Author: Walter Isaacson
Publisher: Simon & Schuster 2021
The most fascinating proteins are the enzymes. Almost all actions that take place in a cell needs to be catalyzed by an enzyme. (See details here.)
Thomas Cech and Sidney Altman discovered in 1980 that some forms of RNA can also act as enzymes. They dubbed these catalytic RNA "ribozymes". Cech and Altman made this discovery by studying "introns". Introns are the parts of DNA that do not code instructions for how to make proteins. When these sequences are transcribed into RNA, they clog things up. So, they have to be sliced out before the RNA can go on its mission to direct the making of proteins. The slicing out of these introns is carried out by a catalyst enzyme. Cech and Altman discovered that there were certain RNA introns that were self-splicing. These are the ribozymes. (Cech and Altman won the Nobel prize for this discovery.)
If some RNA molecules could store genetic information and also act as a catalyst to spur chemical reactions, they might be more fundamental to the origins of life than DNA. DNA cannot naturally replicate themselves without the presence of proteins to serve as a catalyst. Jennifer Doudna and her PhD advisor Jack Szostak were able to engineer a ribozyme that could splice together a copy of itself.
RNA interference was discovered by Andrew Fire and Craig Mello in 1998. As the name suggests there are some small molecules which are capable of messing with the messenger RNA. RNA interference operates by deploying an enzyme known as "Dicer". Dicer snips a long piece of RNA into short fragments. These little fragments then seek out messenger RNA that has matching letters and use a scissors like enzyme to chop it up. (Fire and Mello won the Nobel prize for this discovery.)
Bacteria are single cell organisms. The smallest bacteria are about half a micron in diameter. (a micron is one millionth of a meter.) Viruses are about two hundredth of a micron in diameter. That is a bacterium is about 25 times bigger than a virus. There are more viruses in the world that bacteria. In a milliliter of seawater, you can find 900 million viruses. Viruses attack bacteria. During the billions of years of evolution, bacteria found mechanisms to protect themselves from viruses. Many viruses are composed of DNA. However, the SARS is a coronavirus composed of RNA.
By sequencing a gene in E. Coli bacteria, Francisco Mojica discovered that there were five segments of DNA that are identical to each other in that gene. These repeated sequences, each 29 base pairs long, were sprinkled between normal-looking sequences of DNA. Mojica named these repeated pieces “clustered, regularly interspersed short palindromic repeats” (CRISPR). By studying the tuberculosis bacteria, Rudd Jansen discovered there are genes associated with CRISPRs which encoded directions for making an enzyme. He named these enzymes "CRISPR associated" or Cas enzymes.
Mojica also discovered that the CRISPR segments matched the DNA sequences that were in viruses that attacked E. coli. He found the same thing when he looked at other bacteria with CRISPR sequences. Mojia also found that bacteria with CRISPR sequences immune to the attacks of the viruses with the same sequence. Eugene Koonin extended Mojica's theory by showing that the role of CRISPR-Cas enzymes was to grab bits of DNA out of an attacking virus and insert them into bacteria's own CRISPR.
Koonin thought CRISPR defense system worked through RNA interference.
Biochemistry is the field that studies how chemical molecules in living cells behave. Structural biology studies the structure of molecules such as DNA and RNA.
Jennifer Doudna is a structural biologist. She was interested in how the RNA in some viruses, such as coronaviruses, allow them to hijack the protein-making machinery of the cells. She is also the RNA interference expert at UC Berkeley. A microbiologist at Berkeley contacted Doudna in 2006 to see if she is interested in finding if Koonin's theory is true. That is the first time Doudna heard about CRISPR.
By 2008, scientists have discovered several CRISPR-Cas enzymes. These enzymes were eventually given standardized names such as CRISPR-Cas1, CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13. They have also discovered that CRISPR-Cas enzymes enable the system to cut and paste new memories of viruses, short segments of RNA of an attacking virus, known as CRISPR RNA or crRNA.
