The Code Breaker, page 7
With her husband, Jamie Cate, and son, Andy, in Hawaii, 2003
CHAPTER 8 Berkeley
Going west
In the article that Doudna and her colleagues wrote on their RNA structure discovery, which was published in Science in September 1996, her name is listed last, meaning that she was the principal investigator who headed the lab. Jamie Cate’s name is listed first because he did the most important experiments.1 By then they were more than scientific partners; they had become romantically involved. After her divorce was final, they got married in the summer of 2000 at the Melaka Beach Hotel across the Big Island of Hawaii from Hilo. Two years later, they had their only child, Andrew.
By then, Cate had become an assistant professor at MIT, so they were commuting between New Haven and Cambridge. By train it’s less than three hours, but for a new couple even that was tiresome, so they decided to see if they could get appointments in the same town.2
Yale tried hard to keep Doudna, promoting her to an important professorship. To resolve what is known as the “two-body problem” in academia, it offered Cate a position as well. However, Tom Steitz, the structural biologist who had shown them the techniques of cryocooling, was there doing the same type of research Cate wanted to do, and he felt that would crimp his chance to flourish. “My direct competitor was there,” Cate says. “He’s a great guy, but it would be hard to be in the same institution.”
Harvard offered Doudna a position in the Department of Chemistry and Chemical Biology, which had just been renamed and was growing. She went there as a visiting professor, and on the first day the dean handed her an offer letter for a permanent position. With Cate at MIT, it seemed to be an ideal arrangement. “I was thinking how great it was that I would end up in Boston, back where I was in graduate school and had such a good time,” she said.
It is interesting to imagine how her career would have been different if she had stayed at Harvard. Along with MIT and the jointly managed Broad Institute, the university was a cauldron of biotech research, especially in the field of gene engineering. A decade later, she would find herself in a race to develop CRISPR into a gene-editing tool with various Cambridge-based researchers, including Harvard’s George Church and the men who would become her bitter rivals, Feng Zhang and Eric Lander of the Broad Institute.
Then she got a call from the University of California at Berkeley. Her first reaction was to deflect any offer, but when she told Cate, he was shocked. “You should call them back,” he said. “Berkeley is nice.” When he had been a postdoctoral fellow in Santa Cruz, he had often gone up to the Lawrence Berkeley National Laboratory, which was managed by the university, to do experiments at its cyclotron, a particle accelerator.
When they visited the campus, Doudna was still disinclined to move there. But Cate became more enthusiastic. “I’m more of a western guy,” he says. “I found Cambridge to be uptight. My director at the time always came to work in a bow tie. I was happier at the thought of being at Berkeley, where the energy level was great.” Doudna liked the fact that Berkeley was a public university, and she was easily persuaded. By the summer of 2002, they had moved.
* * *
Their choice of Berkeley is a testament to America’s investment in public higher education. Its roots stretch back to when Abraham Lincoln, in the middle of the Civil War, thought public education was important enough that he pushed through the Morrill Land-Grant Act of 1862, which used funds from federal land sales to establish new agriculture and mechanical colleges.
Among those was the College of Agricultural, Mining, and Mechanical Arts near Oakland, California, founded in 1866, which two years later merged with the nearby private College of California. It became the University of California, Berkeley, and grew into one of the world’s greatest research and learning institutions. In the 1980s, more than half of Berkeley’s funding came from the state. However, since then Berkeley, like most other public universities, has faced reductions. When Doudna arrived, state funding accounted for only 30 percent of Berkeley’s budget. In 2018, state funding was cut again, and it amounted to less than 14 percent. As a result, Berkeley’s undergraduate tuition for a California resident in 2020 was $14,250 per year, more than triple what it was in 2000. Room, board, and other fees raised the total cost to around $36,264. For an out-of-state student, total costs were around $66,000 a year.
