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

CHAPTER 6 FIELD OF DREAMS

One could say that Feng Zhang’s immigrant story—from China to Iowa, Harvard to Stanford, back to Harvard, to MIT, 60 Minutes and scientific celebrity, fame, and a fortune in the making—is the quintessential American Dream. In 1993, Shujun Zhou, a Chinese computer engineer, brought her eleven-year-old son from Shijiazhuang, a heaving city of 10 million people about 150 miles southwest of Beijing, to the United States. They settled in Des Moines, Iowa—a world apart from his bustling birthplace. Driving around the Hawkeye state, Zhang would see a relentless landscape of cornfields and cattle, and highways lined with religious billboards with uplifting signs such as “After you die, you will meet God.” The car radio might have picked up the “Bible Bus,” a twenty-four-hour Christian station. His new home was indeed “a lot more ‘Zen-like,’ a lot more calm,” Zhang jokes. Compared to his home, Des Moines felt “empty.”

Zhang enrolled in the Callanan Middle School and soon became fluent in English. He got his first taste of molecular biology in eighth grade during a Saturday afternoon enrichment program. He joined a group of fellow nerdy middle school students called STING—Science and Technology in the Next Generation, inspired by Star Trek, organized by Ed Pilkington. The first documentary he watched, he jokes, was Steven Spielberg’s Jurassic Park, which for all the revisionist criticism of Michael Crichton’s dinosaur-DNA-locked-in-amber premise, inspired Zhang to imagine the possibilities of engineering and programming biological systems.

Zhang didn’t know it, but Crichton’s storyline gave rise to one of the first documented accounts of genome editing. In his original novel, Crichton included an excerpt of DNA sequence purported to represent dinosaur DNA. But when bioinformatician Mark Boguski idly typed the sequence into his computer, he was crushed to find that Crichton’s dino-DNA actually belonged to a bacterium. After Boguski busted Crichton in a short essay,¹ Crichton contacted Boguski offering both an apology and an invitation to supply a more fitting sample for the sequel. Boguski chose a segment of chicken DNA, representing a living dinosaur descendant, which Crichton included in The Lost World. What the novelist didn’t know was that Boguski had left an easter egg in the sequence: when the genetic code was translated into its corresponding amino-acid code,^I there were four small insertions that, taken together, read:

MARK WAS HERE NIH

In his sophomore year, Zhang got his first intoxicating taste of scientific research, volunteering in a gene therapy lab at the Iowa Methodist Medical Center. He used a virus to ferry a jellyfish gene encoding a green fluorescent protein into human cancer cells. The magical glow that emanated from those cells was early proof that Zhang had green fingers at the bench. His mentor, John Levy, gave Zhang some words to live by: “Try to do something on the sexy side of practical.”² From this point on, Zhang’s future in biological research was pretty much assured. Pilkington observed: “Not everybody is able to fail twenty experiments and then get up and then come up with something brilliant.”³

At nineteen, Zhang placed third in the prestigious Intel Science Talent Search competition, winning a $50,000 scholarship. His mother accompanied him through to the finals, exhorting her son, “Zhan zhi!” (“Stand up straight!”). He excelled at Harvard, matriculating the year before Mark Zuckerberg, earning a degree in chemistry (“the central science”) and physics. Zhang wanted a grounding in the fundamentals that he could apply as he saw fit. Eager to experience Silicon Valley, Zhang moved to Stanford for his PhD. His first choice supervisor, Nobel laureate Steven Chu, had moved on; camped out in Chu’s former office was a new faculty member, Karl Deisseroth.

A psychiatrist by training, Deisseroth had treated many patients with schizophrenia and depression, but was frustrated at how poorly we understand those diseases. He was developing a new technique called optogenetics for studying neuronal activity and neurological diseases. Deisseroth’s brainstorm was to introduce a light-sensitive protein called an opsin into a rodent neuron, affording him the ability to trigger and control its activity.^II

Zhang’s initial task, building on his familiarity with gene therapy, was to introduce the opsin gene, which was derived from a pond-dwelling green algae, into single rat neurons on a tissue culture plate using a recombinant virus. When triggered by light, the newly transfected neurons would emit an electrical signal. Over the next few years, Deisseroth, Zhang, and another grad student, Ed Boyden, progressed from neurons in a dish to rats in a maze. When a New York Times reporter visited the lab one Sunday, Zhang had prepared a show-stopping experiment: inside a white plastic tub was a solitary transgenic brown mouse expressing the opsin gene in a particular type of neuron. Zhang inserted a small metal tube into the mouse’s head, into which he threaded a tiny fiber optic cable. When he flicked on the light switch, the mouse suddenly started spinning in circles; switched off, the mouse stopped moving.⁴ Optogenetics was ready for the big time: a tool to study brain function far more precisely than sticking electrodes into the brain or taking blurry MRI pictures.

