, was concerned that Zhang was in danger of not receiving the recognition he deserved. The press was warming to the story of the Doudna-Charpentier alliance forged in Old San Juan. Jennings took it upon himself to nominate Zhang for his first prize. To be sure, the annual Popular Science magazine “Brilliant Ten” award for scientists and engineers may not rank as one of the world’s most prestigious scientific awards, but Zhang happily joined the class of 2013.² Jennings’s nomination praised Zhang for his work developing gene-editing tools and his willingness to share them widely with his fellow scientists. “These technologies are so fundamental, it’s best to keep them as open as possible,” Zhang said. “If someone had protected the HTML language for making Web pages, then we wouldn’t have the World Wide Web.” Three years later, MIT awarded Zhang tenure. Desimone’s nomination letter to the tenure committee began by saying this was probably the easiest decision the committee would ever have to make.

It is hard to overstate how much the CRISPR world exploded following Zhang’s breakthrough paper in January 2013. Before then, a few dozen CRISPR-related papers were published each year. In the three years following that report, the number rocketed to 3,000. As researchers worldwide gleefully embraced the technology, the competition for prizes and patents intensified.

In January 2016, Cell published an extraordinary perspective by Lander. Titled “The Heroes of CRISPR,” Lander framed this lengthy article as an educational essay paying tribute to the dedicated unsung heroes who had paved the way for the CRISPR gene-editing revolution.³ He wrote eloquently, more in the style of a New Yorker piece than a typically turgid scientific review.

But some suspected that Lander was using this platform to spin the accomplishments of his protégé. The cues were apparent from the opening line of the abstract: “Three years ago [in 2013], scientists reported that CRISPR technology can enable precise and efficient genome editing in living eukaryotic cells.” From the outset, Lander was charting the birth of the CRISPR revolution as Zhang’s landmark paper. Lander appropriately paid tribute to many figures but downplayed the contributions of Charpentier and Doudna, devoting just a few paragraphs to their work compared to a page-and-a-half extolling the life and work of Zhang.

The article also included a map of the world, with the locations of the CRISPR pioneers marked in colored dots, from Japan to Lithuania, Germany to Spain, Boston to Berkeley. But there was something askew. On a closer look, the Atlantic Ocean had been magically compressed, while Greenland and Iceland had been erased completely.^II As a result, the map conveniently centered on Cambridge, Massachusetts, home of the Broad.

Readers also objected to the fact that Lander declared no conflicts of interest: he had no direct financial stake in Zhang’s work but the Broad was embroiled in a fierce patent dispute with Doudna and Charpentier’s respective institutions (see chapter 13). Cell Press, the publisher, said it did not require authors of commentaries to declare any conflicts of interest, although in the circumstances, it wouldn’t have hurt either the editor or author to have included such a note.

The critics pounced. Michael Eisen, an outspoken geneticist and a friend of Doudna’s, posted a scathing rebuttal, branding Lander “The Villain of CRISPR.” “There is something mesmerizing about an evil genius at the height of their craft,” Eisen wrote.⁴ Lander’s masterwork was “so evil and yet so brilliant that I find it hard not to stand in awe even as I picture him cackling loudly in his Kendall Square lair, giant laser weapon behind him poised to destroy Berkeley if we don’t hand over our patents.” He accused “the most powerful scientist on Earth” of scheming to help Zhang win a Nobel Prize and give the Broad Institute the inside track on “an insanely lucrative patent.”

Such a clapback among prominent scientists is both rare and fascinating. Eisen’s objection to Lander’s history lesson was nothing personal against Zhang but a judgement of what he considered the most crucial discovery in the CRISPR timeline. If there was a pivotal step in bringing CRISPR to the genome editing party, Eisen said, it was Doudna and Charpentier’s 2012 demonstration that CRISPR could be adapted into a molecular scissors following a decade of heroic foundational work. “Once you have that, the application to human cells, while not trivial, is obvious and straightforward,” Eisen declared.

Piling on was science historian Nathaniel Comfort, who labeled Lander’s essay a “Whig history” of the CRISPR saga—an effort to rationalize the status quo and spin the establishment’s point of view. Comfort was pleased to see Mojica and others receive some overdue credit. “Too often the early players and the scientists at lesser-known universities become lost to history altogether. But we should also recognize how Lander uses those actors to create a crowd in which to bury Doudna and Charpentier.”⁵

Not surprisingly, neither Doudna nor Charpentier were too thrilled with Lander’s account. “The description of my lab’s research and interactions with other investigators is factually incorrect, was not checked by the author and was not agreed to by me prior to publication,” Doudna said.⁶ Charpentier added that the description of her group’s contributions was “incomplete and inaccurate.”^III By this time, however, the two women were receiving ample opportunities to give their own accounts of the story.

