The Code Breaker: Book Review

The Code Breaker: Jennifer Doudna, Gene Editing and The Future of the Human Race (2021), Walter Isaacson. This was a great book, explaining a difficult subject, introducing both a history and major players in addition to Doudna—and a bit about the processes involved. The Covid pandemic is a recent crisis, one dramatically involving CRISPR (discovered by Doudna and others), both for testing and effective vaccines. Isaacson included this in the book, apparently until close to the publication date.

After I read it, I thought I knew quite a bit about CRISPR. After a couple of weeks, it was fading and I was unable to really explain it coherently. That meant a book review. It doesn’t have much directly related to MVG, except that politicians should stay the hell away from science—of course, Trump and his ilk did not. There are also some points that were generalizable about key concepts like critical thinking skills and how basic science can lead to major discoveries decades later. (I ignore most of the personal information.)

The book actually starts out with the impact of the coronavirus pandemic on Doudna’s Berkeley Lab, when the campus was shut down. “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 developed clustered repeated sequences, known as CRISPRs, that can remember and then destroy viruses that attack them. In other words, it’s an immense system that can adapt itself to fight each new wave of viruses” (p. xiv). Doudna assembled teams to first create corona testing based on CRISPR, who then had to collaborate by Zoom.

RNA does the work of building proteins based on DNA coding. Doudna in 2012 used CRISPR to edit genes (which can potentially cure sickle-cell anemia, cancers, blindness). “The key to innovation is connecting a curiosity about basic science to the practical work of devising tools that can be applied to our lives” (p. xix).

Part One: The Origins of Life.

Chapter 1: Hilo. Watson’s Double Helix memoir inspires Doudna; Isaacson covers Darwin, Mendel, Crick and Watson (and Rosaland Franklin), and beyond. Chemical molecules determine what biological role it can play; chemistry becomes biology as atoms bond to create molecules.

Chapter 2: The Gene. “Darwin and Wallace had a key trait that is a catalyst for creativity: they had wide-ranging interests and were able to make connections between different disciplines” (p. 12). Mendel experimented with what Wilhelm Johansen in 1905 called the gene.

Chapter 3: DNA. Nucleic acids as the “workhorses of heredity;” sugar, phosphates, and bases (adenine, thymine, guanine, and cytosine: A-T and G-C) in RNA and DNA, “Protein-wrapped packages that contain and seek to replicate the genetic material encoded by the nucleic acids” (p. 17): combine genetics, biochemistry and structural biology. Watson studied “phages,” viruses that attack bacteria. Wilkins and Franklin used crystallography and X-ray diffraction. Linus Pauling figured out the structure of proteins using X-ray crystallography, quantum mechanics of chemical bonds, and model building. When the base strands split apart, because they would attract the natural partner, the copying mechanism exists.

Chapter 4: The Education of a Biochemist. In chemistry class, experiments were by recipe. In science, you don’t know the results. Doudna did her graduate work at Harvard, which required working at multiple labs to find the one for dissertation research.

Chapter 5: The Human Genome. The human genome project begun in 1990 was to sequence the three billion base pairs of human DNA and 20,000 genes. Francis Collins was one of the scientists, becoming director of NIH in 2009; the other was Craig Venter, who formed Celera to profit from patenting the discoveries. Some 4,000 diseases were found to be caused by DNA mutations.

Chapter 6: RNA. The goal became to write as well as read genes, involving RNA. “A small segment of DNA that encodes a gene is transcribed into a snippet of RNA, which then travels to the manufacturing region of the cell. There this ‘messenger RNA’ facilitate the assembly of the proper sequence of amino acids to make a specified protein. … Enzymes serve as catalysts. They spark and accelerate and modulate the chemical reactions in all living things” (p. 44).

Chapter 7: Twists and Folds. Structural biology: how chemical reactions cause biological activity. “RNA is made up of very few chemicals [4 chemicals versus proteins which have 20], so it accomplishes complex tasks based on the different ways it is folded” (p. 59). Doudna figured out how RNA could be an enzyme and slice, splice and replicate itself.

Chapter 8: Berkeley. Doudna was interested in how the RNA in some viruses allowed them to hijack the protein-making of other cells like bacteria, RNA interference using “Dicer” which snips RNA into short fragments, then use a scissors-like enzyme to chop it up. She discovered the molecular structure of Dicer. A key finding was that Dicer could be reengineered to turn of various genes. RNA interference could be an option to treat viral infections.