“This is one of those things that only comes around every five or ten years,” Deisseroth told his student.⁵ And he was right: Deisseroth won the Breakthrough Prize in 2015 and helped lay the foundation for President Barack Obama’s $300-million BRAIN Initiative. Deisseroth credited Zhang’s skills as “absolutely essential to the creation of optogenetics.”⁶ Not too many PhD students get to see their handiwork featured in the Times or win a share of a major scientific prize. It was a sign of things to come.

Still in his twenties, Zhang returned to Boston in 2010 and joined the lab of George Church as a Harvard junior fellow. For a gifted scientist intent on developing new tools for studying genetics and the brain, there could be no more inspiring training ground. For twenty-five years, Church had been a driving force in genomics, pursuing the kind of bold, fearless research that appealed to a talent like Zhang. Church and his dozens of students developed new sequencing technology for reading DNA but was increasingly interested in writing entire genomes. In collaboration with stem cell researcher Paola Arlotta, Zhang and a new PhD student, Le Cong, threw themselves into the emerging field of gene editing. The idea was to develop a method to improve the creation of sequence-specific TALENs to target genes and modulate gene activity.⁷ Joining them in a corner of the Wyss Institute were two other postdocs, Prashant Mali and Kevin Esvelt.

Born in Beijing, Le Cong shared Zhang’s love of engineering. He enjoyed tinkering with radio sets and designing computer games as a child. While studying electronic engineering at Tsinghua University, Le Cong’s interests turned to medicine after the death of some close relatives. “We’ve made so much progress in modern medicine, but there’s so much we don’t know,” he told me.⁸ Even simple diseases such as type 1 diabetes can take people’s lives.

Arriving at Harvard on a fellowship, Le Cong quickly bonded with Zhang and was swept up by the infectious lab chatter about the genomics revolution. He wanted to develop tools to engineer the human genome to model brain diseases such as autism, schizophrenia, and bipolar disorder. Although officially Church’s student, Le Cong says that in January 2010, “Feng became my advisor and mentor. We were the only folks interested in working on new tools in gene editing.” It was difficult work: the techniques to synthesize bacterial chromosomes were still rudimentary, and extrapolating to larger mammalian genomes would be even harder.

Across the Charles River in Cambridge, Bob Desimone, the director of the McGovern Institute for Brain Research at MIT, was looking for new faculty. The institute was established by the late Pat McGovern, who had dropped out of MIT to start a publishing company in his basement. That company evolved into IDG (International Data Group), the global publisher of Computerworld, MacWorld, and Bio-IT World. McGovern became a billionaire but ran his enterprise with the warm touch of a family-run business.^III McGovern and his wife Lore proudly donated $350 million to create the eponymous institute.

The faculty search wasn’t going particularly well until Zhang’s name came up. His research credentials were impeccable, the only question was whether his flourishing interest in genome manipulation would be the right fit. Desimone asked MIT’s top stem cell researcher, Rudy Jaenisch, to assess the candidate. Jaenisch’s verdict: “He’s certainly very clever. If he can do 10 percent of what he’s proposing, he’s going to be a star.”

Zhang joined the McGovern Institute in January 2011 with a joint appointment at the nearby Broad Institute. His goal was to continue developing TALENs and other systems to engineer genome mutations to model and devise treatments for autism, Alzheimer’s disease, and schizophrenia. Zhang concedes he might have had a slight case of Imposter Syndrome, but it didn’t linger. Le Cong decided to hitch a ride as well. “We’d been working colleagues, it was wonderful to continue our relationship,” he said. Even though he was still technically Church’s graduate student, they shared a taxi over the Harvard Bridge to start their new adventure at MIT.

In early February, 2011, Zhang paid a return visit to Harvard Medical School. The Broad Institute’s annual Board of Scientific Counselors meeting was at the Joseph Martin Auditorium, a block from Zhang’s old lab. A lecture from a most unlikely source was about to change his life.

