"The Code Breaker"

[fiefoe]

Walter Isaacson organizes his material in a very lucid way, and the biology part is interesting. Too bad once the discoveries are done, politics takes over for the rest of the book.

• It was fitting that a virus-fighting team would be led by a CRISPR pioneer. The gene-editing tool that Doudna and others developed in 2012 is based on a virus-fighting trick used by bacteria, which have been battling viruses for more than a billion years. In their DNA, bacteria develop clustered repeated sequences, known as CRISPRs, that can remember and then destroy viruses that attack them.
• Many creative people—including most of those I have chronicled, such as Leonardo da Vinci, Albert Einstein, Henry Kissinger, and Steve Jobs—grew up feeling alienated from their surroundings.
• In the book, Watson dramatized (and overdramatized) how as a twenty-four-year-old bumptious biology student from the American Midwest he ended up at Cambridge University in England, bonded with the biochemist Francis Crick, and together won the race to discover the structure of DNA in 1953. Written in the sparky narrative style of a brash American who has mastered the English after-dinner art of being self-deprecating and boastful at the same time, the book manages to smuggle a large dollop of science into a gossipy narrative about the foibles of famous professors, along with the pleasures of flirting, tennis, lab experiments, and afternoon tea.
• as Jennifer was growing up he became more aware that the books he assigned to his class were mostly by men. So he added Doris Lessing, Anne Tyler, and Joan Didion to his syllabus.
• He later said that one of the most important lessons his parents taught him was “Hypocrisy in search of social acceptance erodes your self-respect.” He learned it too well. From his childhood into his nineties, he was brutally outspoken in his assertions, both right and wrong,
• Wilkins specialized in crystallography and X-ray diffraction. In other words, he took a liquid that was saturated with molecules, allowed it to cool, and purified the crystals that formed. Then he tried to figure out the structure of those crystals. If you shine a light on an object from different angles, you can figure out its structure by studying the shadows it casts. X-ray crystallographers do something similar: they shine an X-ray on a crystal from many different angles and record the shadows and diffraction patterns. In the slide that Wilkins showed at the end of his Naples speech, that technique had been used on DNA.
• “Suddenly I was excited about chemistry,” Watson recalled. “I knew that genes could crystallize; hence they must have a regular structure that could be solved in a straightforward fashion.”
• “I have never seen Francis Crick in a modest mood.” It was a line that could likewise have been written of Watson, and they admired each other’s immodesty more than their colleagues did. “A youthful arrogance, a ruthlessness, and an impatience with sloppy thinking came naturally to both of us,” Crick recalled.
• The sexist structure at King’s helped keep them apart: there were two faculty lounges, one for men and the other for women, the latter unbearably dingy and the former a venue for elegant lunches.
• Franklin was a focused scientist, sensibly dressed. As a result she ran afoul of English academia’s fondness for eccentrics and its tendency to look at women through a sexual lens,
• There was an exciting consequence of this structure: when the two strands split apart, they could perfectly replicate, because any half-rung would attract its natural partner. In other words, such a structure would permit the molecule to replicate itself and pass along the information encoded in its sequences.
• But like many famous siblings, DNA doesn’t do much work. It mainly stays at home in the nucleus of our cells, not venturing forth. Its primary activity is protecting the information it encodes and occasionally replicating itself. RNA, on the other hand, actually goes out and does real work. Instead of just sitting at home curating information, it makes real products, such as proteins. Pay attention to it. From CRISPR to COVID, it will be the starring molecule in this book and in Doudna’s career.
• In work done in the early 1980s that would win them the Nobel Prize, they made the surprising discovery that some forms of RNA could likewise be enzymes. Specifically, they found that some RNA molecules can split themselves by sparking a chemical reaction. They dubbed these catalytic RNAs “ribozymes,”
• Doudna realized that she would need to learn more about structural biology if she wanted to truly understand how some RNA molecules could reproduce themselves. “To figure out how these RNA do chemistry,” she says, “I needed to know what they looked like.” Specifically, she needed to figure out the folds and twists of the three-dimensional structure of self-splicing RNA... Doudna also sensed that once you figured out the structure of a ribozyme, it might lead to groundbreaking genetic technologies. The citation for the Nobel Prize that Thomas Cech won with Sidney Altman hinted at what this might be: “A futurist possibility is to correct certain genetic disorders. Such a future use of gene shears will require that we learn more about the molecular mechanisms.” Gene shears. Yes, the Nobel committee was prescient.
