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In laboratory-grown human cells, this new gene-editing technology was used to correct the mutations responsible for cystic fibrosis, sickle cell disease, some forms of blindness, and severe combined immunodeficiency, among many other disorders.
CRISPR enables scientists to accomplish such feats by finding and fixing single incorrect letters of DNA out of the 3.2 billion letters that make up the human genome,
In mid-2015, Chinese scientists published the results of an experiment in which they had injected CRISPR into human embryos. The researchers had used discarded, nonviable embryos, but their study was nevertheless a major milestone: the first-ever attempt to precisely edit the DNA of the human germline.
Imagine if someone who learned she carried the mutated copy of the HTT gene, which virtually guarantees early-onset dementia, had access to a CRISPR-based drug that could eliminate the DNA mutations before any symptoms appeared.
After running a battery of tests, the NIH scientists slowly pieced together an explanation for Kim’s serendipitous cure. They concluded that a single cell in her body must have experienced an uncommon and usually catastrophic event called chromothripsis—a recently discovered phenomenon in which a chromosome suddenly shatters and is then repaired, leading to a massive rearrangement of the genes within it. The effects in the body are generally either trivial (if the damaged cell dies immediately) or dire (if the rearranged DNA inadvertently activates cancer-causing genes).
in the 1990s, two New York patients were diagnosed with a genetic disorder called severe combined immunodeficiency (SCID), also known as the “bubble boy” disease because of the sterile environments in which some children have been contained to reduce their exposure to pathogens.
The reason in both cases, scientists determined, was that the patients’ cells had spontaneously corrected the disease-causing mutation in a gene called ADA, and they’d done it without disturbing the remainder of the gene or the chromosome.
The genome—a term coined in 1920 by the German botanist Hans Winkler and probably intended as a portmanteau of gene and chromosome—refers to the entire set of genetic instructions found inside a cell.
The genome is made up of a molecule called deoxyribonucleic acid, or DNA, which is constructed of just four different building blocks. Known as nucleotides, these are the familiar letters of DNA: A, G, C, and T, shorthand for the chemical groups (also known as bases) of adenine, guanine, cytosine, and thymine that distinguish the four compounds. The letters of these molecules are connected in long single strands. Two of these strands come together to form the famous double-helix structure of DNA.
Two strands of DNA wrap around each other along a central axis, with the continuous sugar-phosphate backbone of each one occupying the outside of the helix;
The letter A from one strand always pairs with T on the other strand, and G always pairs with C. These are known as base pairs.
Shortly before cell reproduction, the two strands are separated by an enzyme that “unzips” the double helix right down the middle. After that, other enzymes build a new partner strand for each single strand simply by using the same base-pairing rules, resulting in two exact copies of the original double helix.
DNA, as it turns out, is much like a secret language; each specific sequence of letters provides instructions to produce a particular protein inside the cell. The proteins then go on to carry out most of the critical functions in the body, like breaking down food, recognizing and destroying pathogens, and sensing light.
To transform the instructions contained in DNA into proteins, cells use a crucial—and closely related—intermediary molecule called ribonucleic acid, or RNA, which is produced from the DNA template via a process called transcription. RNA has three of the same letters as DNA, but in RNA, the letter T (for thymine) is replaced with the letter U (for uracil). In addition, the sugar that makes up the backbone of RNA contains one more oxygen atom than the sugar in DNA (hence the name deoxyribonucleic acid). RNA acts as a messenger, ferrying information from the nucleus, where the DNA is stored, to
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Whereas most bacterial genomes exist inside the cell as a single continuous piece of DNA, the human genome is composed of twenty-three distinct pieces, called chromosomes, that range in length from 50 to 250 million letters.
The full set of nuclear chromosomes can be found in almost every cell in the body (red blood cells are an important exception, as they lack a nucleus), but the nucleus isn’t the only place in the cell where DNA is found. The human genome also includes a separate mini-chromosome—just sixteen thousand letters of DNA—located in mitochondria, the energy-producing batteries of the cell.
in sickle cell disease, a genetic disorder of the blood, the seventeenth letter of a gene known as beta-globin is mutated from an A to a T.
When translated into amino acids, this mutation results in the amino acid glutamate being replaced by the amino acid valine in a critical region of the hemoglobin protein, the major oxygen-transporting component of red blood cells. The consequences of this tiny change in the protein—a difference of just ten atoms out of more than eight thousand total—are dire. The mutated hemoglobin molecules stick together and form abnormal filaments that change the shape of the red blood cells, which leads to anemia, increased risk of stroke and infection, and severe bone pain.
Sickle cell disease is an example of a recessive genetic disease. This means that both copies of an individual’s HBB gene must carry the mutation for that person to be affected; if only one copy has the alteration, the nonmutated gene can produce enough normal ...