Part Two: CRISPR.

Chapter 9: Clustered Repeats. In gene sequencing: “five highly homologous sequences of 29 nucleotides were arranged as direct repeats” (p. 71); that, is, identical DNA segments. They also were palindromes. These were in 20 species of bacteria and archaea, later called CRISPR (clustered regularly interspaced short palindromic repeats). Other DNA segments between them were called spacers, which proved to match those of viruses that attacked bacteria—this was part of the bacteria immune system. Viruses called “bacteriophages” attacked the bacteria; unfortunately, these are the largest number of biological entities on earth, some trillion for every grain of sand. “The role of the CRISPR-associated enzymes was to grab bits of DNA out of the attacking viruses and insert them into the bacteria’s own DNA” (p. 76).

Chapter 10. The Free Speech Movement Café. These discoveries were made by microbiologists, who studied living organisms. The next step was controlled experiments in test tubes, the purview of Doudna (a structural biologist): isolate the components of CRISPR and test them in a lab and figure out their structure.

Chapter 11. Jumping In. CRISPR used by bacteria in boiling acid springs. How it folded and twisted became important. “Enzymes are a type of protein. Their main function is to act as a catalyst that sparks chemical reactions in the cells of living organisms, from bacteria to humans. They are catalyzed by enzymes. These include breaking down starches and proteins in the digestive system, causing muscles to contract, sending signals between cells, regulating metabolism, and cutting and splicing DNA and RNA” (p. 86).

“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” (p. 86).

Chapter 12: The Yogurt Makers. Harry Truman established the NSF for basic research. Scientists working for a yogurt maker used CRISPR for starter cultures for fermentation of dairy products using bacteria. Bacteria-destroying viruses were a major threat. They had historical records of DNA sequences of bacteria from the 1980s. “They noticed that bacteria that had been collected soon after a big virus attack had new spacers with sequences from those viruses, indicating that these had been acquired to repel future attacks” (p. 92). They were able to engineer their own spacers to develop immunity. They patented the process.

Chapter 13. Genentech. Genentech was created in 1972 by Cohen and Boyer to use recombinant DNA to create hybrids, creating biotechnology. The idea is to splice DNA fragments from different organisms. First up was synthetic insulin. Doudna was involved but went back to the lab as a research scientist. [Here’s where you appreciate scientists, not the corporate environment.]

Chapter 14: The Lab. Scientific discovery: going for research and building a lab to do research, creating a team. Doudna’s approach was to connect small dots to make big pictures; often using the Socratic method. Sternberg on how to use single-molecule fluorescence method to test behavior of CRISPR-associated enzymes; finding out how RNA-guided proteins find target sequences of invading virus.

Chapter 15: Caribou. Looking to turn CRISPR to medicine. Most 20th century advances in drugs were chemical. Genentech changed focus to biotechnology, manipulating living cells to license discoveries to drug companies. Discoveries from Doudna’s lab from Cas6 patented, especially diagnostic tools.

Government-business-university partnership had produced transistors, microchips, computers, GPS, lasers, the internet and search engines. Now NIH funded Doudna to commercialize through Caribou, initially RNA-protein complexes. Also, a Gates grant for viral infections, HIV, hepatitis, flu.

Chapter 16: Emmanuelle Charpentier. Studied CRISPR using Cas9, focus on “persistence and creativity.” “crRNAs are the small snippet 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. … [Plus] trans-activating CRISPR RNA. … 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” (p. 124). Charpentier hypothesis: the tracrRNA directs the creation of short crRNAs. She and her team discovered that viral-defense had only three components: tracrRNA, crRNA and Cas9 enzyme. She then worked with Doudna to isolate the chemical components in a test tube and figure out how each worked.

Chapter 17: CRISPR-Cas9. Mission to make CRISPR-Cas9 chop up DNA of a virus in a test tube, using Cas9 enzyme and crRNA. Then tracrRNA added, which chomped up the DNA: “crRNA contained a 20-letter sequence that acted as a set of coordinates to guide the complex to a piece of DNA with a similar sequence. The tracrRNA, which had helped create this crRNA, now had the additional role of acting like a scaffold that held the other components in just the right place when they glommed on to the target DNA. Then the Cas9 enzyme began slicing away” (p. 132). The enzyme was adaptable; when a new virus emerged, it learned to recognize and destroy it. The crRNA guide could be modified to target any DNA sequence (used as an editing tool). The next step was to create a “single-guide RNA (sgRNA).”