• She recalled the times she had visited his classroom and seen his excitement at communicating his passions. She also recalled, less happily, the times that she had gotten angry at him because she thought he made snap judgments, some of them prejudiced, about people. Bonds can take different forms, both in chemistry and in life. Sometimes an intellectual bond is the strongest.
• 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.
• 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.
• 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,”
• 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.
• 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
• Until then, CRISPRs had largely been the purview of microbiologists, such as Mojica and Banfield, who studied living organisms. They had come up with elegant theories about CRISPR, some of them correct, but they had not done controlled experiments in test tubes. “At the time, nobody had actually isolated the molecular components of the CRISPR system, tested them in a lab, and figured out their structures,” Doudna said. “So the time was right for biochemists and structural biologists like me to jump in.”3
• Although Wiedenheft was able to sequence the DNA of the viruses, he found himself wanting more. “Once I started peering at the DNA sequences, I realized they were uninformative,” he says. “We had to determine structures, because structures, the folds and shapes, are conserved over a longer evolutionary period than the nucleic acid sequences.” In other words, the sequence of letters in the DNA did not reveal how it worked; what was important was how it folded and twisted, which would reveal how it interacted with other molecules.
• In Conti’s lab, Jinek developed a passion for the star molecule of this book, RNA. “It’s such a versatile molecule—it can do catalysis, it can fold into 3D structures,” he later told Kevin Davies of the CRISPR Journal. “At the same time, it’s a carrier of information. It’s an all-rounder in the world of biomolecules!”
• By 2008, scientists had discovered a handful of enzymes produced by genes that are adjacent to the CRISPR sequences in a bacteria’s DNA. These CRISPR-associated (Cas) enzymes enable the system to cut and paste new memories of viruses that attack the bacteria. They also create short segments of RNA, known as CRISPR RNA (crRNA), that can guide a scissors-like enzyme to a dangerous virus and cut up its genetic material. Presto! That’s how the wily bacteria create an adaptive immune system!
  The notation system for these enzymes was still in flux in 2009, largely because they were being discovered in different labs. Eventually they were standardized into names such as Cas1, Cas9, Cas12, and Cas13.
• Science can be the parent of invention. But as Matt Ridley points out in his book How Innovation Works, sometimes it’s a two-way street. “It is just as often the case that invention is the parent of science: techniques and processes are developed that work, but the understanding of them comes later,” he writes. “Steam engines led to the understanding of thermodynamics, not the other way round. Powered flight preceded almost all aerodynamics.”
• They then accomplished something very useful: they showed that they could engineer this immunity by devising and adding their own spacers. The French research facility was not approved for genetic engineering, so Barrangou did that part of the experiments in Wisconsin. “I showed that when you add sequences from the virus into the CRISPR locus, the bacteria develops immunity to that virus,” he says.5 In addition, they proved that CRISPR-associated (Cas) enzymes were critical for acquiring new spacers and warding off attacking viruses. “What I did was knock out two Cas genes,” Barrangou recalls. “That wasn’t easy to do twelve years ago. One of them was Cas9, and we showed when you knock it out you lose the resistance.”
  They used these discoveries in August 2005 to apply for and get one of the first patents granted for CRISPR-Cas systems. That year Danisco started using CRISPR to vaccinate its bacterial strains.
• In other words, CRISPR did not work through RNA interference, which had been the general consensus when Banfield first approached Doudna. Instead, the CRISPR system targeted the DNA of the invading virus.
  That had a holy-cow implication. As Marraffini and Sontheimer realized, if the CRISPR system was aimed at the DNA of viruses, then it could possibly be turned into a gene-editing tool. That seminal discovery sparked a new level of interest in CRISPR around the world. “It led to the idea that CRISPR could be fundamentally transformative,” Sontheimer says. “If it could target and cut DNA, it would allow you to fix the cause of a genetic problem.”
• They had also established the essential role of another part of the complex: CRISPR RNAs, known as crRNAs. These are the small snippets of RNA that contain some genetic coding from a virus that had attacked the bacteria in the past. This crRNA guides the Cas enzymes to attack that virus when it tries to invade again. These two elements are the core of the CRISPR system: a small snippet of RNA that acts as a guide and an enzyme that acts as scissors.
  But there was one additional component of the CRISPR-Cas9 system that played an essential role—or, as it turned out, two roles. It was dubbed a “trans-activating CRISPR RNA,” or tracrRNA, pronounced “tracer-RNA.” Remember this tiny molecule; it will play an outsized role in our tale. That’s because science is most often advanced not by great leaps of discovery but by small steps. And disputes in science are often about who made each one of these steps—and how important each really was. This would turn out to be the case for the discoveries involving tracrRNA.
