Note: this page is in progress. I continue to read the book. This page needs to be edited but these are my preliminary notes as I read the book.
The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race, Walter Isaacson, c. 2021.
UC- Berkeley's newest supercomputer named after Jennifer Doudna: link here.
A Nobel Prize winner.
The Book
Introduction: Into the Breach.
Jennifer Doudna: superstar at Berkeley
- co-inventor of CRISPR -- gene editing technology
- her world changed: robot competition canceled for her son, Covid -- March 12, 2020
- the next day, she led a Berkeley team to decide what role they would play in the Covid outbreak
- their first meeting: sat 6 feet apart
- it would be their first and last meeting
- from now on: Zoom and Slack
CRISPR: human technology to do what bacteria have been doing for millennia to defeat viruses. Amazing. Page xvi.
CRISPR:
- 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.
- in other words, it's an immune system that can adapt itself to fight each new wave of viruses -- just what we humans need in an era that has been plagued, as if we were still in the Middle Ages, by repeated viral epidemics.
Steps Doudna took:
- created a coronavirus testing lab;
- postdoc Jennifer Hamilton, was one of the leaders of that team/lab
- created a team to develop new types of coronavirus tests based on CRISPR
- based on lessons learned by starting a company some years earlier that used CRISPR for detecting viral disease
- settled on ten projects
Cross-country competitor: Feng Zhang
- her rival since 2012
- China-born and Iowa-raised researcher at the Broad Institute of MIT and Harvard
CRISPR:
- already science fiction becoming real
- November, 2018: a young Chinese student who had been to some of Doudna's gene-editing conferences used CRISPR to edit embryos and remove a gene that produces a receptor HIV;
- that led to the birth of twin girls, the world's first "designer babies."
A digression: is CRISPR being use in embryo research to "prevent/cure/ameliorate" sickle cell disease?
After two billion years, humans can now edit genes. Time for a discussion with ChatGPT.
Doudna:
- a graduate student in the 1990s
- other biologists were more interested in DNA
- Doudna: RNA
- wow! What a choice!
- Covid-19; seasonal flu viruses: RNA
- if I read this correctly, it was already called CRISPR when Doudna was doing her research -- but Doudna was studying "the CRISPR system" that bacteria were using in their battles against viruses;
- but now Doudna was using that same CRISPER system to edit genes;
- this reminds me of Jensen Huang pivoting with GPUs -- taking chips that were used for gaming to using those same chips for AI -- amazing!
AI prompt: The CRISPR system was already being used by bacteria to fight viruses when Doudna used the CRISPR system to edit genes. So, Doudna did not discover / invent CRISPR. She used the bacterial CRISPR system to edit genes. That was her breakthrough. Is that correct? And, of course, if accurate, that would raise issues with regard to patents. Is that accurate? Your thoughts?
Gemini:
Who discovered CRISPR if Doudna did not?
Does seasonal flu (e.g., Type A -- H1N1 -- use CRISPR CAS to target human DNA? [This was either a typo on my part or is evidence of how much I misunderstood CRISPR at this time.]
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How CRISPR Works
But looks at this. Cascade mentioned in the book, p. 111.
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By Chapter
Introduction: Into the Breach
Part One: The Origins of Life
Chapter 1:
Chapter 2:
Chapter 3:
Doudna read The Double Helixi while in sixth grade
Pomona College; good program in chemistry and biochemistry
in fall of 1981 -- I was in Grand Forks AFB, first operational assignment as pediatrician / flight surgeon
summer after her freshman year, family friend Don Hemmes, University of Hawaii, biologist
graduate school: Harvard; met Jack Szostak; DNA, yeast
Chapter 5: The Human Genome
From Walter Isaacson's book on Jennifer Doudna:
Chapter 5: The Human Genome
- Doudna working in Jack Szostak's lab
- 1986: Harvard, genetics; Mass General Hospital
- began working at Harvard as a molecular biologist in 197
- simultaneously started his own independent research laboratory at the Sidney Farber Cancer Institute (now the Dana-Farber Cancer Institute) in Boston. He later became a full professor at Harvard in 1988 before eventually transitioning his academic lab to the University of Chicago.
- while at Harvard, 1986, Human Genome Project
- 3 billion pairs in human DNA; 20,000 genes
- Cold Spring Harbor Laboratory: James Watson and his son Rufus Watson;
- the younger, schizophrenic; led the elder to starting what would later become the human genome project;
- in the 1940s, phages -- Salvador Luria and Max Delbrück led a study group on phages that included the young Watson. He returned as director, Cold Spring Harbor, 1968 - 2007.
- Human Genome Project
- launched 1990
- Watson: first director
- major players: Eric Lander, Craig Venter, Francis Collins
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Eric Lander
p. 39
****************
Craig Venter
p. 39
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James Watson
Query:
How did James Watson end up at Cold Spring Harbor Laboratory in the 1940s?
Reply:
Back to Chapter 5, and Human Genome Project; formally launched in 1990. -- p. 39. See above the box above.
