More on this book
Community
Kindle Notes & Highlights
Conversely, deletions are the culprit in the most common type of cystic fibrosis, a life-threatening genetic disease that primarily affects the lungs; the deletion of three letters of genetic code in the CFTR gene results in a protein that lacks an important amino acid and does not function properly.
In 2001, after herculean efforts and at a cost of more than three billion dollars, the first draft of the genome was published.
Since the completion of the Human Genome Project, the process of DNA and whole-genome sequencing has become staggeringly quick, cheap, and effective.
Scientists have precisely identified well over four thousand different kinds of DNA mutations tha...
This highlight has been truncated due to consecutive passage length restrictions.
Homologous recombination occurs most famously during the formation of egg and sperm cells, when the two sets of chromosomes we inherit from our parents are pared down to just one, to be combined with a second set during sexual reproduction. In this process of elimination, cells select a blend of the paternal and maternal chromosomes; each pair of chromosomes engage in their own version of sex, exchanging large chunks of DNA in a way that increases genetic diversity.
Homologous recombination, in other words, might allow scientists to precisely paste genes into matching sites in the genome—a dramatic improvement over the randomness of gene splicing with viruses.
Cells, it seemed, could do most of the hard work of modifying their genomes all by themselves. This meant that scientists could deliver genes more gently, without using viruses to ram new DNA into the genome.
Nucleases are enzymes that cut apart nucleic acids; some cut RNA, others cut DNA. Endonucleases cut RNA or DNA somewhere within the strands, as opposed to exonucleases, which cut exclusively from the ends. Some endonucleases are highly toxic to cells because they cut just about any piece of DNA they find, regardless of its sequence. Other endonucleases are highly specific and cut only certain sequences, and many more fall somewhere in the middle.
The results of Jasin’s experiment were astounding. She succeeded in inducing a whopping 10 percent of the cells to precisely repair a mutated gene by homologous recombination, a success rate that seems low now but that was hundreds of times higher than what scientists had managed to achieve previously. It was the most promising evidence yet that this process might allow scientists to rewrite the code of the genome without the risk of illegitimate recombination or random splicing from retroviral vectors.
Introduce a double-strand break in the right place, and cells would practically do the work for you.
These next-generation gene-editing systems had three critical requirements: They had to recognize a specific, desired DNA sequence; they had to be able to cut that DNA sequence; and they had to be easily reprogrammable to target and cut different DNA sequences.
Scientists needed to be able to pluck the correct nuclease off the shelf or at least have a way of generating it on demand.
Srinivasan Chandrasegaran, a professor at Johns Hopkins University, realized that instead of building nucleases from scratch, finding new ones in nature, or remaking I-SceI, he could take a hybrid approach by selecting pieces of proteins that existed naturally and combining them. Such chimeric nucleases would fulfill the first two requirements of a gene-editing nuclease: they would be able to recognize and cut a specific sequence of DNA.
To do the targeting, he harnessed a family of ubiquitous, naturally occurring proteins called zinc finger proteins, so named because they recognized DNA using fingerlike extensions held together by zinc ions and arranged side by side, just like the fingers of a hand. Because these zinc finger proteins were built of multiple repeated segments arranged in tandem, with each segment recognizing a specific three-letter DNA sequence, it seemed likely that scientists could redesign the proteins to recognize various DNA sequences by combining segments in different ways.
Next, working in fruit flies, Carroll’s lab programmed a new ZFN to target a gene involved in body pigmentation called yellow and showed that this strategy could produce a precise genetic alteration in a whole organism. This was a profoundly significant development for gene editing.
In many ways, the foundations of molecular genetics were laid by experiments done with these bacterial viruses.
In 1977, Fred Sanger and his colleagues succeeded in determining the complete DNA genome sequence of a phage called ΦX174. Twenty-five years later, the same phage would again become famous: its genome was the first to be synthesized entirely from scratch. Bacteriophages aren’t just popular laboratory pets, though; they are also the most prevalent biological entity on our planet—by a long shot. They are as ubiquitous in the natural world as light and soil, and they can be found in dirt, water, our intestines, hot springs, ice cores, and just about anywhere else that supports life.
Scientists estimate that there are somewhere on the order of 1031 bacteriophages on earth; that’s ten million trillion trillion, or a one with thirty-one zeros after it.