  It turns out that tracrRNA performs two important tasks. First, it facilitates the making of the crRNA, the sequence that carries the memory of a virus that previously attacked the bacteria. Then it serves as a handle to latch on to the invading virus so that the crRNA can target the right spot for the Cas9 enzyme to chop.
• The study of CRISPR would become a vivid example of the call-and-response duet between basic science and translational medicine. At the beginning it was driven by the pure curiosity of microbe-hunters who wanted to explain an oddity they had stumbled upon when sequencing the DNA of offbeat bacteria. Then it was studied in an effort to protect the bacteria in yogurt cultures from attacking viruses. That led to a basic discovery about the fundamental workings of biology. Now a biochemical analysis was pointing the way to the invention of a tool with potential practical uses. “Once we figured out the components of the CRISPR-Cas9 assembly, we realized that we could program it on our own,” Doudna says. “In other words, we could add a different crRNA and get it to cut any different DNA sequence we chose.”
• Researchers were able to devise proteins that could serve as a guide to get the cutting domain to a targeted DNA sequence. One system, zinc-finger nucleases (ZFNs), came from fusing the cutting domain with a protein that has little fingers shaped by the presence of a zinc ion, which allow it to grasp on to a specified DNA sequence. A similar but even more reliable method, known as TALENs (transcription activator–like effector nucleases), came from fusing the cutting domain with a protein that could guide it to longer DNA sequences.
  Just when TALENs were being perfected, CRISPR came along. It was somewhat similar: it had a cutting enzyme, which was Cas9, and a guide that led the enzyme to cut a targeted spot on a DNA strand. But in the CRISPR system, the guide was not a protein but a snippet of RNA. This had a big advantage. With ZFNs and TALENs, you had to construct a new protein guide every time you wanted to target a different genetic sequence to cut; it was difficult and time consuming. But with CRISPR you merely had to fiddle with the genetic sequence of the RNA guide. A good student could do it quickly in a lab.
• Tall and gangly, George Church looks like, and actually is, both a gentle giant and a mad scientist. He is one of those iconic characters who is equally charismatic on Stephen Colbert’s TV show and in his bustling Boston lab amid a gaggle of adoring researchers. Always calm and genial, he has the amused demeanor of a time traveler who is eager to get back to the future. With his wild-man beard and halo of hair, he looks like a cross between Charles Darwin and a woolly mammoth, an extinct species that he wants, perhaps out of a vague sense of kinship, to resurrect using CRISPR.1
• Zhang had barely heard of CRISPR, but ever since his seventh-grade enrichment class he had learned to perk up at the mention of enzymes. He was particularly interested in those enzymes, known as nucleases, that cut DNA. So he did what any of us would do: he googled CRISPR.
• It’s part of the atmosphere at the Broad. Whiteboarding is like a sport, the way foosball is in less rarefied offices.
• In this competition to adapt CRISPR into a gene-editing tool in humans, Zhang and Doudna came into the arena from different routes. Zhang had never worked on CRISPR. People in that field would later refer to him as a latecomer and interloper, one who jumped on CRISPR after others had pioneered the field. Instead, his specialty was gene editing, and for him CRISPR was simply another method to get to the same goal, along the lines of ZFNs and TALENs, though much better. For her part, Doudna and her team had never worked on gene editing in living cells. Their focus for five years had been on figuring out the components of CRISPR. As a result, Zhang would end up having some difficulty in sorting out the essential molecules in a CRISPR-Cas9 system, while Doudna’s difficulty would be figuring out how to get the system into the nucleus of a human cell.
• The excitement over the potential of CRISPR provoked all of the major players to begin square dancing, forming groups and swapping partners in the quest to create companies that would commercialize CRISPR for medical applications.
• The most vibrant and viral responses came from one of Doudna’s high-octane colleagues at Berkeley, genetics professor Michael Eisen. “There is something mesmerizing about an evil genius at the height of their craft, and Eric Lander is an evil genius at the height of his craft,” he wrote and posted publicly a few days after the article appeared. He called the piece “at once 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.”
• The modern biotechnology industry was born a century later, when a Stanford attorney approached Stanley Cohen and Herbert Boyer and convinced them to file for a patent on the method they had discovered for manufacturing new genes using recombinant DNA. Many scientists, including Paul Berg, the discoverer of recombinant DNA, were horrified at the idea of patenting a biological process, but the royalties that flowed to the inventors and their universities quickly made biotech patents popular. Stanford, for example, made $225 million in twenty-five years by granting hundreds of biotech companies non-exclusive licenses to the Cohen-Boyer patents.