Chapter 18: Science, 2012. The paper describing the project used Dropbox to communicate, with their changes tracked. Doudna and Jinek in California, Charpentier and Chylinski in Umea, then submitted it to Science. It talked about how crRNA and tracrRNA worked to bind the Cas9 protein onto the target DNA; also describing the structure of the two Cas9. “It was the first time researchers had isolated the essential components of the system and discovered their biochemical mechanisms. In addition, the paper contained a potentially useful invention: the single guide RNA” (p. 139).

Chapter 19: Dueling Presentations. Other scientists were working on similar projects as Doudna/Charpentier, but “the Doudna-Charpentier paper, published online on June 28, 2012, galvanized an entire new field of biotechnology: making CRISPR work in the editing of human genes” (p. 149).

Part Three: Gene Editing

Chapter 20: A Human Tool. Recombinant DNA: “The road to engineering human genes began in 1972 when Professor Paul Berg of Stanford discovered a way to take a bit of the DNA of a virus found in monkeys and splice it to the DNA of a totally different virus” (p. 153). Then, discoveries to clone them in the millions. “Gene therapy involved delivering into the patient’s cells some DNA that had been engineered to counteract the faulty gene that caused the disease” (p. 153).

“The goal was to edit the flawed sequence of DNA in the relevant cells of the patient. Thus was born the endeavor called gene editing…. [one of the keys] causing a break in both strands of the DNA double helix. … The invention of gene editing required two steps. First, researchers had to find the right enzyme that could cut a double-strand break in DNA. Then they had to find a guide that would navigate the enzyme to the precise target in the cell’s DNA. … Researchers were able to devise proteins that could serve as a guide to get the cutting domain to a targeted DNA sequence.” (p.154).

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” (p. 155). CRISPR worked in bacteria, which do not have a nucleus. Could it work in human cells, that had a nucleus? Doudna’s lab and others around the world went to work. This proved to be fairly easy.

Chapter 21: The Race. This was called a “healthy rivalry.” Discoveries plus credit for discovery: papers, grants, prizes. Big ones were Darwin-Wallace, Newton-Leibniz, and Pauling-Watson, Crick. Rivals were Doudna, Feng Zhang and the Broad Institute of MIT & Harvard, and George Church of Harvard.

Chapter 22: Feng Zhang. He ran a lab at the Broad Institute near MIT.

Chapter 23: George Church. Church ran a Harvard lab.

Chapter 24: Zhang Tackles CRISPR. The Broad Institute “was founded in 2004 by Eric Lander. … Lander envisioned the Broad as a place where different disciplines would work together. This required a new type of institution, one that fully integrated biology, chemistry, mathematics, computer science, engineering, and medicine” (p. 175). Zhang had never worked on CRISPR (his field was gene editing), but jumped in. Of course, Doudna had never worked on gene editing.

Chapter 25: Doudna Joins the Race. “She had never experimented with human cells, nor had she ever engineered gene-editing tools such as TALENs. … She jumped into what she knew would be a crowded race to take their discoveries about CRISPR-Cas9 and turn it into a tool that would work in human cells. Doudna realized, correctly, that using CRISPR to edit human genes was the next breakthrough waiting to happen” (p. 187). Students had experience, including Alexandra East. It looked like the step to human cells was not difficult.

Chapter 26: Photo Finish. “Biochemistry does not always predict what will actually happen in living cells” (p. 191). Zhang used “codon optimization,” three-letter snippets of DNA. Paper accepted Dec. 12, 2012. Church submitted his paper about the same time and accepted on the same date, Dec. 12.

Chapter 27: Doudna’s Final Sprint. Doudna heard about Zheng and Church and rushed into print. This was a scientific discovery with many groups about the same time. Doudna’s paper appeared on Jan. 29, 2013. Similar papers also showed up, a total of five.

Chapter 28: Forming Companies. Exploring the business potential of CRISPR. Doudna and Church formed CRISPR Therapeutics, then an epic patent battle. A group formed that included Zhang, but Zhang and Broad received a patent first. Three companies competed.

Chapter 29: Mon Amie. Doudna and Charpentier received the Breakthrough Prize in 2014.

Chapter 30. The Heroes of CRISPR. History weaponized: Eric Lander wrote “The Heroes of CRISPR which emphasized Zhang and minimized Doudna. Colleagues came to Doudna’s defense; also called “Whig history;” that is, as a political tool.