George Church wasn’t thrilled with the media narrative, either. After all, his paper appeared in the same issue of Science as Zhang’s, but as the Nobel Prize is awarded to a maximum of three recipients, there is a tendency during prize season to search for the holy trinity. In CRISPR circles, that trio was usually Charpentier, Doudna and Zhang. Church aired his frustration a couple of years later to The Scientist. He wasn’t trying to take anything away from Doudna and Charpentier, pioneers who deserved credit for getting gene-cutting to work. “The spark that [they] had was that CRISPR would be a programmable cutting device.” But getting it to do precision editing was another matter. Indeed, Church argued that his human cells were a more accurate system than the aberrant culture cells that Zhang’s group had used.^IV In terms of credit, Church said, “you could say two and two. But to oversimplify that back down to three is like consciously omitting one.”⁷

Church later told me it wasn’t so much to cement his own place in history, whatever that matters, but the “egregious omission” of the postdocs who did the work—his and others. “I felt that Martin Jínek had been left out of the story, and Prashant Mali, and Luhan Yang, and Le Cong. You just never heard of them.”⁸

Doudna did not make a habit of putting on her “out of office” email message but her travel schedule soon became packed with prize ceremonies, media interviews, and keynote lecture invitations. Her talks were polished and accessible, generously crediting Charpentier and her colleagues. Despite her rapidly growing profile, she wasn’t thinking about writing a book until she received a surprise invitation from Max Brockman, the son of a leading New York literary agent, John Brockman. Doudna’s initial proposal, co-written with graduate student Samuel Sternberg, was a little dry, with references to Thomas Kuhn’s The Structure of Scientific Revolutions. As Sternberg admitted later: “What kind of person on the street was going to read that?”⁹

But public interest was intensifying. That was driven home when Sternberg accepted an invitation to breakfast at a Mexican restaurant in Berkeley from a woman who asked if he would be interested in starting a company to deliver CRISPR gene editing to future parents. Sternberg had no interest in that particular venture, but it supported giving the proposal another shot. The result was A Crack in Creation, published in spring 2017, which tells Doudna’s personal story, although she deftly sidestepped any commentary or controversy on the patent dispute.¹⁰

In various permutations, Charpentier, Doudna, and Zhang have hoovered up almost every major science prize, with two conspicuous exceptions: the Lasker Award, which is often referred to as America’s Nobel Prize and the Nobel Prize. Those appear to be a sure thing, but to whom and for what is a topic of much speculation.

The two women have shared the “Nobel Prizes” of Japan, Spain, Israel, and Canada (with Zhang), to name a few. The most lucrative award was the Breakthrough Prize, created by Silicon Valley billionaires including Priscilla Chan and Mark Zuckerberg (Facebook), Sergey Brin (Google) and his ex-wife Anne Wojcicki (23andMe), and Dick Costolo (Twitter). At a black-tie awards ceremony in November 2014, Doudna and Charpentier received their awards from Hollywood actress Cameron Diaz. Charpentier flashed her Gallic humor on stage. “It’s kind of surreal to receive the prize from Cameron,” she said, then turned to Costolo: “Three powerful women… I was just wondering if you’re Charlie?”

Two years later, Charpentier and Doudna joined Barrangou, Horvath, and Zhang for the annual Canada Gairdner Awards, the most prestigious Canadian scientific honor. At the banquet dinner, it is a tradition for each awardee to choose their own walk-up music as they head to the stage to accept their award. Zhang naturally chose John Williams’s stately theme from Jurassic Park. He thanked his parents for their sacrifices on his behalf and his wife for keeping him company in the lab on late nights and for the birth of their daughter. Horvath selected a jazzy rendition of the Mission: Impossible theme. He joked that his scientific career began working on sauerkraut and quoted a famous French proverb: “Impossible n’est pas français.” Barrangou chose “Happy” by Pharrell Williams, mugging shamelessly for the cameras as he shimmied up to the stage in his trademark cowboy boots. Charpentier, by contrast, selected a moody slice of French electronica by Daft Punk.

The most interesting speech was by Doudna, who selected Billie Holiday’s “On the Sunny Side of the Street.” She thanked her students, colleagues and mentors, as well as her two special guests, husband-and-wife Harvard Medical School professors George Church and Ting Wu, for inspiring her when she was a student at Harvard. She also paid tribute to Church’s under-recognized work in CRISPR. “His work has had a huge impact on the gene editing field over the years, including adapting the CRISPR-Cas system for gene editing in mammalian cells.”¹¹ (Some might wonder if she wasn’t throwing some shade in the direction of Zhang and Lander, who was also a guest at the dinner.) Then she announced that she was donating her $100,000 award to the nonprofit organization for genomics education cofounded by Wu and Church.