"The Central Dogma" -- a phrase he later regretted -- Frances Crick
From CRISPR to COVID, RNA will be the STARRING molecud in this book and in Doudna's career.
Ribozymes: RNA that acts like enzymes -- p. 44
Thomas Cech and Sidney Altman: studying introns.
KEY: Jack Szostak switches from DNA in yeast to RNA based on Cech and Altman's revelation of ribozymes.
Doudna stays on. First student of Szostak's to study RNA.
Big question: how life began.
"the RNA world" -- p. 47
1991 paper for Nature -- a "tour de force," but it took the next two decades to learn about the ribozymes.
So, now were near the 2020s.
Team player, also George Church, p. 48.
Jim Watson: first time she met Jim Watson, 1987 at Cold Spring Harbor Laboratory; conference hosted by Jim Watson. "How did life begin?"
She was 23; went because Szostak unable to attend. First of many meetings with Jim Watson.
Post-doctoral work: how ribozymes replicate themselves. Similar to Rosalind Franklin.
Moved on. Left Jack Szostak. Structural biochemistry with Thomas Cech who had now at the University of Colorado in Boulder and was using X-ray crystallography in order to explre each nook and cranny of the structure of RNA. -- p. 53. Wow.
Earned her PhD in Szostak's lab in 1989; then moved to Boulder.
In January 1988 she married a Harvard Medical School student, Tom Griffin, who wanted to move to Boulder! They did but he didn't have her passion and they divorced amicably some years later.
Working to learn the 3-D structure of RNA; very, very difficult because RNA was not big enough to work with. -- p. 56. About the only one working with RNA. Graduate student Jamie Cate helped.
Tom and Joa Steitz, Yale, were on sabbatical for a year at Boulder; working similar problem. Invited Doudna to Yale to join them.
Jamie Cate went along. The two of them, a graduate student, Cate, and the professor, on a path towards tenure, Doudna, solved the problem: the 3-D structure of RNA -- and how it could slice, splice and replicate.
Chapter 8: Berkeley
Paper published, September, 1996.
Cate and Doudna get married, 2000; one son, Andrew. in Hawaii.
She at New Haven (Yale) and he at MIT (Boston, area).
She the headed to Harvard where her rivals would be: George Church, Feng Zhang, and Eric Lander. Bitter rivals.
Cate and Doudna moved to UC-Berkeley. Berkeley wanted Doudna.
At Berkeley. 2002. China: SARS. RNA. Renamed SARS-CoV-1 in 2022.
Doudna became interested in RNA interference. Interfers with mRNA. -- p. 65.
RNA interference; started with purple petunias; Craig Mello and Andrew Fire coined the term "RNA interference." Explained how it worked in worms. Nobel prize.
Dicer. page 66.
The chapter ends with her work on RNA interference, which humans don't have, and the introduction of a Spanish researcher who discovered a new phenomenon.
Osaka University in Japan: Yoshizumi Ishino was a student. Discovered the repetitive clustered nonsensical segments of DNA in E. coli, but didn't pursue, and the spacers.
Francisco Mojica, graduate student at the University of Alicante on the Mediterranean coast of Spain discovered the function of these sequences. Mojica working with archaea.
1990 discovered the sequences and discovered Ishino's paper. Mojica published in 1995; thought it had something to do with replication; wrong.
2001: researcher Ruud Jansen of Utrect University in the Netherlands agreed to the new name with acronym, CRISPR.
Jansen identified CRISPER-associated (Cas) enzymes. p. 73.
Mojica discoveres the spacers were parts of viruses that attacked bacteria. Key discovery, p. 74.
Difficult to get published; not published until 2005, two years after discovery.
They thought the immunity was working through RNA interference so not excited. That's when Doudna got the call.
Chapter 9: The Free Speech Movement Café
Doudna had published her paper on Dicer. Got a call from Berkeley professor Jillian Banfield.
Banfield kept finding CRISPRS. She contacted Doudna.
Doudna had not yet heard of CRISPR!!! -- p. 79.
- 19
Part Three: Gene Editing
Chapters 20 - 31
Part Four: CRISPR in Action
Chapters 33-34
Part Five: Public Scientist
Chapters 35 - 36
Part Six: CRISPR Babies
Chapters 37 - 39
Part Seven: The Moral Questions
Chapters 40 - 43
Part Eight: Dispatches from the Front
Chapters 44 - 47
Part Nine: Coronavirus
Chapters 48 - 56
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Chapter 12: The Yogurt Makers
Page 90.
Studying CRISPR with the goal of improving ways to make yogurt and cheese:
Rudolphe Barrangou in North Carolina;
Philippe Horvath in France
apparently both worked for Danisco, a Danish food ingredient company that makes starter cultures which initiate and control the fermentation of dairy products.
Question: at this point, around 2008, Doudna was studying CRISPR at UC-Berkeley. What did they know about CRISPR at this point; I need to go back and re-read the very early part of CRISPR. Later: this was in 1987 -- need to go back and re-read that section; repeating units accidentally discovered; scientist who discovered them didn't pursue what he had found; Doudna did. She thought this bacterial immune system had to a be a big deal.
