The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race

Nonfiction | Biography | Adult | Published in 2021

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Chapters 9-12Chapter Summaries & Analyses

Chapter 9 Summary: “Clustered Repeats”

Yoshizumi Ishino, a PhD student at Osaka University in Japan, was sequencing a gene in the bacteria E. coli when he found an oddity in the DNA he sequenced: five sequences were identical. These repeat pairs, each composed of 29 base pairs, occurred between normal stretches of DNA. Ishino didn’t pursue the matter further. Four years later, in 1990, Francisco Mojica, a graduate student of Spain’s University of Alicate, was sequencing the DNA of a variant of unicellular organism called archaea that thrive in extremely salty water. Mojica found 14 identical DNA sequences repeating regularly, all of them palindromes, reading the same forward and backward. To see if any other scientist had discovered similar findings, Mojica conducted painstaking research and found Ishino’s paper. He wondered, why did bacteria and archaea, totally different species with little genetic material to spare, have “spacer” sequences of repetitive DNA? Mojica found few takers to fund his investigation.

Mojica initially named the spacer sequences “short regularly spaced repeaters,” or SRSR, but the acronym was unmemorable. One evening a more apt name came to him: CRSISPR, meaning “cluster regularly interspaced short palindromic repeats.” Mojica and his collaborator Ruud Jansen—of Utrecht University in the Netherlands—formalized the name, and it stuck. By 2003, the sequences of many microorganisms were available online under the Human Genome Project. While running the spacer sequences of E. coli through databases, Mojica was surprised to find similar sequences in some viruses. Excited, Mojica came to a very important conclusion: Bacteria had immune systems that remembered viruses, and bacteria developed corresponding spacer sequences to prime themselves against future virus attacks.

What Mojica had discovered was a weapon in the age-old war between bacteria and viruses. Bacteria-eating “phages” are the most common viruses on earth, almost 10 to the power of 31 in number. For 3 billion years bacteria have been fighting ever-evolving phages through different methods, including spacer sequences. Mojica found that bacteria without CRISPRs did get infected by viruses. However, surviving bacteria incorporated some of the virus’s DNA, passing on acquired immunity to the next generation. Elegant and astonishing as Mojica’s findings were, he struggled to get them published. His paper was finally published in 2005, two years after his discovery, in the Journal of Molecular Evolution. Mojica’s paper heralded a host of publications about CRISPR, including one by Eugene Koonin, a researcher at the US National Center for Biotechnology Information. Koonin found that CRISPR-associated enzymes grabbed bits of DNA from attacking viruses and inserted them into the bacteria’s DNA. However, Koonin and other scientists were wrong in assuming that the CRISPR defense system worked through RNA interference, and that bacteria used the bits of DNA to interfere with the messenger-RNA of attacking viruses.

Chapter 10 Summary: “The Free Speech Movement Cafe”

Jillian Banfield, an Australian scientist working on bacteria living in salty, acidic climates, was one of those who thought CRISPR-defense worked with RNA interference. Since Doudna was working on RNA, Banfield reached out to her, and the two women exchanged their findings at the trendy Free Speech Movement Cafe in Berkeley. Before Doudna and Banfield met, CRISPRs had mainly been studied in living cells by microbiologists like Mojica. No one had actually isolated the molecular components of CRISPR in a lab. As Doudna told Isaacson, the time was ripe for biochemists and structural biologists to join the fray.

Chapter 11 Summary: “Jumping In”

Doudna wanted to collaborate with Banfield, but she had no one in her lab to work on CRISPR—not until Blake Wiedenheft walked in for an interview. His chief interest was learning the structure of DNA, how it folded and twisted and interacted with other molecules. Wiedenheft dedicated himself to studying CRISPRs at Doudna’s lab and soon struck up a friendship with Martin Jinek, the lab’s crystallography expert. Jinek had so far focused on recreating RNA interference in vitro, or in a test tube, so he could isolate the enzymes essential to interfering with the expression of a gene. He had successfully crystallized the structure of one particular enzyme. Now under Doudna’s guidance, Jinek and Wiedenheft would dissect the CRISPR system into individual components to see how each worked. Wiedenheft started with CRISPR-associated enzymes, or Cas.

As discussed in Chapter 6, enzymes are proteins that catalyze chemical reactions in living cells. From breaking down starches and proteins for digestion to cutting and splicing DNA and RNA, enzymes start off most important biochemical reactions. By 2008, scientists had discovered some enzymes adjacent to CRISPR sequences in bacterial DNA. These Cas enzymes enabled bacteria to cut and paste memories of attacking viruses, and they also guided a “scissor-like enzyme” to shred the genetic material of a dangerous virus. Doudna’s team decided to focus on the enzyme called Cas1 (Cas are standardized by names such as Cas1, Cas9, and so on), since Cas1 occurs in all bacteria with a CRISPR system. With Jinek’s help, Wiedenheft isolated, cloned, and crystallized the Cas1 gene. The duo found that Cas1 has a distinct fold, which serves as the mechanism to cut snippets of DNA from invading viruses. Thus, the fold was key to the memory-forming stage of the bacteria’s immune system. This was the first explanation of a CRISPR mechanism based on analyzing its structure.