In a photograph from that evening, Barrangou stands at the center of the CRISPR quintet, his boots putting him a head taller than his peers. (Also in the group was another awardee, Anthony Fauci, recognized for global health.) The Gairdner was the undoubted highlight of his career, recognition for a landmark study that fermented the CRISPR revolution. Doudna and Charpentier were deservedly recognized for developing the single-guide RNA technology—the tipping point as he calls it.¹² “Single-guide RNA is an invention—it’s novel, not obvious, not natural. They didn’t just recapitulate it like Virgis. They engineered it, they designed it.” But genome editing is not until 2013, when “George and Feng and Luciano and Jin-Soo Kim and eventually Jennifer show that.”

Horvath shared the Massry Prize with Doudna and Charpentier in 2015 and Harvard’s Alpert Prize with Barrangou, Charpentier, Doudna, and Šikšnys. “I have mostly been in the shadow of Charpentier and Doudna, but not for the Bower,” he told me. The 2018 Bower Award and Prize for Achievement in Science was perhaps his greatest honor. Created in 1824, prizes have been bestowed on more than 2,000 scientists and inventors, including Tesla, Edison, Einstein, Hawking, Church, and Bill Gates. Horvath’s citation read:

For the foundational discovery of the role of CRISPR-Cas as a microbial system of adaptive immunity that has been developed as a powerful tool for precise editing of diverse genomes.

Horvath said his awards had created “some stress” among his colleagues, but I also sense that he feels his contributions haven’t been adequately recognized. “I recognize that the real interest is in the gene-editing aspect, and it’s possible that we’ll forget those who discovered the natural bacterial system and remember only the final developers of tools that allow this revolution,” he told me. Horvath also spares a thought for Jínek and Chyliński, the bench scientists who led the CRISPR breakthrough in 2012. “Maybe their defect is not to be women,” he said in a flash of political incorrectness. “Currently there is a demand for women in science. There is a positive discrimination for women in science. It’s good,” he says quickly, “but there might be some drawbacks to that. As soon as you have a woman who is at this level who gets the recognition, it’s obvious.”

A fun ritual each September is to predict the next group of Nobel laureates. For CRISPR, it is surely a matter of when, not if. A maximum of three people can share the award for each category. And you have to be alive.^V Some might argue that Nobels have already been awarded for gene targeting, the forerunner of genome editing, shared by Mario Capecchi, Oliver Smithies and Martin Evans in 2007 (for “introducing specific gene modifications in mice”). Perhaps the prize will go to the disciples of genome editing “before CRISPR”, whom we’ll meet in the next chapter.

Barrangou thinks people are asking the wrong question: it’s not when, or whom, but for which discovery. In other words, which committee? Chemistry or Medicine? If the award goes for chemistry, then the development of the sgRNA favors Doudna and Charpentier, but a strong case can be made for Šikšnys, who shared the Kavli Prize, or Jínek, who performed the signature sgRNA experiments. If it’s for the discovery of Cas9, then maybe Moineau. If the award is given for physiology or medicine, then it must go for genome editing, most likely Zhang and Church. “George was right there!” says Barrangou. “He’s been written off the books of history for no reason. You can’t keep George out of that, that’s crazy.”¹³ But for all its potential, CRISPR-Cas—indeed the entire field of genome editing—still has to prove itself as a game-changing, life-saving therapeutic.

Luciano Marraffini’s key role in helping Zhang kick-start his CRISPR program in 2012 was omitted from Lander’s “heroes” narrative. The affable Argentine’s technical expertise was central to the gene-editing discovery but, with the exception of the 2017 Albany Prize, has largely fallen under the radar. At the Albany Prize ceremony, Marraffini shared the stage with Mojica, who was asked to reflect on life as the grandfather of CRISPR. It was like adopting a child. “You give it a nice name—CRISPR,” he said. “You’re very proud of this child. It feels like [someone] that belongs to you, even though it’s not true. You try to look after them.” After ten years, the child becomes “a very clever person,” and then “a very important person.” Overall, he said, “I feel full of joy, I feel happy, I feel proud.”¹⁴

As for the Nobel speculation, Mojica wishes it could be put to rest. “If I get it, I will disappear from the planet,” he says.¹⁵ Of course, if he gets it, there is not the slightest chance of that happening.