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Chapter 13: Genentech
Autumn, 2008: Jillian Banfield tells Doudna she was worried that the most important discoveries had already been made, and it was now time for Doudna to move on.
Doudna demurred. "I knew there was some kind of adaptive immunity going on and wanted to know how it worked."
So, if I'm reading this correctly, researchers understood how the system worked and how bacteria protected themselves from viruses. BUT APPARENTLY, Doudna felt there was more -- she was thinking there was too much "energy" invested in this system not to have a "deeper meaning." She thought that "deeper meaning" might be immunity of some sort; an "immune system" of some sort that would rival what eukaryotic cells have and what researchers have been able to devise.
Which raises the question: do eukaryotic cells have something similar to CRISPR?
Which leads to the next question: why doesn't RNAi protect humans from seasonal flu?
- interferon is primary mammalian response against viruses, but
- some have suggested that interferon itself may shut down the RNAi system.
Wow.
And then there's the "interferon storm" issue.
Of course this raises even more questions.
Back to the book. Chapter 13:
- Wow. Genetech. Mental breakdown for Doudna.
- Returns to Berkeley after a two-month absence while preparing her move to Genetech, which, of course, did not happen.
Chapter 14: the new team under Doudna when she "returned" to Berkeley.
After a single mention in the book, Kaihong Zhou was never mentioned again.
Chapter 15: Caribou
p. 114: Stanford and its start after WWII.
Chapter 16: Emmanuelle Charpentier
p. 119.
tracrRNA -- Cas9 -- crRNA
The process of unraveling the roles of tracrRNA began in 2010, p. 125.
Charpentier in the process of moving from Vienna to Umeå, Sweden. Thirteenth largest city in Sweden; serves as port city for northern Sweden; on the Gulf of Bothnia, the northernmost part of the Baltic Sea. Yeah, this is really far north. From wiki:
Before leaving Vienna, she found a volunteer, Elitza Deltcheva, a young student from Bulgaria, who was willing to pursue work on tracrRNA. This little team discovered the three components: tracrRNA - Cas9 enzyme - crRNA, p. 125.
Puerto Rico, March, 2011 -- Doudna and Charpentier meet! Page 127.
The foursome that made one of the most important advances in modern science:
- Doudna, UC-Berkeley
- Charpentier, Umeå, Sweden (previously Vienna where some of her researchers remained)
- Martin Jinek,
- Doudna's lab, had been working on Cas1 and Cas6
- Krzysztof Chylinski,
- Polish, in Charpentier's lab in Vienna, who had worked on Charpentier's earlier Cas9 paper
Chapter 17: CRISPR-Cas9
"fast-growing" annual CRISPR conferece, July 2011; UC-Berkeley
first time Jinek has personally met Chylinski
Chylinski did the all-important tracrRNA experiment -- solved the problem
p. 132 -- how the three-component system worked -- tracrRNA had a dual role
graphic: p. 133
the three-component system was a nice-to-know how bacteria protected themselves from viruses, but Doudna recognized immediately how Cas9 could be used as a tool to edit DNA
"call-and-response duet between basic science and translational medicine."
the eureka moment -- p. 133
sgRNA: "single-guide RNA" -- bottom of page 134
the key: middle paragraph p. 135 -- something able to patent!!!
Doudna's collaboration with Charpentier had produced two significant advances
signing the laboratory notebook!
Chapter 18: Science, 2012
the paper, sent to Science, June 8, 2012
six authors: Jinek, Chylinski, Ines Fonfara, Michael Hauer, Doudna, and Charpentier.
Again: the all-important invention -- the single-guide RNA.
Paper formally accepted Wednesday, June 20, 2012 -- p. 140.
Chapter 19: Dueling Presentations
Virginijus Šikšnys, Vilnius University, Lithuania.
Walter Isaacson really provides a nice summary of the presentations at the June 21, 2012: Šikšnys vs Charpentier.
Jinek and Chylinski made the two presentations.
Again, the crux: the single-guide technology p. 148.
Erik Sontheimer.
Many could have claims to CRISPR technology but at this point, one must seriously look at Erik Sontheimer.
Last page, chapter 19, p. 149:
Doudna, Sontheimer, Brrangou and Šikšnys went out to dinner together -- they knew it was important, but lots of important work is always being done. They knew there was something very, very important they had discovered, but probably very, very giddy, appropriately competitive, but no one thinking of a Nobel Prize.
Part Three: Gene Editing
Chapter 20: A Human Tool
"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."
He had manufactured what he dubbed "recombinant DNA."
Page 164, writing on Feng Zhang, Walter Isaacson writes:
Instead of pursuing computer science, however, Zhang became a forerunner of what will, I think, soon be common among aspiring geeks: his interests shifted from digital tech to biotech. Computer code was something his parents and their generation did. He became more interested in genetic code.
Later, Zhang, "I was excited to discover that animals could be a programmable system. That meant human genetic coding could be programmable as well." It was more exciting than Linux. -- p. 164.
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