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we now knew, 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. As Blake put it, CRISPR functioned like a molecular vaccination card:
experiments to prove that CRISPR RNAs target the DNA of invading genetic parasites. He also showed that this targeting likely relied on base-pairing interactions—the only process that could account for the specificity with which CRISPR hunted its prey.
calcium ions play a crucial role in its development by signaling the fungal cells to grow in response to nutrients.
cas genes, that flanked the CRISPR regions of bacterial genomes and that appeared to contain the code for special types of proteins called enzymes.
we know the genetic code that cells use to translate the four letters of DNA into the twenty letters of protein, biologists can determine the amino acid sequence of the protein that a gene will produce just by looking at the original DNA sequence.
by comparing that amino acid sequence to other, related proteins that are better understood, scientists can make informed predictions about the functions of many different genes.
Whether she or he is studying a gene from a microbe, a plant, a frog, or a human, a biochemist will often begin by cloning that gene into an artificial mini-chromosome, called a plasmid, and then engineering a specialized strain of E. coli to accept that plasmid as part of its own genome. By piecing together the gene of interest with other synthetic DNA instructions, the biochemist can trick E. coli into not only churning out dozens of copies of that plasmid per cell but also dedicating the majority of its resources to converting the gene of interest into thousands of copies of the protein
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like Cas1, it functioned as a chemical cleaver. In the case of Cas6, however, its job was to specifically and methodically slice the long CRISPR RNA molecules into shorter chunks that could be used to target phage DNA.
Cas3 didn’t cut the DNA just once; it chewed it up into hundreds of pieces. Once Cascade recruited Cas3 to the site of a CRISPR RNA–viral DNA match, Cas3 started shuttling along the phage genome at a rate of over three hundred base pairs per second, slicing up the DNA and leaving the lengthy phage genome as a jumble of scraps in its wake. If simpler nucleases were like pruning shears, Cas3 was like a pair of motorized hedge clippers. Its speed and efficiency were stunning.
We were amazed to see just how diverse CRISPR was. In 2005, researchers had identified nine different types of CRISPR immune systems. By 2011, that number had decreased to three—but within these basic types there were thought to be ten subtypes. And by 2015, the classification would change yet again to include two broad classes comprising six types and nineteen subtypes.
In Type I systems like E. coli and P. aeruginosa, the Cas3 enzyme—that motorized hedge clipper—chewed the DNA to shreds.
The Type II system found in S. thermophilus, by contrast, was more restrained and precise. Canadian scientists Sylvain Moineau and Josiane Garneau, working with the Danisco team, had succeeded in trapping phage genomes from infected cells as they were being destroyed by the CRISPR immune system. In a process typical of simpler nucleases, whatever was doing the cutting in S. thermophilus operated more like a pair of scissors, clipping the DNA apart at exactly the site where the letters of the viral genome matched the letters of the CRISPR RNA.
Monitoring the DNA-cutting reaction in a test tube required a sensitive detection method, since there was no way to directly visualize the DNA being cut. At fifty letters long, the DNA double helix would be just seventeen nanometers, or seventeen-billionths of a meter, long, roughly one-thousandth the width of a human hair.
sickle cell disease—the condition in which a single DNA mutation interferes with red blood cells’ ability to ferry oxygen through the body. Members of his lab had been using CRISPR to target and cut the mutated beta-globin gene, catalyzing reversion of the faulty letter A at position 17 back to the correct letter, T.
biology.
First, a temporary copy of DNA, called messenger RNA (or mRNA), is made in the cell’s nucleus. Like a strand of DNA, the mRNA is a chain of letters, and its sequence matches the sequence of the DNA it copied (the only major exception being that T gets replaced by U). The mRNA is exported out of the cell’s nucleus and delivered to a protein-synthesizing factory called a ribosome, which translates the four-letter language of RNA (A, G, C, and U) into the twenty-letter language of proteins (the twenty amino acids). This translation proceeds according to the genetic code, a cipher in which every
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TYR gene is widely distributed among animals, plants, and fungi; it produces a protein called tyrosinase that is involved in synthesizing melanin, an important pigment. TYR mutations in humans lead to a deficiency in tyrosinase and cause type I albinism, a genetic condition associated with vision defects, pale skin lacking pigmentation, and red eyes.
Stanley demonstrated that a deactivated version of CRISPR had its own uses for manipulating the genome. Rather than introducing permanent genetic changes by editing DNA, the deactivated CRISPR allowed scientists to make temporary changes that would not alter the underlying genetic information of a cell but nevertheless affected how genetic information was expressed. In particular, he transformed CRISPR into a gene-expression controller that could turn genes on or off or dial them up or down, much like a dimmer adjusts lighting.
Recently, food scientists at a Minnesota company called Calyxt used the TALEN gene-editing technology to alter two soybean genes, generating seeds with a drastic reduction in the unhealthy fatty acids and an overall fat profile similar to that of olive oil. They accomplished this without causing any unintended mutations and without introducing any foreign DNA into the genome.
The lengthy cold storage required to increase potatoes’ shelf life can lead to cold-induced sweetening, a phenomenon in which starches are converted into sugars such as glucose and fructose. Any cooking process involving high heat—necessary to make french fries and potato chips—converts these sugars into acrylamide, a chemical that is a neurotoxin and a potential carcinogen.
Since 1994, when the first commercially grown GMO plant approved for human consumption was introduced—a slow-ripening tomato variant known as the Flavr Savr—well over fifty GMO crops have been developed and approved for commercial cultivation in the United States, among them canola, corn, cotton, papaya, rice, soybean, squash, and many more. In 2015, 92 percent of all corn, 94 percent of all cotton, and 94 percent of all soybeans grown in the United States were genetically engineered in this way.
