“Precision editing of any genome is within reach.”27
I. The late Roger Hendrix, a renowned microbiologist, came up with the estimate of 1031 phage (10,000,000,000,000,000,000,000,000,000,000) on the planet, making them the most prevalent biological entity.
II. In 2020, Rotem Sorek’s group at the Weizmann Institute of Science in Israel reported a new bacterial back-up anti-phage defense system called retrons.
III. PAM stands for Protospacer Adjacent Motif. Different Cas enzymes recognize different PAMs, ranging from three to six bases. The most commonly used Cas9, from Streptococcus pyogenes, recognizes a triplet sequence, NGG, where N can be any of the four bases.
IV. Cas9 actually has two active sites, providing two separate cutting actions, one for each strand of the double helix.
V. ZFN, zinc finger nuclease; TALEN, transcription activator-like effector nuclease (see chapter 8).
VI. It will never catch on, but Patrick Harrison, a geneticist at Trinity College, Dublin, came up with a modified definition of CRISPR, a mnemonic that explains the editing/repair process: Cut—Resect—Invade—Synthesis—Proofread—Repair. On Last Week Tonight, comedian John Oliver had his own irreverent definition: Crunchy-Rectums-In-Sassy-Pink-Ray-Bans.
CHAPTER 3 WE CAN BE HEROES
Great moments in technology and science can emerge from the most unlikely sources. In 1966 Playtex, the company behind the iconic Cross Your Heart bra, entered a NASA competition to design the spacesuit for the first Apollo moon landing. The suits had to be able to withstand pressure and extreme temperature swings. They also had to be flexible, an attribute that Playtex handsomely demonstrated by filming one of its technicians playing American football for hours while sporting a spacesuit. Thus, it came to pass that four Playtex seamstresses sewed the twenty-one-layer A7L spacesuit that Neil Armstrong fashioned on the lunar runway.1
From the Sea of Tranquility on the moon to the salterns of Santa Pola off the Mediterranean. I’m visiting Alicante, a popular tourist resort on the Costa Blanca in southeast Spain. It is an unlikely candidate for one of the more extreme habitats of life on planet earth. But drive south about fifteen miles, you reach Las Salinas de Santa Pola. Salterns, or salt flats, are as the name suggests, a network of rectangular lagoons characterized by extreme salt concentrations resulting from intense sun and wind. At the perimeter, where the water meets land, the salt crystallizes out, forming a crusty white band like the rim of a perfect margarita.
This place has ecological, historical, and commercial significance. Flamingos and other wildlife abound. A watchtower dates back to the 16th century, where the lookouts of King Felipe II kept watch against the Moors. It looks like a nature reserve, but this is an industrial salt mine. Each lake is the size of a football field, concentrating the salt in stepwise fashion. Today, Bras del Port extracts on average 4,000 tons of salt daily from the Mediterranean Sea. A veritable mountain of salt sits ready for distribution: about 60 percent will be used for water treatment, the rest is for food.
For Francisco Mojica, a microbiologist at the University of Alicante, the study of the halophilic life that thrives in this peculiar habitat is his passion. Where he once toiled in obscurity, today he struggles to escape the media spotlight. In 2017, the leading Spanish newspaper, El País, speculated whether Mojica would make it from the salterns to the Nobel spa.
Mojica has kindly agreed to drive me in his unflashy Volkswagen Passat to las Salinas. It is a journey he makes quite frequently, usually in the company of photographers or film crews who direct him to survey the pristine pink waters or hold a flask of salt water up to the Spanish sun as if for the very first time, like admiring a glass of rioja. In a delicious irony, Mojica never actually collected samples for his own research because as a young graduate student, there were already samples in the lab, taken by his boss a decade earlier.
Mojica first came to the salterns after he had finished military service in 1989 and was looking for a research position. He was offered a PhD position in the microbiology department at the local university in a lab that studied a microbe called Haloferax. “I didn’t have an interest in particular with these organisms. My boss decided the issue of my thesis work,” he told me as we stroll around the salterns.2
Haloferax is not a bacterium (although confusingly it used to be called Halobacterium) but belongs to a distinct group of single-cell organisms called Archaea. To the naked eye there is little to distinguish the two clades. But that belies an evolutionary chasm of some three billion years. The appreciation that Archaea are not just a superficial off-branch of prokaryotes but an entirely separate “third domain” of life is due to the seminal work of evolutionary biologist Carl Woese. DNA sequencing revealed striking genetic differences between Archaea and bacteria, like comparing the operating systems of a Mac and a PC. Ed Yong put it nicely: “It was as if everyone was staring at a world map, and Woese had politely shown that a full third of it had been folded underneath.”3
The water is pink and the salty crusts of the lagoons are drizzled with pinkish red bands that teem with microscopic life. The source of the reddish hue is the production of carotenoids, part of the microbial defense mechanism against salt and sunlight. “It’s like a sunscreen,” Mojica laughs. The color changes with salinity: red shifts to pink as the salt concentration rises from 10 to 30 percent. The same chemicals give the flamingos their trademark pink plumage as they feed on the tiny brine shrimp that, like the Haloferax, thrive in these salty waters.
The salt-loving Archaea of Alicante are by definition extremophiles—lifeforms that are adapted to live in unusually harsh habitats, whether it be underwater volcanic vents, parched deserts, or frozen tundra. For Haloferax, the level of salt in regular seawater just doesn’t cut it: they require ten times as much salt to thrive. Trying to replicate those conditions in the lab is extremely difficult, thus they remain poorly understood compared to their bacterial distant cousins. The two main Haloferax species here are H. mediterranei and H. volcanii (the latter named not because they’ve mistaken a salt lake for a volcanic vent but after the Israeli scientist who discovered them, Benjamin Volcani). I can also smell the presence of anaerobic bacteria responsible for the strong sulfurous odor that wafts over the water.
Mojica’s obsession with the salt-loving microbes of Santa Pola is the embodiment of basic research. “It was knowing by knowing, to expand knowledge,” he says.4 Buried in the circular genetic code of Haloferax, Mojica reasoned, must lie a clue to explain its love of salt. This was not so straightforward: the first complete microbial genome sequence wasn’t reported until 1995, by Claire Fraser and Craig Venter’s group, which also decoded the first complete Archaea genome two years later. Mojica’s lab was not a flashy genome center with the latest DNA sequencing hardware. In the early 1990s, sequencing for many researchers was still a cumbersome manual process, which involved making a large gel sandwich between two glass plates, then separating radioactively labeled DNA fragments by size in an electric current. From the resulting ladder imaged on an X-ray film, Mojica could spell out the corresponding DNA sequence.
In one of his first sequencing attempts, in August 1992, Mojica saw something so surprising, he assumed he’d messed up the experiment. He saw weird repetitive sequences, each about 30 bases long, which he duly noted in his first paper.5 “We were absolutely lucky,” he said, after sequencing less than 1 percent of the Haloferax genome. “It was the first paper where CRISPR was taken seriously!” he says.I Mojica also showed that the repeats were surprisingly transcribed into RNA, suggesting they had some sort of function.