Chapter 31: Patents. Patents were first issued by Venice in 1474. They are included in Article 1 of the Constitution. Patenting biological processes started with recombinant DNA. Generally, the patent filer and university get the loot. Doudna and Charpentier received patents in 2019. Lawsuits still pending.

Part Four: CRISPR in Action

Chapter 32: Therapies. Sickle cell, using patient blood with CRISPR-Cas9 gene editing. Affordability a problem. Cancer, with China in the lead. Treatment and a detection tool. Blindness. Potential for Alzheimer’s, heart disease, a couple of dozen trials. Doudna among the CRISPR researchers shifting to detection and treatment tools.

Chapter 33: Biohacking. Josiah Zayner on DIY genetic kits. Potentials for hackers and terrorists.

Chapter 34: DARPA and Anti-CRISPR. DARPA makes the military the largest source of money for CRISPR research, including ways to block a CRISPR editing system, dubbed “anti-CRISPRs.” Some 50 anti-CRISPR proteins discovered.

Part Five: Public Scientist.

Chapter 35: Rules of the Road. Utopians vs. bioconservatives. Genetic engineering as a duty or ethically problematic. Most universities and funders agreed to rules. What about increased inequality. It started with recombinant DNA in the 1970s, then in vitro fertilization.

Chapter 36: Doudna Steps In. The Hitler nightmare. What guidelines would be needed?

Part Six: CRISPR Babies.

Chapter 37: He Jiankui, the Eager Entrepreneur. He used the technology to diagnose genetic conditions in early-stage human embryos. Then he edited the genes in human embryos of twins to prevent AIDS. It was not medically necessary. He expected celebrity status.

Chapter 38: The Hong Kong Summit. He made a 2018 presentation. He had not followed international guidelines. Problems were also safety, clinical testing, ethics and social consensus.

Chapter 39: Acceptance. The political and public reaction to CRISPR babies was mixed, but relatively favorable. Commissions were created including one by the WTO. What about “genetic tourism?” He Jiankui was put on trial in 2019 in China, sent to jail for three years.

Part Seven. The Moral Questions. “If scientists don’t play God, who will?” (James Watson).

Chapter 40: Red Lines. “Is there an inherent goodness to nature? … Should the rich be able to buy the best genes?” (p. 336). The big concern was changes to DNA in early-stage embryos which will be inherited. About two-thirds of prenatal-diagnosed Down’s fetuses result in abortions. What about treatments like Downs versus enhancements like height or IQ. Also, preventions.

Chapter 41: Thought Experiments. Positional good like enhanced height versus absolute good like resistance to viruses. Then all the questions about the meaning of life and happiness.

Chapter 42: Who Should Decide? The fundamental competing perspectives on morals are (1) individual rights (with personal liberty and choice) going back to John Locke and (2) justice based on what’s good for society: important for such issues as vaccinating kids for school or wearing masks, which favor societal benefits (Mill’s utilitarianism is used as a rationale). Or this can be argued as a social contract issue (Locke can be viewed as a contract theory, also Robert Nozick; on the other side would be John Rawls, including his veil of ignorance).

Chapter 43: Doudna’s Ethical Journey. “Because there are still huge risks that may be unknown, she feels that CRISPR should be used only when it is medically necessary and there are no good alternatives” (p. 369).

Part Eight: Dispatches from the Front.

Chapter 44: Quebec. New DNA system discovered called transposons or “jumping genes,” which can jump from one place to another on chromosomes. CRISPR can be used as a guiding system.

Chapter 45: I Learn to Edit. How-to chapter.

Chapter 46: Watson Revisited. “Jefferson Conundrum: To what extent can you respect a person for great achievements when they are accompanied by reprehensible failings” (p. 390). Watson was outspoken about racial inferiority and other controversial topics.

Chapter 47: Doudna Pays a Visit. Watson: “The reason that CRISPR is the most important discovery since DNA’s structure is that it not only describes the world, as we did with the double helix, but makes it easy to change the world” (p. 396).

Part Nine: Coronavirus.

Chapter 48: Call to Arms. February 2020: “She began thinking about what she and her colleagues should be doing to fight the pandemic” (p. 401). The plan was to create teams with multiple specialties, also involving Innovation Genomics Institute. “Viruses are deceptively simple little capsules of bad news. 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 SARS-SoV-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” (p. 403). In an electron microscope a protein spike on the virus looks like a crown (corona), which binds to human cells. The Chinese posted the full genetic sequence, which was modeled.