Red grapefruits created by neutron radiation, seedless watermelons produced with the chemical compound colchicine, apple orchards in which every tree is a perfect genetic clone of its neighbors—none
2004, a team of physicians from Berlin published a remarkable study describing a boy who was extraordinarily muscular at birth, with bulging thigh and upper-arm muscles. The child continued to develop abnormally pronounced muscles through age four and could perform incredible feats of strength, like extending his arms while holding a three-kilogram dumbbell in each hand. Given his condition’s resemblance to the double muscling in cows and mice, and given the history of unusual strength in his family, the physicians suspected that genetics could explain his physique. After some molecular
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Chinese scientists working with goats targeted the myostatin gene as well as a growth factor gene known to control hair length.
CRISPR offers the greatest hope to treat monogenic genetic diseases—those caused by a single mutated gene.
Two of the promising disease targets for ex vivo CRISPR therapies are sickle cell disease and beta-thalassemia.
Believe it or not, some lucky people are naturally resistant to HIV. These individuals lack thirty-two letters of DNA in the gene for a protein called CCR5, which is located on the surface of white blood cells—those cells that form the bedrock of the body’s immune system. CCR5 proteins are one of the parts of the cell’s surface that the HIV virus latches onto in the initial stage of its invasion. This specific, thirty-two-letter deletion causes the CCR5 protein to be truncated and prevents it from making its way to the cell surface. Without CCR5 proteins to attach to, HIV molecules can’t
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In people of African and Asian descent, the thirty-two-letter CCR5 deletion is virtually nonexistent, but it’s fairly prevalent among Caucasian people; 10 to 20 percent of Caucasians possess one copy of the mutated gene, and homozygous individuals—those who possess two copies—are completely resistant to HIV.
These CCR5-lacking individuals are otherwise completely healthy and even experience a reduced risk of certain inflammatory diseases; the missing protein doesn’t cause any adverse effects. Just about the only known risk to not having it is a possible increase in susceptibility to the West Nile virus.
Duchenne muscular dystrophy can arise from any one of several mutations in the DMD gene—the largest human gene known—which encodes a protein called dystrophin. This protein helps muscle cells contract, and the lack of a functional dystrophin protein is the underlying problem for DMD patients. Males are disproportionately affected; since the DMD gene is found on the X chromosome and males possess only one X chromosome (paired with a paternally inherited Y chromosome), a single mutated copy of DMD leaves them wholly devoid of healthy dystrophin. Females, however, have two X chromosomes and thus
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And then there are in vivo delivery strategies that won’t use viruses at all. Building on advances in nanotechnology—the science of fabricating submicroscopic structures—researchers are exploring the use of lipid nanoparticles to ferry CRISPR throughout the body. Resistant to degradation and easy to manufacture, these delivery vehicles also have the benefit of releasing the Cas9 protein and its guide RNA into the patient’s body in a regimented way. Viruses (and their CRISPR cargo) can persist in cells for a long time, which—as I’ll explain—can cause problems in the editing process, but lipid
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A revolutionary form of cancer treatment, immunotherapy is a departure from the three main types—surgery, radiation, and chemotherapy—that doctors have historically employed. Unlike these older approaches, cancer immunotherapy aims to use a patient’s own immune system to hunt down and destroy dangerous cells. In a complete paradigm shift, immunotherapy targets not the cancer, but the patient’s own body, empowering it to fight cancer on its own. The core idea behind cancer immunotherapy is to tweak the human immune system, specifically the T cells, its primary foot soldiers. By rewiring these
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Layla Richards, is the first human whose life was saved by therapeutic gene editing.
in Junjiu Huang’s lab at Sun Yat-sen University in Guangzhou, China. Huang and his colleagues had injected CRISPR into eighty-six human embryos. The target in this study was the gene responsible for producing beta-globin,
prestigious journals Nature and Science had both rejected Huang’s manuscript, partly because they had ethical objections to the experiments it described. Many scientists agreed that the research had been pursued prematurely, and others wondered about the motivations behind it. Harvard researcher George Daley told the New York Times that the attention scientists were sure to receive for editing the human germline might be “the sort of deranged motivation that sometimes prompts people to do things.”
editing an embryo’s CCR5 gene might make the resulting human resistant to HIV but more susceptible to the West Nile virus. Correcting the two mutated copies of the beta-globin gene in people who suffer from sickle cell disease would rid them of the illness but also deprive them of the mutation’s protection against malaria.
Researchers now suspect that people who carry one copy of the mutated gene that causes cystic fibrosis (which requires two copies) have an increased defense against tuberculosis, an infectious disease that accounted for 20 percent of all European deaths between 1600 and 1900.
gene variants implicated in neurodegenerative diseases like Alzheimer’s may have benefits, such as improved cognitive function and better episodic and working memory in young adults.
mutations in a gene called LRP5 endow individuals with extra-strong bones;
mutations in a gene called ABCC11 are associated with lower levels of armpit odor production (and, oddly, the type of earwax an individual produces);
mutations in a gene called DEC2 are associated with a lower requirement of daily sleep.
One of these liminal examples of germline editing involves the gene PCSK9, which produces a protein that regulates a person’s level of low-density lipoprotein cholesterol (the “bad” cholesterol), making the gene one of the most promising pharmaceutical targets to prevent heart disease—the leading cause of death worldwide.
in a long list of countries that includes Canada, France, Germany, Brazil, and Australia, clinical interventions in the human germline are expressly prohibited, with criminal sanctions that range from fines to lengthy prison terms. In other countries, such as China, India, and Japan, these interventions are forbidden, but with guidelines that are not legislative and thus less enforceable. In the United States, the current policy might be considered restrictive: there are no outright bans,