RNA interference
Doudna’s study of RNA structure led her to a field that would become unexpectedly relevant later in her career: viruses. Specifically, she was interested in how the RNA in some viruses, such as coronaviruses, allow them to hijack the protein-making machinery of cells. During her first semester at Berkeley, in the fall of 2002, there was an outbreak in China of a virus that caused a severe acute respiratory syndrome (SARS). Many viruses are composed of DNA, but SARS was a coronavirus that instead contained RNA. By the time it died out after eighteen months, it had killed close to eight hundred people around the world. It was officially known as SARS-CoV. In 2020, it had to be renamed SARS-CoV-1.
Doudna also became interested in a phenomenon known as RNA interference. Normally, the genes encoded by the DNA in cells dispatch messenger RNAs to direct the building of a protein. RNA interference does just what the name implies: small molecules find a way to mess with these messenger RNAs.
RNA interference was discovered in the 1990s, partly by researchers who were trying to make petunias more purple by juicing up the flower’s color genes. But the process ended up suppressing some of the genes, leading to mottled and speckled petunias. Craig Mello and Andrew Fire coined the term “RNA interference” in a 1998 paper and later won the Nobel Prize when they discovered how the phenomenon works in the nematode, a tiny worm.3
RNA interference operates by deploying an enzyme known as “Dicer.” Dicer snips a long piece of RNA into short fragments. These little fragments can then embark on a search-and-destroy mission: they seek out a messenger RNA molecule that has matching letters, then they use a scissors-like enzyme to chop it up. The genetic information carried by that messenger RNA is thus silenced.
Doudna set about to discover the molecular structure of Dicer. As she had done with self-splicing RNA introns, she used X-ray crystallography to map its twists and folds, which she hoped would show how it worked. Until then, researchers did not know how Dicer was able to cut RNA into precisely the right letter sequences to silence a specific gene. By studying the Dicer structure, Doudna showed that it acted like a ruler that had a clamp at one end, which it used to grab on to a long RNA strand, and a cleaver at the other end, which it used to slice the segment at just the correct length.
Doudna and her team went on to show how a particular domain of the Dicer enzyme could be replaced in order to create tools that would silence other genes. “Perhaps the most exciting finding of this study is that Dicer can be reengineered,” their 2006 paper noted.4 It was a very useful discovery. It permitted researchers to use RNA interference to turn off a wide variety of genes, both to discover what each gene does and to regulate its activity for medical purposes.
In the age of coronaviruses, there is another role that RNA interference may play. Throughout the history of life on our planet, some organisms (though not humans) have evolved ways to use RNA interference to fight off viruses.5 As Doudna wrote in a scholarly publication back in 2013, researchers hoped to find ways to use RNA interference to protect humans from infections.6 Two papers published in Science that year gave strong evidence that it might work. The hope then was that drugs based on RNA interference might someday be a good option for treating severe viral infections, including those from new coronaviruses.7
* * *
Doudna’s paper on RNA interference appeared in Science in January 2006. A few months later, a paper published in a little-known journal described a different virus-fighting mechanism that exists in nature. It was by an obscure Spanish scientist who discovered the mechanism in microorganisms like bacteria, which have a far longer and even more brutal history fighting viruses than we humans do. At first, the handful of scientists studying this system assumed that it worked through RNA interference. They would soon discover that the phenomenon was even more interesting.
PART TWO CRISPR
The scientist does not study nature because it is useful.
He studies it because he takes pleasure in it,
and he takes pleasure in it because it is beautiful.