Doudna and her team decided to concentrate on CRISPR-Cas1. It is the only Cas enzyme that appears in all bacteria that have CRISPR systems. Martin Jinek, Rachel Haurwitz, Blake Widenheft, Kaihong Zhou are the original members of the Doudna's team. Their main tool was the X-ray crystallography. Cas1 is easy to get crystallize to be used in X-ray crystallography. They discovered that Cas1 has a distinct fold that enabled it to cleave invading viruses and add snippets of code into the bacteria's CRISPR array. They published this finding in 2009, their first CRISPR paper.
On the other side the Atlantic Ocean, Emmanuelle Charpentier in Umea, Sweden was studying Cas9. Krzysztof Chylinski and Elitza Deltcheva are the members of Carpentier's team. The reason for choosing Cas9 was that if you deactivate Cas9 in bacteria, then the CRISPR system no longer cutup the invading viruses. They have discovered that crRNA (of a previously known virus) guides the Cas enzymes to attack the virus if it tries to invade again. They have also identified another major component of the CRISPR-Cas9 system that played an essential role. It was named trans-activating CRISPR RNA or tracrRNA. tracrRNA facilitated the process of making crRNA.
Charpentier's team discovered that the CRISPR-Cas9 system accomplished its viral-defense system using only three components: tracrRNA, crRNA, and Cas9 enzyme. They published this finding in 2011. Carpentier knew that tracrRNA helped to create crRNA, but she did not know any other role played by the tracrRNA.
When the Carpentier's work was published, Daudna contacted Charpentier and offered to work together to find exactly how CRISPR-Cas9 system worked. The two teams agreed to work together. Jinek at the Doudna's lab found out that without the tracrRNA, the crRNA guide does not bind to the Cas9 enzyme. Working together the two labs discovered the precise mechanisms of each of the three essential components of the CRISPR-Cas9 system.
In addition to help creating crRNA, the two teams discovered that tracrRNA has another role. It serves as a handle to latch onto the invading virus.
Here is the summary. The crRNA guides the Cas9 to a virus DNA that contains a letter sequence that is similar to the crRNA sequence. Then tracrRNA holds onto the virus DNA at the right place so that the Cas9 enzyme can cut it to pieces.
They instantaneously recognized that this mechanism can be used for gene editing. They engineered in the lab a new RNA they called single-guide RNA (sgRNA) that performed both the guiding and the holding onto tasks. This simplified the gene editing process.
They published this finding in Nature on August 17, 2012. The Nature article. Doudna and Carpentier won the Nobel prize for this finding.
This book, written as a biography of Doudna, takes you from her childhood to the winning of the Nobel price. In the interim, the author provides details of all findings in chronological order, introduce all major players, activities of their rivalries, and how science progress amid the intense competition.
I will finish this note adding some information about the existing coronavirus vaccines.
The genetic material of SARS-Cov-2 virus (coronavirus) is RNA. For SARS-Cov-2, the human receptor is a protein known as Angiotensin-converting enzyme 2 (ACE2). The disease caused by SARS-Cov-2 virus is called COVID-19.
Vaccines work by stimulating a person's immune system. Usually, a deactivated components of a targeted virus are injected as a vaccine. The purpose of this is to stimulate the immune system to produce antibodies to attach the virus. However, in 2020 gene editing techniques were used to create RNA vaccines. An injected messenger RNA (mRNA) instructs cells to make part of spike protein that is on the surface of the coronavirus. The cells then become vaccine manufacturing facilities.
Pfizer and Moderna vaccines are RNA vaccines. They delivered the mRNA inside tiny oily capsules known as lipid nanoparticles that were injected by a syringe. GCACGUAGUGU is the mRNA snippet used by Pfizer and Moderna.
Chinese Sinovac vaccine is an old fashion dead virus vaccine.
Oxford added spikes of the coronavirus to a harmless adenovirus that causes flu in chimpanzees. This was then injected by a syringe. This is known as the AstraZeneca vaccine.
Johnson and Johnson added spikes of the coronavirus to a harmless human adenovirus to create their vaccine.
This is an excellent book and I recommend this book to any reader. It is very well written and very easy to read.
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