There were lots of moving parts to do research, from grant writing to setting up zoom calls, plus lawyers to share discoveries with other researchers. Doudna set up 10 projects to develop a diagnostic test and find ways to destroy the virus.

Chapter 49: Testing. The CDC messed up their first Covid test, because of a defective chemical compound. “In normal circumstances, hospitals and university labs can devise their own tests to use their facilities, as long as they do not market them” (p. 407). But the FDA declared a public health emergency, requiring authorization which was not quickly forthcoming. The WHO delivered diagnostic tests around the world. Anthony Fauci stepped in and urged the FDA to allow them to use their own tests. The Trump administration failed in widespread testing, so it was up to university and private research labs.

Chapter 50: The Berkeley Lab. The old approach was using swabs to test for polymerase chain reactions. The alternative was using CRISPR technology. Berkeley lab did both. Tests were available by April.

Chapter 51: Mammoth and Sherlock. “Cas12a, which had a special property. It could be targeted, like Cas9, to find and cut a specified sequence of DNA. But it didn’t stop there. Once it cleaved the double-stranded DNA target, it went into an indiscriminate cutting frenzy, chopping up any single-stranded DNA that was nearby. … Jamie Cate suggested that this property could be harnessed to create a diagnostic tool. This was combined with a “reporter molecule” causing a glowing signal. That could detect a particular virus or bacteria or cancer” (p. 422). They called in DETECTR.

Chapter 52: Coronavirus Tests. At-home tests using CRISPR developed in May 2020. A device called STOP-COVID could be adapted for any virus.

Chapter 53: Vaccines. Isaacson became part of the clinical trials, which injected a snippet of RNA. “Safe viruses can be used as a delivery system, or vector, to transport material into the cells of patients” (p. 437). J&J used a human adenovirus as the delivery mechanism, Oxford which partnered with AstraZeneca. Or your can deliver the genetic code using DNA or RNA into human cells. “RNA vaccines deliver their payloads inside tiny oily capsules, known as lipid nanoparticles, that are injected into the muscles of the upper arm” (p. 440).

“It took Moderna only two days to create the desired RNA sequences that would produce the spike protein, and thirty-eight days later it shipped the first box of vials to the NIH to begin early-stage trials. … Moderna had been working for ten years to perfect lipid nanoparticles” (p. 442). Two RNA vaccines were approved in December 2020. This was the first time an RNA vaccine had been approved.

“Throughout human history, we have been subjected to wave after wave of viral and bacterial plagues. The first known one was the Babylon flu epidemic around 1200 BC. The plague of Athens in 429 BC killed close to 100,000 people, the Antonine plague in the second century killed ten million, the plague of Justinian in the sixth century killed fifty million, and the Black Death of the fourteenth century took almost 200 million lives, close to half of Europe’s population” (p. 447).

“The invention of easily reprogrammable RNA vaccines was a lightning-fast triumph of human ingenuity, but it was based on decades of curiosity-driven research into one of the most fundamental aspects of life on planet earth: how genes encoded by DNA are transcribed into snippets of RNA that tell cells what proteins to assemble. Likewise, CRISPR gene-editing technology came from understanding the way that bacteria use snippets of RNA to guide enzymes to chop up dangerous viruses. Great inventions come from understanding basic science. Nature is beautiful that way” (p. 447).

Chapter 54: CRISPR Cures. “Most deaths from COVID-19 came from organ inflammation due to unwanted immune-system responses” (p. 449). “There are hundreds of viruses that can infect people, but there’s only a handful that have available drugs. That’s in part because viruses are so different from each other” (p. 451). There are “conserved sequences” of RNA which are the same in many viruses. This makes it easier to target them for treatment. One approach can detect up to 169 viruses at a time.

Chapter 55: Cold Springs Harbor Virtual. “The most important next step will be innovations in ‘microfluids,’ which involves channeling tiny amounts of liquid in a devise, and then connecting the information to our cell phones. That will allow us all to test our saliva and blood for hundreds of medical indicators” (p. 460).

Chapter 56: The Nobel Prize. Doudna got the call early in the morning on October 9, 2020. She and Charpentier won the chemistry award: “This year’s prize is about rewriting the code of life” (p. 470).

“Before the pandemic, communication and collaboration between academic researchers had become constrained. Universities created large legal teams dedicated to staking a claim to each new discovery … and guarding against any sharing of information that might jeopardize a patent application” (p. 473). Papers went to free servers for distribution.

Epilogue.