Michael Gilmore is an expert on antibiotic resistance in bacteria. In 2007, while at a microbiology conference in Pisa, Italy, he was impressed by a poster presented by Danisco on the link between phage immunity and DNA repeats with a weird name: CRISPR. Gilmore wanted one of his new postdocs to study CRISPR. One promising candidate, Luciano Marraffini, opted instead for a position in Chicago. In stepped Kelli Palmer who, with colleagues at the Broad Institute, began comparing the sequences of multidrug-resistant strains of bacteria with older strains. She made a striking observation: genomes from multidrug-resistant clinical strains dating back to the 1970s were larger and generally lacked CRISPR. By comparison, the other isolates still possessed CRISPR.

Gilmore’s deduction was that “the repeated introduction of various antibiotics since the ’40s not only selected for resistance to those antibiotics, but it selected for the ability to acquire [drug] resistance with enhanced facility.” In other words, bacteria were gaining a selective advantage by losing their CRISPR defense system. That might leave them vulnerable to phages, but it would make it easier for them to acquire new genetic elements that could confer antibiotic resistance (via a mechanism called horizontal gene transfer). The overuse of antibiotics has resulted in bacteria effectively lowering their genome defenses to make it easier to acquire resistance from other microbes.⁹ The genomes of clinical isolates of some bacteria might be 25 percent larger than those of commensal strains.¹⁰

Zhang’s ears pricked up as Gilmore casually mentioned that bacteria contain CRISPR and its attendant nucleases. The acronym had appeared in several top journals by this time, but Zhang had to google the term to learn more about it. The next day, Zhang flew to Miami to attend a conference,^IV but hunkered down in his hotel room instead, devouring the CRISPR literature. The more he read, the harder it was to contain his excitement. He read a CRISPR review article by Horvath and Barrangou in Science¹¹ and the “amazing” 2010 Nature paper by Moineau’s group that showed that the type II CRISPR-Cas system cleaves phage DNA.¹² But Zhang wasn’t interested in bacterial immunity or how to make a cheesier pizza. His focus was genome editing—a technique that could work in animal models and ultimately in humans. If, as Moineau’s paper suggested, you could use RNA and CRISPR to target DNA, this would be much easier than either ZFNs or TALENs.

On Saturday February 5, Zhang fired off an email to Le Cong: “Take a look at this,” he wrote, including a link to the Horvath-Barrangou review. “Maybe we can test in mammalian system.” Le Cong replied, “It should be very cool to test in mammalian systems.” Two days later, Zhang emailed: “Hey let’s keep this confidential. This can completely replace any kind of [zinc finger] system. I ordered the Cas genes for synthesis. We should be able to test them… I’ve done a patent search.”

On February 13, Zhang filed a “memorandum of invention,” an internal Broad Institute document that summarized his new invention: multiplexed genome engineering. The idea, which he said originated just nine days earlier at Gilmore’s lecture, was a clear statement that Zhang thought CRISPR might complement, or even replace, the current methods of gene editing, ZFNs and TALENs.¹³ Zhang judged that CRISPR had the makings of a programmable gene-editing technology that could be targeted to almost any DNA sequence.

In a short McGovern Institute video filmed at the end of 2011, Zhang discussed his research goals. Coming from an engineering background, he said, “I think about how to take things apart, put them together, and then try to fix it.” Using the same approach, Zhang hoped “to understand disease mechanisms and be able to fix the brain.” The main tools in his arsenal were the TALEN proteins.¹⁴ He exuded a quiet fearlessness, as if nothing was beyond his reach. But there was no mention yet of CRISPR or the potential to edit human DNA.

Zhang and Le Cong’s early efforts using Cas9 did not work as planned. Two key modifications were required to get Cas9 to work in human cells, as I mentioned earlier: one was codon optimization to make the gene appear less foreign to a human cell. The other was to add a nuclear localization signal, a motif that helps ferry DNA into the cell nucleus (obviously not an issue in bacteria as they lack a nucleus). But for some reason, the S. thermophilus Cas9 wasn’t behaving nicely. Zhang needed a new system and a Cas expert. The man he was looking for—who had almost been a colleague of Gilmore’s—was just down the road in New York City.

Shortly before 10:00 P.M. on January 2, 2012, Zhang sent a short email to Marraffini at the Rockefeller University. The Argentine had received flattering faculty offers from MIT and Yale, but the prospect of living in cosmopolitan New York was irresistible. And Rockefeller’s rich tradition in microbiology and genetics proved a perfect fit. Zhang didn’t beat about the bush:

Dear Luciano,

Happy new year! My name is Feng Zhang and I am a research [sic] at MIT. I read many of your papers on the Staphylococcus CRISPR system with great interest and I was wondering if you would be interested in collaborating to develop the CRISPR system for applications in mammalian cells.