—Henri Poincaré, Science and Method, 1908
Francisco Mojica
Erik Sontheimer and Luciano Marraffini
CHAPTER 9 Clustered Repeats
Francisco Mojica
When Yoshizumi Ishino was a student at Osaka University in Japan, his PhD research included sequencing a gene in E. coli bacteria. It was 1986, and gene sequencing was a laborious process, but he eventually succeeded in determining the 1,038 base pairs of DNA that made up the gene in question. In a long paper on the gene that he published the following year, he noted in the last paragraph an oddity that he did not consider important enough to mention in the paper’s abstract. “An unusual structure was found,” he wrote. “Five highly homologous sequences of 29 nucleotides were arranged as direct repeats.” In other words, he found five segments of DNA that were identical to each other. These repeated sequences, each twenty-nine base pairs long, were sprinkled between normal-looking sequences of DNA, which he called “spacers.” Ishino had no idea what these clustered repeats were. In the last line of his paper, he wrote, “The biological significance of these sequences is not known.” He didn’t pursue the topic.1
The first researcher to figure out the function of the repeated sequences was Francisco Mojica, a graduate student at the University of Alicante on the Mediterranean coast of Spain. In 1990, he began working on a PhD dissertation on archaea, which, like bacteria, are single-cell organisms without a nucleus. The archaea he was studying thrive in salt ponds that are ten times saltier than the ocean. He was sequencing regions that he thought might explain its love of salt when he spotted fourteen identical DNA sequences that were repeated at regular intervals. They seemed to be palindromes, meaning they read the same backward and forward.2
At first he assumed that he had screwed up the sequencing. “I thought it was a mistake, because sequencing was hard back then,” he says with a hearty laugh. But by 1992, when his data kept showing these regularly spaced repeats, Mojica wondered if anyone else had found something similar. Google did not yet exist, nor did online indexes, so he manually sorted through citations for the word “repeat” in a set of Current Contents, a printed index of scholarly papers. Because this was in a previous century, when very few publications were online, whenever he found a listing that looked promising, he had to go to the library to find the relevant journal. Eventually he found Ishino’s paper.
The E. coli bacterium that Ishino studied is a very different organism from Mojica’s archaea. So it was surprising that they both had these repeated sequences and spacer segments. This convinced Mojica that the phenomenon must have some important biological purpose. In a paper he published in 1995, he and his thesis advisor dubbed them “tandem repeats,” and they guessed, incorrectly, that they might have something to do with cell replication.3
After doing two quick postdoctoral stints, one in Salt Lake City and the other at Oxford, Mojica returned in 1997 to the University of Alicante, which was just a few miles from where he was born, and launched a research group to study these mysterious repeated sequences. It was difficult to get funding. “I was told to stop obsessing about repeats, because there were a lot of those type of phenomena in organisms, and mine were probably nothing special,” he says.
But he knew that bacteria and archaea have small amounts of genetic material. They cannot afford to waste a lot of it on sequences that have no important function. So he kept trying to figure out the purpose of these clustered repeats. Perhaps they helped shape the DNA structure or formed loops that proteins could latch on to. Both of those speculations also proved wrong.
The name “CRISPR”
By then, researchers had found these repeated sequences in twenty different species of bacteria and archaea, and many different names for them had sprouted. Mojica became dissatisfied with the name his dissertation advisor had foisted on him, “tandem repeats.” The sequences were interspaced, not in tandem. So he renamed them, initially, “short regularly spaced repeats,” or SRSR. Though more descriptive, it was an unmemorable name with an unpronounceable acronym.
Mojica had been corresponding with Ruud Jansen of Utrecht University in the Netherlands, who was studying these sequences in tuberculosis bacteria. He had been calling them “direct repeats,” but he agreed that they needed to come up with a better name. Mojica was driving home from his lab one evening when he came up with the name CRISPR, for “clustered regularly interspaced short palindromic repeats.” Although the clunky phrase was almost impossible to remember, the acronym CRISPR was, indeed, crisp and crispy. It sounded friendly rather than intimidating, though the dropped “e” gave it a futurist sheen. When he got home, he asked his wife what she thought of the name. “It sounds like a great name for a dog,” she said. “Crispr, Crispr, come here, pup!” He laughed and decided it would work.