While the developers of genome editing rack up accolades, the task of improving the original CRISPR system and expanding the CRISPR toolbox marches on. The potential of CRISPR has inspired thousands of new researchers around the world to study the fundamental biology of CRISPR and apply it in a host of settings including new forms of therapy.¹⁶

In CRISPR circles, the original Cas9 still commands the lion’s share of the attention. But researchers including Banfield and Koonin are mining the astonishing diversity of microbial life on planet earth to identify unknown organisms and catalogue the diversity of CRISPR immune systems and Cas genes therein. Scientists have wasted no time in adapting some of these systems for new research and diagnostic purposes.

One early and profound addition to the toolbox was to take the molecular scissors and immediately blunt the blades to mute Cas9’s DNA cutting function. That may sound counterintuitive, but the RNA programmability of Cas9 serves a multitude of purposes beyond cutting DNA. Cas9 can be used to ferry many kinds of molecules to a specific spot in the genome to modulate gene expression up or down (CRISPR activation or interference). This non-cutting Cas9, described by Stanley Qi and colleagues, is called “dead Cas9.”¹⁷ Its applications include base editing,¹⁸ a new riff on CRISPR genome editing in which Cas9 is used not to cut DNA but to position different enzymes to nick the DNA and perform pinpoint chemistry on a specific base. (We’ll return to this in chapter 22.)

The original Cas9 protein from S. pyogenes (SpCas9) is made up of 1,366 amino-acid building blocks, which makes for a tight squeeze when packing this molecule into the limited cargo space of the most popular gene therapy vector, the adeno-associated virus (AAV). There are many flavors of Cas9 derived from other microbes, some significantly smaller than SpCas9, others that recognize a different PAM site. Many of these provide new tools, increasing the options for the CRISPR engineer. Doudna’s team also found a way to put Cas9 on pause, holding it in a locked formation with the molecular equivalent of a plastic zip tie, which can be snipped as required to release the nuclease.¹⁹ This affords researchers more control in exactly where—or when—they unleash Cas9, reducing the chance of undesirable off-target effects.

In 2016, almost a decade after they first talked CRISPR over coffee, Doudna and Banfield unearthed a trove of new CRISPR tools from a metagenomic sampling of microbes that have not yet been cultured in the laboratory.²⁰ Among the highlights were two diminutive Cas nucleases, named CasX and CasY. CasX is only 60 percent the size of SpCas9. It cuts DNA in much the same manner, even though it bears no sequence similarity to SpCas9, suggesting it evolved quite independently. And unlike SpCas9, it is derived from a bacterial species that does not naturally infect humans, so in principle it would not have any of the potential immunogenicity concerns.

Another interesting Cas scalpel is Cas12 (formerly known as Cpf1), in particular Cas12a, discovered by the Zhang lab.²¹ Unlike Cas9, this enzyme produces a staggered cut—slicing the two strands of the helix in different places, rather than a clean cut. It is a small protein and doesn’t require a tracrRNA. While independently investigating the properties of Cas12a, members of the Zhang and Doudna labs were shocked to find that Cas12a did something quite different to single-stranded DNA—it didn’t so much cut DNA as shred it.²² Another new addition to the toolbox, Cas13, did much the same thing to RNA. If the detection of a specific DNA or RNA molecule could be coupled to some sort of chemical signal, the groups would have a simple diagnostics platform.

That’s exactly what the two groups did. Two of Doudna’s students, Janice Chen and Lucas Harrington, helped create Mammoth Biosciences to commercialize their diagnostics system, dubbed DETECTR. (Chen’s brother is world champion figure skater Nathan Chen.) Meanwhile, two of Zhang’s protégés, Omar Abudayyeh and Jonathan Gootenberg, joined Zhang, Pardis Sabeti, and the cofounders of Sherlock Biosciences; you don’t need to be a pipe-smoking detective to know what their system is called.^VI

Here’s an example: let’s say we want to program Cas12 to detect the SARS-CoV-2 virus responsible for the COVID-19 pandemic. A series of guide RNAs are designed to recognize certain sequences that have been amplified from the coronavirus genome. But once Cas12 recognizes that sequence, a new enzymatic property is switched on, such that it will cut (and keep cutting) any single-stranded DNA molecules in the vicinity. By adding reporter molecules that light up when cut, the presence of even trace amounts of the virus can be detected using a simple color assay on a paper strip.²³

Similarly, the Cas13 family can be used to detect infections such as flu, dengue, and Zika, and of course COVID-19.²⁴ Once activated, Cas13 exhibits what Zhang calls “collateral RNase activity”—it keeps cutting RNA. By supplying a suitable quantity of chemically tagged RNA reporter molecules, his team has the basis of a simple, portable detection system that can work on urine, blood, or saliva. The presence of virus will switch on Cas13, cutting the RNA reporters and releasing a fluorescent marker that can be read on a simple paper strip much like a pregnancy test.