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One example is WHIM syndrome, in which the one thousandth letter of the CXCR4 gene is mutated from a C to a T; the mutant gene creates a hyperactive protein that dominates the functioning of the healthy gene.
the neurodegenerative disorder known as Huntington’s disease is due to a mutation of the HTT gene in which the same three letters of DNA get repeated too many times. This causes brain cells to produce abnormal proteins that gradually damage them.
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. Other diseases occur when segments of a gene are inverted (that is, when they appear in reverse order) or when segments or even entire chromosomes are mistakenly duplicated or deleted.
In 2001, after herculean efforts and at a cost of more than three billion dollars, the first draft of the genome was published.
Scientists have precisely identified well over four thousand different kinds of DNA mutations that can cause genetic disease.
As a tool, viral vectors are astoundingly reliable; researchers working with viral vectors can get genes into target cells with nearly 100 percent efficiency.
A full 8 percent of the human genome—over 250 million letters of DNA—is a remnant of ancient retroviruses that infected ancestors of our species millennia ago.
Gene therapy, by its very nature, is also ineffective for a wide range of genetic conditions that aren’t caused by missing or deficient genes. Such conditions can’t be fixed by simply delivering new genes into cells.
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.
The challenge was coming up with a viable enzyme that would cut the genome in one specific place out of billions of possible options. To solve that problem, Jasin cleverly stole a piece of molecular machinery from yeast: the I-SceI endonuclease.
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.
The I-SceI endonuclease that Jasin selected was one of the most specific endonucleases known at the time, requiring a perfect match of eighteen consecutive DNA letters for it to cut a given segment. Selecting a highly discriminating endonuclease was critical; if Jasin had chosen an enzyme that was too promiscuous, it would cut the genome all over the place, not only making the results more difficult to interpret, but potentially harming the host cell. With specificity for eighteen letters in a row, though, I-SceI would cut just one sequence of DNA out of the more than fifty billion possible
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there was a fundamental problem: the vast majority of these enzymes recognized sequences that were only six or eight letters long—far too short to be useful. Those sequences occurred tens of thousands or even hundreds of thousands of times in the human genome, meaning that even if the nuclease could stimulate homologous recombination in one gene, it would shred up nearly the entire genome in the process. The cell would be destroyed before it ever had a chance to initiate DNA repair.
This is to cut a DNA strand.
a paradigm-shifting study that presented a solution to this problem took place in 1996. 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.
in spite of their promise, ZFNs were never widely adopted outside of a handful of labs. The researchers who used them had extensive experience in protein engineering, collaborations with the few labs that already had such experience, or deep pockets with which to pay the hefty price for the designer nucleases.
That
technology—at least, the first version of it—was discovered in 2009 and came from studies of novel types of proteins found in Xanthomonas, a pathogenic plant-infecting bacterium. Called transcription activator–like effectors, or TALEs, these proteins are remarkably similar to zinc finger proteins in their construction: they’re built out of multiple repeating segments in which each segment recognizes a given area of DNA. But there is a difference: whereas each finger of the zinc finger proteins recognizes a three-letter sequence of DNA, each segment in TALEs recognizes just a single letter of
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my lab had just determined the three-dimensional structure—the precise location of every single atom—of a molecule of ribonucleic acid, or RNA, which formed part of a larger molecule called a self-splicing ribozyme. In the 1980s, Tom Cech, my postdoctoral adviser at the University of Colorado, Boulder, had been awarded the Nobel Prize for his discovery of self-splicing ribozymes.
RNA interference, a molecular system that plant and animal cells use to suppress the expression of particular genes and that organisms also use during their immune responses.
CRISPR—referred to a region of bacterial DNA and that the acronym stood for “clustered regularly interspaced short palindromic repeats.”
there seemed to be an inverse correlation between the number of DNA sequences in a bacterium’s CRISPR that matched viral DNA, and the number of viruses that could infect the CRISPR-containing bacterium; the more matches, the lower the threat of infection.
In 1917, two years after Twort’s paper was published, bacterial viruses were rediscovered by a Canadian-born physician named Félix d’Herelle.
about 20 percent of bacterial infections are treated with phages in Georgia today—but after antibiotics were discovered and developed in the 1930s and 1940s, this treatment quickly lost momentum, especially in the West.
Scientists estimate that there are somewhere on the order of 1031 bacteriophages on earth;
A single teaspoon of seawater contains five times more phages than there are people in New York City.
They cause roughly a trillion trillion infections on earth every second, and in the ocean alone, about 40 percent of all bacteria die every day as a result of deadly phage infections.
All phages are built out of a durable protein exterior, called a capsid, inside of which genetic material is packaged. Phage capsids come in dozens of different shapes, all of which have been optimized to safeguard the viral genome and effectively deliver the genetic material into bacterial cells, where it can multiply and spread.
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
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a single phage can wipe out an entire bacterial population in a matter of hours.
bacteria unleash enzymes called restriction endonucleases to chop up any DNA that lack those markings, effectively purging any phage genes that managed to penetrate the cell wall.
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.