Would it be possible to schedule a phone call with you in the next few days?¹⁵

Marraffini had never heard of Zhang but a quick Google search left him suitably impressed. He replied ninety minutes later and said yes. “We have been working on a ‘minimal’ CRISPR system that could be useful… Happy 2012!” They sealed the collaboration in a phone call the next day. A week later, after receiving a nudge from Zhang, Marraffini emailed an eight-page document that highlighted DNA sequences and other useful information about CRISPR in S. pyogenes. He closed with a five-step plan to produce the “minimal” Cas system that he believed could be used to edit human genes. The relevant materials, including the gene template for Cas9 and the tracrRNA, followed by mail.

Zhang was a man in a hurry. He was working in the trenches with his team, his weapons of choice a row of personalized pipettors with FENG taped across each one. On January 12, he told Marraffini he’d already identified a pair of target sites in a human gene (AAVS1) and was preparing to express the bacterial genes in mammalian cells. Around this time, he was included in an $11 million NIH grant application filed by the Broad Institute’s then deputy director, David Altshuler.¹⁶ The main idea was to use genome editing to engineer stem cell models for type 2 diabetes and other diseases. Zhang proposed using the four components described by Charpentier—the CRISPR array, Cas9, the tracrRNA, together with another enzyme (RNase III)—to reconstitute the active CRISPR complex in human cells.

Within a few months, Zhang had enough data to show he could deliver the CRISPR-Cas9 machinery into the nucleus of mouse or human cells (by flanking Cas9 with nuclear localization signals) and target a gene of choice. Marraffini also had preliminary results showing that Cas9 could target a human gene sequence. They also found an isoform of the tracrRNA that was expressed in human cells. Zhang toyed with the idea of writing up these preliminary results—potentially the first demonstration of CRISPR gene editing in animal cells—but decided against it. “I [wanted] to wait until we have a paper that can make a significant difference, not just to be first with something,” he said.¹⁷

In late June, Zhang saw the names of Doudna and Charpentier on a CRISPR paper in Science. The consensus was that this was a landmark paper, but Zhang was uncharacteristically dismissive. “I didn’t feel anything,” he told WIRED. “Our goal was to do genome editing, and this paper didn’t do it.”¹⁸ But the report impacted Zhang’s team in at least two important respects. First, it showed that competition in CRISPR was heating up. “We didn’t think we got scooped, but we knew people would jump on the topic so we had to speed up,” Le Cong told me.¹⁹ Second, it introduced the idea of the single-guide RNA (sgRNA). Zhang and Le Cong didn’t hesitate to put this idea into action with the help of a new recruit with something to prove.

Fei Ann Ran was born in Szechuan, China, and moved with her family to Pasadena, California, when she was ten years old. She traveled to the East Coast for college and enrolled for her PhD at Boston Children’s Hospital. But more than halfway through, Ran suffered a devastating setback: her supervisor, geneticist Laurie Jackson-Grusby, ran out of funding and closed her lab. “She left,” Ran told me in a cafe on Longwood Avenue, the central artery of the Harvard Medical School campus. “She decided not to publish any of our work. I was in my fifth year of grad school. It was a pretty terrifying time.”²⁰

A member of Ran’s PhD committee, Harvard chemistry professor Greg Verdine, suggested she contact Zhang, whom Verdine remembered as a star student. Ran didn’t know much about genome editing or Zhang. A friend told her Zhang was famous for his work on TALENs. But she didn’t know what TALENs were, either—she misheard this as “Talent.” Luckily, she fit the bill. One of Zhang’s postdocs, Neville Sanjana, was developing a method to treat an inherited brain disorder called Angelman syndrome^V by building a TALEN activator to switch on a silent gene. Ran made one TALEN but soon switched to working on the easier CRISPR system.

In July, Zhang gave a public lecture at the Broad Institute on “Engineering the Brain” in which he discussed the potential of genome editing to understand the brain and potentially treat incurable brain disorders. There was still no mention of CRISPR. Meanwhile, Ran was devouring the CRISPR literature and most of all hoping to salvage her PhD. “I was so single-minded on getting this to work in mammalian cells,” she said. “Feng and Le had been doing a lot of work in mammalian cells. There was no question it couldn’t work but how could it work robustly?” Le Cong and Ran were keen to try the new sgRNA approach, but it didn’t perform as well as adding the crRNA and tracrRNA separately. By incrementally extending the tail of the tracrRNA, they were able to boost the gene-editing efficiency.