The reliability of simple, cheap, one-stop diagnostic tests have a bad reputation following the debacle of Theranos, the theatrically overhyped Silicon Valley unicorn launched by Elizabeth Holmes that crashed from a $9 billion valuation to bankruptcy following great investigative reporting by John Carreyrou.²⁵ Unlike Theranos, which only belatedly published a single peer-reviewed study,²⁶ the Doudna and Zhang teams have already laid out the science and technology behind their diagnostics discoveries in a series of top-tier publications.

The portability of Mammoth’s and Sherlock’s kits could find huge markets—at home for the flu, in hospitals for antibiotic resistance, and in the field where outbreaks of coronavirus and other viruses emerge. Chen’s group has shown DETECTR can accurately detect HPV samples in a fraction of the time of a conventional test. Led by Harvard’s Sabeti, the SHERLOCK test has already shown promising results in detecting cases of Lassa fever in Nigeria, dengue in Senegal, and Zika virus in Honduras. Both companies are actively adapting their platforms to detect the COVID-19 virus. Beyond diagnostics, applications beckon in areas from food security and agriculture to bioterrorism. The Zhang lab has already applied SHERLOCK to gene detection in plants, pointing to an array of applications in detecting pathogens or pests.²⁷

In Toronto, Joseph Bondy-Denomy “found something amazing that we never expected,” said his PhD supervisor Alan Davidson.²⁸ He discovered anti-CRISPRs, a growing family of viral proteins that are able to disarm or neutralize bacterial CRISPR defenses—the rocks and paper to CRISPR’s scissors. Erik Sontheimer described a means to use anti-CRISPRs to restrict genome editing to a tissue of choice. Harvard Medical School’s Amit Choudhary is identifying small chemicals that can fine tune Cas enzyme activity. DARPA has launched a program called Safe Genes to fund research into anti-CRISPRs, and Bondy-Denomy wasted little time in cofounding a company, Acrigen Biosciences, to make gene editing safer and more efficient.

Other toolbox additions include Cas3, a DNA shredder to generate large deletions; a system called EvolvR to introduce mutations and evolve a specific target region; and systems that engineer programmed DNA insertions at a target site. Similar ideas were developed in parallel by Samuel Sternberg’s group at Columbia University, and the Zhang lab. Sternberg adapted a CRISPR system from Vibrio cholera to develop a programmable system based on transposons (parasitic jumping genes) to insert a custom DNA sequence at a specific site in the genome.²⁹ The system offers an appealing alternative to genome engineering without breaking DNA or triggering the cellular DNA damage response.

Scientists are just starting to appreciate some of these new tools, but these are just the tip of the iceberg. Zhang says about 150,000 microbial genomes have been sequenced, but we only understand something about the defense systems in about one third of them. There are so many more secrets yet to be revealed from the sequences of our microbial ancestors, which have had a mere billion years to innovate and evolve.

I. My editorial accompanying the hemochromatosis gene paper was entitled Definitely Maybe, a clever nod I thought to Oasis’s debut album and the line Kirstie Alley exhaled in Cheers after Ted Danson first plants a kiss on her. It was criminally edited to “A Definite Maybe.” (I’m still upset 25 years later.)

II. While Lander was playing at plate tectonics, he could have scratched off the UK as well. For a country that has contributed so much to our understanding of evolution and molecular biology—Darwin, Fleming, Crick, Rosalind Franklin, Sydney Brenner, Fred Sanger, Alec Jeffreys, Paul Nurse, and more—the origins of the CRISPR revolution surprisingly sidestepped the UK.

III. Some commentators also accused Lander of diminishing the contributions of the women at the center of the CRISPR story in favor of their male rivals. This was silly; Lander has mentored many superb female scientists, including Stacey Gabriel, Jill Mesirov, Pardis Sabeti, Anne Carpenter, and Aviv Regev, who in 2020 was recruited to lead R&D at Genentech.

IV. Zhang used cultured 293 cells, a kidney cell line.

V. There is an exception: if you die but nobody on the Nobel selection committee knows at the time of the announcement, you may still be awarded the Prize on a technicality. This happened to Ralph Steinman in 2011, who passed away three days before the winners were revealed.

VI. SHERLOCK stands for Specific Hypersensitive Enzymatic Reporter unLOCKing.