On November 21, 2001, the name was anointed in an email from Jansen in reply to Mojica’s suggestion. “Dear Francis,” he wrote, “What a great acronym is CRISPR. I feel that every letter that was removed in the alternatives made it less crispy, so I prefer the snappy CRISPR over SRSR and SPIDR.”4
Jansen formalized the decision in a paper he published in April 2002, which reported his discovery of genes that seemed to be associated with CRISPRs. In most organisms that had CRISPRs, the repeated sequences were flanked by one of these genes, which encoded directions for making an enzyme. He named these “CRISPR-associated,” or Cas, enzymes. 5
A virus defense
When Mojica began sequencing the DNA of his salt-loving microbes in 1989, gene sequencing was a slow process. But the Human Genome Project, which was just getting started, eventually spawned new high-speed sequencing methods. By 2003, when Mojica focused on figuring out the role CRISPRs played, the genomes of close to two hundred bacteria had been sequenced (as well as those of humans and mice).
That August, Mojica was on holiday in the beach town of Santa Polo, about twelve miles south of Alicante, staying at the house of his wife’s parents. That was not his idea of a good time. “I really do not like sand or being on a beach in the summer when it is hot and crowded with people,” he says. “My wife would be lying on the beach getting a suntan, and I would head off and drive up to my lab in Alicante for the day. She had fun on the beach, but I had more fun analyzing sequences from E. coli bacteria.”6 Spoken like a dedicated scientist.
What fascinated him were the “spacers,” those regions of normal-looking DNA segments that were nestled in between the repeated CRISPR segments. He took the spacer sequences of E. coli and ran them through databases. What he found was intriguing: the spacer segments matched sequences that were in viruses that attacked E. coli. He found the same thing when he looked at other bacteria with CRISPR sequences; their spacer segments matched those of viruses that attacked that bacteria. “Oh my goodness!” he exclaimed at one point.
One evening, when he was sure about his discovery, he explained it to his wife after he got back to the beach house. “I just discovered something really amazing,” he said. “Bacteria have an immune system. They’re able to remember what viruses have attacked them in the past.” She laughed, admitted she didn’t quite understand, but said she believed it must be important because he was so excited. He replied, “In a few years, you’ll see this thing that I’ve just discovered will be written about in newspapers and in history books.” That part she did not believe.
* * *
What Mojica had stumbled upon was a battlefront in the longest-running, most massive and vicious war on this planet: that between bacteria and the viruses, known as “bacteriophages” or “phages,” that attack them. Phages are the largest category of virus in nature. Indeed, phage viruses are by far the most plentiful biological entity on earth. There are 1031 of them—a trillion phages for every grain of sand, and more than all organisms (including bacteria) combined. In one milliliter (0.03 ounces) of seawater there can be as many as 900 million of these viruses.7
As we humans struggle to fight off novel strains of viruses, it’s useful to note that bacteria have been doing this for about three billion years, give or take a few million centuries. Almost from the beginning of life on this planet, there’s been an intense arms race between bacteria, which developed elaborate methods of defending against viruses, and the ever-evolving viruses, which sought ways to thwart those defenses.
Mojica found that bacteria with CRISPR spacer sequences seemed to be immune from infection by a virus that had the same sequence. But bacteria without the spacer did get infected. It was a pretty ingenious defense system, but there was something even cooler: it appeared to adapt to new threats. When new viruses came along, the bacteria that survived were able to incorporate some of that virus’s DNA and thus create, in its progeny, an acquired immunity to that new virus. Mojica recalls being so overcome by emotion at this realization that he got tears in his eyes.8 The beauty of nature can sometimes do that to you.
It was an astonishing and elegant discovery, one that would have great repercussions. But Mojica had a ridiculously difficult time getting it published. He submitted a paper to Nature in October 2003 entitled “Prokaryotic Repeats Are Involved in an Immunity System.” In other words, CRISPR systems were a way that bacteria acquired immunity to viruses. The editors did not even send it out for review. It did not contain, they incorrectly judged, much that wasn’t in previous CRISPR papers. They also declared, with more validity, that Mojica had not presented any lab experiments showing how the CRISPR system worked.