Chapter 12 Summary: “The Yogurt Makers”

Scientific discoveries are not just made by scientists but also by inventors working in industry. Rodolphe Barrangou and Philippe Horvath—two scientists at Danish dairy giant Danisco—had a special interest in CRISPRs, since viruses are the natural enemy of bacteria that ferment milk into yogurt and cheese. Danisco had a vast collection of starter bacterial cultures for different cheeses and curds. Using computational biology to study the sequences of bacteria in Danisco’s huge database, Barrangou and Horvath found that bacteria collected after a big viral attack had new spacers, indicating immunity. This confirmed Mojica and Kooner’s hypothesis. However, Barrangou and Horvath went a step further and added sequences from a virus to the bacteria’s CRISPR locus to produce spacers. They also proved that Cas enzymes were essential to CRISPR formation by knocking out Cas genes. When the Cas genes, such as those of the Cas9 enzyme, were neutralized, the bacteria lost its ability to acquire immunity. Barrangou and Horvath successfully received a patent to use the CRISPR-Cas system to vaccinate bacteria. They also published their findings in Science in 2007, attracting Banfield’s attention.

Soon Barrangou and Banfield decided to start annual CRISPR conferences, which proved to be a great incubator for ideas. While the first conference was held in 2008, Luciano Marraffini and Erik Sontheimer of Northwestern University, Chicago, showed that DNA was the main target of the CRISPR system. Thus, unlike what Kooner and Banfield had assumed, CRISPR did not work through RNA interference. Marraffini and Sontheimer’s discovery had a “holy cow implication” that CRISPR technology could be used to edit DNA and thus repair the source of genetic problems. However, Marraffini and Sontheimer’s application to patent CRISPR as a gene-editing tool was rightfully rejected. Though they had the idea, Marraffini and Sontheimer did not know the technique with which the CRISPR enzyme cut DNA. To learn that mechanism, the molecules had to be studied in vitro rather than in silico, or in living cells, as Marraffini and Sontheimer had done. Studying molecules and processes in vitro was Doudna’s specialty. However, before Doudna could turn her attention to CRISPR, she took a brief career detour.

Chapters 9-12 Analysis

Chapter 9 introduces CRISPR. Interestingly, Isaacson traces the discovery of CRISPRs from Ishino’s observations in 1986, amplifying the text’s theme that scientific discoveries are rarely epiphanies experienced by one researcher. Ishino’s work informed that of Mojica, which informed Doudna’s research. This intergenerational relay race also plays out across geographies, sprinting from Japan to Spain to the United States. Though the individual scientists worked in their bubbles, they are linked by the spirit of discovery.

Chapters 9-12 also cover many important themes, such as the relationship between science and industry, and the importance of collaborations. In Chapter 12, Isaacson discusses the “linear model of innovation.” Propagated by Vannevar Bush, an MIT engineering dean in 1945, the linear model argues that no invention happens in isolation. Instead, every new innovation is founded on new discoveries and concepts painstakingly researched by scientists. The linear model is right in the sense that the work of basic researchers like Mojica has enabled the world to invent gene-editing technologies. However, the model errs in assuming that all inventions are a linear outcome of scientific research, when the fact is science too sometimes follows inventions. For instance, it was the steam engine that led to the understanding of thermodynamics rather than the other way around. Nowhere is the iterative interplay between science, invention, and business more apparent than in the parallel work of Doudna and Banfield and a couple of food scientists, Barrangou and Horvath.

As Isaacson notes, Barrangou and Horvath worked for a company rather than a research lab. Yet, by 2007, they had authored an important paper on CRISPR, which attracted Banfield’s attention. And it was Banfield who introduced Doudna to CRISPRs. Work in applied science often enables inventors to solve for immediate problems, an aspect the linear model elides. In fact, assuming that corporate involvement in science is always bad is erroneous. The “iterative dance among basic scientists, practical inventors, and business leaders” (99) has led to many great inventions, from the transistor to CRISPR.

The spirit of collaboration emerges as another important motif, exemplified by Banfield and Doudna’s meeting at the Free Speech Movement Café and the partnership between Barrangou and Horvath. There is special emphasis on all-women pairs like Doudna and Banfield. Not only does this signify a change in the dominant patriarchal order, but it also foreshadows the singular achievement of Doudna and Emmanuelle Charpentier—the first women duo without a male copartner to win the Nobel Prize for Chemistry.