Ran found the atmosphere in the lab exhilarating despite the long hours, the group working around the clock almost in shifts. Le Cong still technically belonged to Church, but had a special bond with Zhang, “almost a father-son relationship.” The group would eat an early dinner and a late-night supper, frequently Chinese takeout, in the kitchen adjacent to Zhang’s laboratory. From the Broad’s tenth floor, Ran could gaze across the Charles River and admire the Boston skyline—the twin peaks of the Hancock Building and the Prudential Center, the sparkling floodlights of Fenway Park. But the only nightlife that mattered was in the lab. “I was the later crew, but Feng was the omni crew,” she said. “He’d show up before everybody and leave after everybody.” His wife, Yufen Shi, whom Zhang met at Stanford and married in 2011, would often wait patiently for him in his office.

Occasionally there was time to relax outside the lab. Shortly after her arrival, Ran accompanied her colleagues on a summer camping trip to nearby Harbor Island. Most of the group slept in a twenty-person tent. In a lab photo from one of those group activities, Zhang’s youthful appearance blends in with his students and postdocs. Anyone not knowing him would be hard pressed to pick out the professor. Zhang displayed a childlike excitability, desperate to see everyone’s latest results. “It was like taking a kid to a candy store,” Ran said. She kept improving the gene-editing efficiency and showed they could edit more than one gene at a time. The experiments worked routinely, unlike the frustrations with TALENs.

The final question was could they get CRISPR-Cas9 to edit genes inside human cells? Ran and Le Cong took Cas9, made a new guide RNA, and put everything into human cells growing in a petri dish. “And then we waited… and then we waited,” she recalled.²¹ A few days later, they sequenced the genome of those cells. “We could see the scars of DNA damage and repair—in other words, mutations—exactly where we thought they’d be. This was really exciting!”²² Zhang was excited, too. “I want to see!” he said. He told her: “Isn’t this cool that you’re one of the only people in the world to see this?”

As noted earlier, many groups were desperately interested in applying CRISPR to genome editing, not least members of the Church lab. Before Zhang left, Prashant Mali had been working on TALENs and dabbling with other gene-editing technologies. “CRISPR was just on our list of nucleases,” Church told me. “But we were always looking for precision editing. And we wanted to do it in human cells.”²³ Kevin Esvelt had joined the Church lab after a successful PhD with Harvard chemist David Liu. He’d got a rudimentary version of CRISPR working in the lab, which he was using like antivirus software to prevent infections from stray viruses. Intrigued by Esvelt’s experience working with Cas9 in bacteria, Mali asked for his help getting CRISPR to work in mammalian cells. Esvelt had his doubts, assuming Doudna had an insurmountable lead. But Mali persisted. “This is going to be so big that if we discover one tiny little piece that the other groups miss, it will be worth it.”²⁴

As it turned out, that “one tiny piece” was demonstrating the need for the whole sgRNA—truncating it reduced activity. With help from another talented grad student named Luhan Yang, Church submitted their paper to Science in late October. It not only demonstrated “facile, robust, and multiplexable human genome engineering” but also predicted that more than 40 percent of human gene sequences were amenable to genome editing using CRISPR. As a courtesy, he emailed Doudna to let her know his group had extended her results to editing in normal human cells. As far as he knew, that was a first.

Of course, it wasn’t. Three weeks earlier, Zhang had submitted his group’s manuscript to the same journal. Le Cong and Ran were credited as co–first authors, and Marraffini and his student Wenyan Jiang were also included. Barrangou was one of the referees helping Science judge the merits of those and similar results from other teams. Barrangou judged that the reports from Church and Zhang demonstrating CRISPR editing in human cells were the most significant of the bunch. Of the two, Feng’s paper was perhaps the better but “they’ve got to take both, back to back. The single-guide RNA technology is the tipping point. The real credit should go to George and Feng to show genome editing in human cells.”²⁵

Science published Zhang’s article alongside Church’s paper on January 3, 2013.^{26, 27}, Jin-Soo Kim’s demonstration of CRISPR gene editing in human cells came out four weeks later, in Nature Biotechnology.²⁸ Jínek and Doudna presented their human cell success in eLife. There were also reports from Keith Joung (Massachusetts General Hospital) working on zebrafish²⁹ and Marraffini, squeezed out by Science, who published his studies on bacteria in March.³⁰

