The Code Breaker

by Walter Isaacson

curiosity-driven science is the seed corn that eventually leads to new technologies and innovations.

Basic research is the pacemaker of technological progress.”

“Steam engines led to the understanding of thermodynamics, not the other way round. Powered flight preceded almost all aerodynamics.”

Cases where innovation precede explanation

in silico.

Experiment performed within a computer setting.

I once asked Steve Jobs what his best product was, thinking he would say the Macintosh or iPhone. Instead he said that creating great products is important, but what’s even more important is creating a team that can continually make such products.

“You’re never completely at home, and that can drive you. It can challenge you not to seek being comfortable.”

That helped her honor the maxim often preached by Louis Pasteur himself: Be prepared for the unexpected.

Researchers had shown that if you deactivated Cas9 in bacteria, the CRISPR system no longer cut up the invading viruses.

crRNA guides the Cas enzymes to attack that virus when it tries to invade again.

CRISPR system: a small snippet of RNA that acts as a guide and an enzyme that acts as scissors.

It was dubbed a “trans-activating CRISPR RNA,” or tracrRNA,

tracrRNA performs two important tasks.

The two tasks of tracrRNA are: 1) It helps in the creation of crRNA(guides having the genetic copy of a former virus) 2) It helps in acting as a handle on the virus DNA, for the Cas9 enzymes to latch on to and destroy.

tracrRNA, crRNA, and the Cas9 enzyme.

"Le Grande Armée" of bacterial immunity.

Together this foursome would make one of the most important advances in modern science.

Jennifer Doudna, Emmanuelle Charpentier, Martin Jinek and Krzysztof Chylinski.

the crRNA guide could be modified to target any DNA sequence you might wish to cut. It was programmable. It could become an editing tool.

“single-guide RNA” (sgRNA).

An amalgam of crRNA(guide) and tracrRNA(handle)

There was no attempt to change the DNA of the patient; it was not gene editing.

Difference between gene editing and gene therapy: Gene therapy: Involves creating a specific DNA that is introduced into the organism, helping in curing the faulty DNA or gene. Gene editing: Involves actively and directly altering the DNA present within a cell.

The first trial came in 1990 on a four-year-old girl with a genetic mutation that crippled her immune system and left her at risk for infection. Doctors found a way to get functioning copies of the missing gene into the T cells of her blood system. The T cells were removed from her body, given the missing gene, and then reintroduced into her body. This led to a dramatic improvement of her immune system and allowed her to live a healthy life.

The goal was to edit the flawed sequences of DNA in the relevant cells of the patient.

break in both strands of the DNA double helix, known as a double-strand break.

in one of two ways.

1) Non homologous end joining: At the site of strand break, the DNA simply ties the broken strands together, not worrying about the missing templates. Very sloppy. 2) Homology-directed repair: Here, splicing occurs by a more accurate method, by taking into consideration, the matching templates of the missing DNA sequence

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

enzymes that can cut DNA or RNA are called “nucleases.”

FokI enzyme,

Acts as a nuclease as well as a guide.

But in the CRISPR system, the guide was not a protein but a snippet of RNA.

The precursors of sgRNA(CRISPR-Cas), were ZFNs and TALENs. Both were declared redundant, due to the reqiurement of a protein for chopping, as compared to an RNA (sgRNA). Proteins are hard to create and use, stymieing the process of gene editing.

Competition drives discovery.

To take an example relevant to these days, the Japanese bacteriologist Kitasato Shibasaburō and his Swiss rival Alexandre Yersin both rushed to Hong Kong in 1894 to investigate the pneumonic plague epidemic and, working with different methods, discovered the responsible bacteria within days of each other.

But it is our contemporary counterpart to the race between Pauling and the team of Watson and Crick to discover the structure of DNA.

"Rivalries are not created by warriors, rather by the mere townspeople"

“He told me that whenever you face a tough question in biology, just say ‘Enzymes.’ It’s the correct answer to most questions in biology.”

nuclear location signal

A amino acid sequence that "tags" a protein in the cytoplasm for import(entry) into the nucleus.

Codons are the three-letter snippets of DNA that provide instructions for the specific arrangement of amino acids, which are the building blocks used to make proteins.

Republic of Venice in 1474 passed a statute giving the inventors of “any new and ingenious device” the exclusive right to profit from it for ten years, people have been wrestling over patents.

Congress passed the Bayh-Dole Act, which made it easier for universities to benefit from patents, even if the research was funded by the government.

Doudna and Zhang could have followed the example of Jack Kilby of Texas Instruments and Robert Noyce of Intel who, after five years of wrangling, agreed to share the patent rights for the microchip by cross-licensing their intellectual property to each other and splitting the royalties, which helped the microchip business grow exponentially and define a new age of technology.

Don’t fight over divvying up the proceeds until you finish robbing the stagecoach.

First accomplish the feat, then decide how the credit gets shared.

The doctors then used CRISPR-Cas9 to disable a gene that produces a protein, known as PD-1, which stops the cell’s immune response. Cancer cells sometimes trigger the PD-1 response, thus protecting themselves from the immune system.

a gene known as P53 encodes for a protein that suppresses the growth of cancerous tumors. It helps the body respond to damaged DNA and prevents cancerous cells from dividing. Humans tend to have one copy of this gene, and cancers proliferate if something goes wrong with it. Elephants have twenty copies of this gene, and they almost never get cancer.

the gene APOE4 raises the risk of the devastating disease of Alzheimer’s.

PCSK9, encodes for an enzyme that facilitates the creation of LDL, the “bad” cholesterol.

Defense Advanced Research Projects Agency (DARPA), which is the Pentagon’s well-funded research arm, launched a program called Safe Genes to support ways to defend against genetically engineered weapons.

bacteria developed CRISPR systems to ward off viruses, but then the viruses developed a way to shut down those defenses.

The anti-CRISPRs could be engineered to regulate gene-editing systems. That would be useful for medical applications that needed to time-limit a CRISPR edit, and they could be used as a defense against systems created by terrorists or malevolent enemies.

“Men ought not to play God before they learn to be men.”

Preimplantation diagnosis involves fertilizing an egg with sperm in a Petri dish, doing tests on the resulting embryosI to determine their genetic characteristics, and then implanting into a woman’s womb the embryo with the most desired traits.

“reprogenetics” to describe the use of technology to determine which genes a child would inherit.

Oviedo Convention was intended to be a legally binding treaty designed to prohibit the use of biological advances in ways that threatened human dignity. It barred genetic engineering in humans except “for preventive, diagnostic or therapeutic reasons and only where it does not aim to change the genetic make-up of a person’s descendants.”

In the article, researchers at a university in Guangzhou described how they used CRISPR-Cas9 in eighty-six non-viable zygotes (precursors to an embryo) to cut out a mutated gene that causes beta thalassemia, a deadly blood disorder like sickle-cell anemia.

germline editing should be allowed, among them: “absence of reasonable alternatives, restriction to preventing a serious disease or condition,” and a few others that were not insurmountable in the foreseeable future.

Germline(in vivo) gene editing must only be ethically permitted in cases of preventing a congenital disease of the zygote, or "absence of any other relative alternative".

“Man has the opportunity to get into the genetic code created by either nature, or as religious people would say, by God,” he said. “One may imagine that scientists could create a person with desired features. This may be a mathematical genius, an outstanding musician, but this can also be a soldier, a person who can fight without fear or compassion, mercy or pain.”

Vladimir Putin's take on using CRISPR-Cas9 germline gene editing for militarial purposes.