The obvious choice was E. coli, the lab workhorse. But which CRISPR system? It turns out S. thermophilus has four different CRISPR systems—all have a cluster of spacers but differ in the architecture and adjacent Cas genes. Šikšnys chose the simplest (type II) system with the smallest number of Cas genes, including one called Cas9, known to be required for phage resistance. With his intimate knowledge of DNA-cutting enzymes, Šikšnys noticed a couple of spots in the Cas9 structure that resembled catalytic active sites he’d observed in restriction enzymes. It was a sign that Cas9 might have interesting DNA-cutting properties of its own.
The Lithuanian’s entry into the CRISPR timeline was one of many new dots on the map. But it took five years to connect the dots from yogurt and pizza starter cultures to the brink of a universal gene-editing technology. The next steps were to answer some practical questions: How were the CRISPR spacers captured from the phage invaders and stitched into the bacterial genome? And how were they weaponized to target and destroy incoming phage?
Moineau’s group first described a key recognition sequence,16 the critical first touchpoint of Cas9 alighting on DNA. As phages mutated or “escaped” CRISPR-mediated destruction, Moineau catalogued a series of single-base mutations in the CRISPR repeats as well as this recognition motif, which Mojica dubbed the proto-spacer adjacent motif, or PAM.17
Over the next few years, researchers around the world began piecing together the molecular details of the CRISPR immune system. It was like the British children’s party game pass the parceclass="underline" at each round the music stopped with a different participant peeling off another layer of wrapping before revealing the gift. After documenting CRISPR in her own microbial samples,18 Banfield reached out to Barrangou to propose they organize a meeting of CRISPR disciples. That summer in 2008, about thirty scientists gathered at Berkeley’s Stanley Hall. Members of Doudna’s lab, located on the seventh floor of the same building, dropped in. Barrangou put the wine and beer costs on his corporate credit card.
The action next shifted to the Netherlands. Studying CRISPR in E. coli, John van der Oost, a microbiologist at Wageningen University, and Stan Brouns showed that the CRISPR spacers are first transcribed into a long contiguous RNA, which is then sliced into discrete CRISPR RNAs corresponding to individual spacers. This RNA then forms a complex with Cas proteins that targets the corresponding phage.19 (The E. coli CRISPR is of the type I variety, in which the role of Cas9 is played by a complex of five proteins known as the Cascade complex.) The result was a CRISPR milestone, duly recognized a decade later when van der Oost shared Holland’s most prestigious science award, the Spinoza Prize. “I don’t think John’s work will ever be forgotten,” says Koonin, the unofficial master record-keeper of CRISPR gene evolution.20
In Chicago, Erik Sontheimer and his Argentine postdoc, Luciano Marraffini, designed some clever experiments using Staphylococcus epidermis to settle another big question: does CRISPR-Cas target the viral RNA—mimicking RNAi—or DNA? Marraffini suspected it would be more efficient for bacteria to dispose of viral infections if they cleaved DNA, taking a machete to the viral genome in one fell swoop. He was correct.21 Practically speaking, they wrote, “the ability to direct the specific addressable destruction of DNA that contains any given 24-48 [base] target sequence could have considerable functional utility, especially if the system can function outside of its native bacterial context.”
Sontheimer and Marraffini saw a glimmer of clinical relevance—the possibility “to impede the ever-worsening spread of antibiotic resistance genes and virulence factors in staphylococci and other bacterial pathogens.” It was one small step on the road to precise CRISPR genome editing, albeit one that exclusively used the delete button. “We were the first to recognize and explicitly articulate the possibility that CRISPR could be repurposed for genome engineering,” said Sontheimer.22 But the celebration was short-lived: Sontheimer’s patent application was denied for lack of experimental evidence, as was an ambitious grant application that was years ahead of its time. (As we shall see in chapter 12, it was the beginning of a contentious legal battle for inventorship.)
As interest in CRISPR grew, additional types of CRISPR systems were discovered. At the University of Georgia, Michael Terns showed that type III CRISPR systems target RNA rather than DNA. Meanwhile, Doudna published her first papers on CRISPR with Wiedenheft and Haurwitz.
In 2010, Moineau, who has been fascinated by phages since he first saw them under an electron microscope, took center stage. “If you go to the ocean and take water in the palm of your hands, you have more viruses in your hands than there are humans on this planet,” he says.23 His forte was the phages that infect S. thermophilus—the key ingredient for yogurt and cheese. Moineau says we eat more than one sextillion (1021) S. thermophilus cells per year.
Moineau’s interest is the ongoing arms race between phage and bacteria. Still collaborating with Horvath, Barrangou, and the Danisco team, Moineau’s lab demonstrated that CRISPR RNAs cleaved their DNA targets directly, literally cutting a circular DNA molecule (a plasmid inside a bacterium) in one spot into a linear fragment, producing clean blunt ends in the vicinity of the PAM sequence.24 Moineau published the story in Nature, one of the highlights of his career.
Meanwhile, researchers continued to expand the family of Cas genes by combing through the expanse of microbial life on earth, much of the credit belonging to Koonin and Makarova. The number of different CRISPR classes and subtypes has grown increasingly complex, with six classes known by 2020. Fortuitously, the Strep bacteria studied by Horvath and Barrangou have a type II system, the most rudimentary of CRISPR systems. And that was huge.
The first time I emailed Emmanuelle Charpentier, in early 2017, I received an immediate response. It was an “out of office” message that said:
Due to my full schedule associated with attendance of prize ceremonies, I will not be able to reply to your email…
Well, that was a first. She wasn’t kidding. Charpentier has won dozens of prestigious awards since 2012. Asked about the distraction of being a semi-permanent fixture on the awards circuit, she told Le Figaro: “It’s very exotic! Let’s say that’s not the reason I did research. As a researcher, I like being isolated in my laboratory with my team.”25 Her PhD supervisor said she was so resourceful, she could start a lab on a desert island.26 It didn’t quite come to that, but after two decades of nomadic existence—moving from the United States to Austria to Sweden—she has found a home in Berlin. Time will tell if she can match the success she earned in 2012.
Charpentier may have a lower public profile in the United States than Doudna or Zhang, but it is a different story in Europe. A 2015 profile in Le Monde dubbed her “the charming little monster” of genetic engineering, with “the air of a mockingbird, perched in the forest of the best-rooted authorities of science in France, Europe, and America.”27 She is called “pugnacious and courageous,” inspired by three muses—curiosity, daring, and freedom. The following year, the same newspaper, in an article titled “The new icons of biology,” hailed Charpentier and Doudna as the “Thelma and Louise” of biomedical research, acquiring scientific honors and prizes like a squirrel harvests hazelnuts.28