Incredibly, there are many, many more phages on earth than there are bacteria for them to infect; as abundant as bacteria are, bacterial viruses outnumber them ten to one.
in the ocean alone, about 40 percent of all bacteria die every day as a result of deadly phage infections.
These viruses are lethal by design, having evolved over billions of years to infect bacteria with brutal efficiency.
Some phages have elegant icosahedral (twenty-sided) geometries; others have spherical capsids attached to long tails.
The viruses’ modi operandi, like their appearances, are diverse but invariably, and ruthlessly, effective. Some viral genomes are packed so tight in the capsids that the genetic material explodes into the cell as soon as the protein shell is breached, releasing internal pressure like an uncorked bottle of champagne. Once the genome makes its way inside the cell, it can
hijack the host by one of two possible pathways. In the parasitic, or lysogenic, pathway, the viral genome insinuates itself into that of its host, where it can stay buried for many generations, waiting for the right moment to strike. By contrast, in the infectious, or lytic, pathway, the genome commandeers its host’s resources immediately, directing the bacterium to produce viral proteins instead of bacterial proteins and replicate the viral genome many times over until the cell violently bursts open from the mounting pressure and scatters fresh phages that infect neighboring cells. Through
...more
Bacterial cells have even developed methods to sense an oncoming infection and commit suicide before it can progress—a selfless way of protecting the greater bacterial community.
Could CRISPR be yet another antiviral defense mechanism?
Computational analyses by Ruud Jansen and his colleagues in the Netherlands—the same team that initially coined the CRISPR acronym back in 2002—had identified a set of genes that almost always flanked the CRISPR regions in bacterial chromosomes. These weren’t the repeat sequences or the spacer sequences within the CRISPR DNA but a separate set of genes entirely.
these CRISPR-associated genes, or cas genes, seemed full of exciting potential. Comparisons with known genes suggested that cas genes coded for specialized enzymes whose functions might include unzipping the two strands of the DNA double helix or slicing up RNA or DNA molecules, just like the DNA-cutting function of restriction endonucleases.
it seemed very possible that, by digging deeper into these and other aspects of CRISPR, we might uncover a treasure-trove of new enzymes—and that these proteins too could have major biotechnology potential. That was it. I was hooked.
Instead of focusing on viral genes that promoted infection, we had to hunt down the genes in bacteria that blocked infection—the ones associated with CRISPR.
scientists at Danisco,
Their study showed, using genetics, that CRISPR was indeed a bacterial immune system—although
Streptococcus thermophilus, one of the key probiotics involved in producing yogurt, mozzarella cheese, and numerous other dairy products. Humans ingest well over a billion trillion live S. thermophilus cells a year, and the annual market value of cultures of the bacterium is more than forty billion dollars.
phage infection, the most common cause of production losses and incomplete fermentation.
A single drop of raw milk contains anywhere from ten to one thousand virus particles, making phage eradication simply impossible.
Philippe Horvath and his team at Danisco France,
wondered what made some strains of S. thermophilus more resistant to phage infection than others.
he’d observed that, although the repeat sequences of CRISPR (the shaded black diamonds in Jill’s sketch) were always the same, the spacer sequences (Jill’s numbered squares) were highly variable from one strain to the next.
The Danisco researchers had revealed another way bacteria fought viruses—a fifth weapons system.
bacteria had in CRISPR a remarkably effective form of adaptive immunity, one that allowed the bacterial genome to steal snippets of phage DNA during an infection and use it to mount a future immune response.
CRISPR functioned like a molecular vaccination card: by storing memories of past phage infections in the form of spacer DNA sequences buried within the repeat-spacer arrays, bacteria could use this information to recognize and...
This highlight has been truncated due to consecutive passage length restrictions.
obscure biology of CRISPRs,
CRISPR immune response required DNA sequences in the bacterial genome and the phage genome to match up perfectly,
it was clear that this immune system was targeting the phages’ genetic material for destruction—but how?
speculation that RNA might coordinate the recognition and destruction phases of bacteria’s antiviral response.
how CRISPR RNA molecules were produced inside the cell.
(Remember that RNA is a molecular cousin of DNA, made up of virtually the same letters, except the letter T in DNA is replaced with the letter U in RNA.)
Since RNA is chemically so similar to DNA, it can create double helixes of its own
CRISPR might indeed be similar to the RNA interference