• doctors used a tiny hair-width tube to inject three drops of fluid containing CRISPR-Cas9 into the lining that contains light-sensing cells directly beneath the retina of the patient’s eyes. A tailored virus was used as the delivery vehicle to transport the CRISPR-Cas9 into the targeted cells. If the cells are edited as planned, the fix will be permanent, because unlike blood cells, the cells of the eye do not divide and replenish themselves.
• “Why is Eric so intent on publicly pushing for a moratorium?” Margaret Hamburg, the co-chair of the World Health Organization group, asked me. It was a sincere question. Lander’s reputation was such that even when he did something that seemed straightforward, others suspected his motives. The call for a moratorium, she felt, seemed like showboating; it was unnecessary, since both the WHO and the national academies were already embarked on figuring out proper guidelines rather than calling a halt to germline editing.
• Instead of being acclaimed a national hero, as he had fantasized, He Jiankui was put on trial at the end of 2019 in the People’s Court of Shenzhen. The proceedings had many elements of a fair trial: he was permitted to have his own attorneys and to speak in his own defense. But the verdict was not in doubt since he had pleaded guilty to the charge of “illegal medical practice.”
• In the case of gene editing, I think the germline is indeed a real line. There may not be a razor-sharp line differentiating it from other biotechnologies, but as Leonardo da Vinci taught us with his sfumato, even slightly blurry lines can be definitive. Crossing the germline takes us to a distinct new realm. It involves engineering a genome rather than nurturing one that was produced naturally, and it introduces a change that will be inherited by all future descendants.
  Nevertheless, this doesn’t mean the germline should never be crossed. It simply means that we can view the germline as a firebreak that gives us a chance to pause, if we decide we ought to, the advance of genetic engineering techniques.
• Another line we might consider, in addition to that between somatic and germline editing, involves the distinction between “treatments” designed to fix dangerous genetic abnormalities and “enhancements” designed to improve human capacities or traits.
• Take Miles Davis. The pain of sickle cell drove him to drugs and drink. It may have even driven him to his death. It also, however, may have driven him to be the creative artist who could produce Kind of Blue and Bitches Brew. Would Miles Davis have been Miles Davis without sickle cell?
• That doesn’t answer the question of whether we should allow genetic enhancements. But as we grope for a set of principles to include in our moral calculus, the distinction does point to a factor we should consider: favoring enhancements that would benefit all of society over those that would give the recipient a positional advantage.
• Viruses are deceptively simple little capsules of bad news.I They are just a tiny bit of genetic material, either DNA or RNA, inside a protein shell. When they worm their way into a cell of an organism, they can hijack its machinery in order to replicate themselves. In the case of coronaviruses, the genetic material is RNA, Doudna’s specialty. In SARS-CoV-2, the RNA is about 29,900 base letters long, compared to more than three billion in human DNA. The viral sequence provides the code for making a mere twenty-nine proteins.
• A few days later, the FDA responded by requiring him to do more trials to see if the test he was using inadvertently detected the MERS and SARS viruses, even though they had been dormant for years and he had no samples of those viruses to test. When he called the CDC to see if he could get a sample of the old SARS virus, it refused. “That’s when I thought, ‘Huh, maybe the FDA and the CDC haven’t talked about this at all,’ ” Greninger told reporter Julia Ioffe. “I realized, Oh, wow, this is going to take a while.”

Genetic vaccines:
* One method for doing this is by taking a harmless virus and engineering into it a gene that will make the desired component. As we all now know, viruses are very good at worming their way into human cells. That is why safe viruses can be used as a delivery system, or vector, to transport material into the cells of patients.

* Instead of engineering the gene for the component into a virus, you can just deliver the genetic code for the component—as DNA or RNA—into human cells. The cells thus become a vaccine-manufacturing facility... The idea was that if it could get inside the nucleus of a cell, the DNA could very efficiently churn out many strands of messenger RNA to go forth and oversee the production of the spike protein parts, which serve to stimulate the immune system. DNA is cheap to produce and do not require dealing with live viruses and incubating them in chicken eggs. <> The big challenge facing a DNA vaccine is delivery. How can you get the little ring of engineered DNA not only into a human cell but into the nucleus of the cell?

* An RNA vaccine has certain advantages over a DNA vaccine. Most notably, the RNA does not need to get into the nucleus of the cell, where DNA is headquartered. The RNA does its work in the outer region of cells, the cytoplasm, which is where proteins are constructed. So an RNA vaccine simply needs to deliver its payload into this outer region.