Ran was on vacation when she got the news of her first major publication, which more or less secured her PhD. Six months of hard work at the cutting edge of science had made up for five years of fruitless labor and frustration. Her reaction was more relief than jubilation. She remained in the lab for another year before writing up her thesis. “I have this great suggestion,” Zhang told her at one point. “Download Arno Pro. It’s a great font!” Ran still has it on her computer. “That’s how I got my PhD, with the help of Feng’s font,” she laughs. She didn’t see much of him as she finished in the lab, and learned about the birth of his first child from an unusual source. While receiving the referee reports for a subsequent paper, the journal editor’s email said, “Congratulations on Ingrid.” Ran smiles: “We learned about it from a journal editor!”

Some would argue that Zhang and Church were merely confirming the obvious, extrapolating from Doudna and Charpentier’s work in bacteria to engineer human DNA. After all, several teams reported success within a couple of months of each other. But getting the system to work in human cells was a major step forward in developing a new type of genetic therapy. It also set the stage for a massive legal dispute about the invention of genome editing (see Chapter 13). Any doubts that Cas9 could work in human and other animal cells had been well and truly answered.

One might ask what if eLife, located in Cambridge, England, had expedited review and publication of Doudna’s follow-up paper and published it before the New Year. Shepherding reviewers over the festive break isn’t easy, especially when Brits typically log off from Christmas to New Year. By the time Doudna’s paper was published, few took notice. A few years later, Doudna was asked: Why did Zhang and Church demonstrate CRISPR gene editing in human cells before her? “They were absolutely set up to do that kind of experiment,” she acknowledged. “They had all the tools, the cells growing, everything was there. For us, they were hard experiments to do because it’s not the kind of science we do. What speaks to the ease of the system was that a lab like mine could even do it.”³¹

The media still didn’t fully grasp the implications of the genome editing papers from Zhang and Church.^VI One of the first to comment was author and columnist Matt Ridley, who marveled in the Wall Street Journal at scientists’ ability to precisely edit a single base.³² Two months later, Forbes science correspondent Matthew Herper surmised, “The [Cas9] protein could change biotech forever.” Herper’s story focused on Church, who had a much bigger profile than his former fellow, and said CRISPR was “spreading like wildfire.” Not only was Zhang not quoted, but ironically, he also fell victim to an editing error: his name was misspelled “Zheng.”³³

Steadily, interest in CRISPR from scientists and media alike picked up. Geneticist Konrad Karczewski recapped the buzziest terms of 2013 in a game of catchphrase bingo popular at conferences. “CRISPR” was included along with nanopores, big data, and Myriad (for the blockbuster supreme court ruling banning gene patents).³⁴ A report from the Harvard labs of Chad Cowan and Kiran Musunuru compared CRISPR and TALENs side by side, with CRISPR winning convincingly.³⁵ “It was a surprisingly important paper,” said T. J. Cradick,³⁶ the former head of genome editing at CRISPR Therapeutics, not least because it gave venture capitalists the green light to explore CRISPR’s commercial potential.³⁷ The Boston Globe covered CRISPR for the first time in a story about the launch of local biotech company, Editas Medicine, featuring Zhang, Doudna, and Church among the cofounders. Science recognized CRISPR in its annual “Breakthrough of the Year” awards—albeit as a runner-up to cancer immunotherapy.³⁸ CRISPR achieved top billing two years later, having “matured into a molecular marvel.”³⁹

I. In the single letter code, each of the twenty amino acids, encoded by a three-letter codon of DNA, is represented by a single letter. For example, M-A-R-K stands for methionine-alanine-arginine-lysine.

II. This idea was initially proposed by Francis Crick, who spent the final three decades of his life at the Salk Institute studying the brain and consciousness. Crick called for a new technique that could probe brain function at the single-cell level, and even suggested that this level of control could be achieved through light.

III. Every Christmas, McGovern toured every IDG office location, meeting with each employee in person to hand them a cash gift and thank them for their specific contributions to the company, which he knew cold.

IV. The Miami 2011 Winter Symposium, “Epigenetics in Development and Disease.” co-organized with Nature Publishing Group.

V. Angelman syndrome is an example of a class of genetic diseases called imprinting disorders, in which mutations effectively silence one copy of a gene, depending on whether it was inherited from the mother or father.

VI. Two industry publications ran reports based on press releases: Genetic Engineering & Biotechnology News (GEN) covered the Zhang paper, while Genomeweb highlighted the Church lab report.