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Agustus 24, 2017

beautiful: robot dna, genom BUATAN V kanker manusia … 170212_240817

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time.com : With the usual mix of anticipation and apprehension, Kaitlyn Johnson is getting ready to go to her first summer camp. She’s looking forward to meeting new friends and being able to ride horses, swim and host tea parties. She’s also a little nervous and a little scared, like any 7-year-old facing her first sleepaway camp.

But the wonder is that Kaitlyn is leaving the house for anything but a medical facility. Diagnosed with leukemia when she was 18 months old, her life has been consumed with cancer treatments, doctors’ visits and hospital stays.

Acute lymphoblastic leukemia is the most common cancer among young children, accounting for a quarter of all cancer cases in kids, and it has no cure. For about 85% to 90% of children, the leukemia can, however, be effectively treated through chemotherapy.

If it is not eliminated and comes back, it is, more often than not, fatal. Rounds of chemotherapy can buy patients time, but as the disease progresses, the periods of remission get shorter and shorter. “The options for these patients are not very good at all,” says Dr. Theodore Laetsch, a pediatrician at the University of Texas Southwestern Medical Center.

When Kaitlyn’s cancer wasn’t controlled after three years and round after round of chemotherapy drugs, her doctors had little else to offer. “They said, ‘This did nothing, it didn’t touch it,'” says Kaitlyn’s mother Mandy, a dental assistant from Royce City, Texas. “My stomach just dropped.” Kaitlyn could receive a bone-marrow transplant, but only about half of those procedures are successful, and there was a good chance that she would reject the donor cells. If that happened, her chances of surviving were very small.

In a calculated gamble, her doctors suggested a radical new option: becoming a test subject in a trial of an experimental therapy that would, for the first time, use gene therapy to train a patient’s immune system to recognize and destroy their cancer in the same way it dispatches bacteria and viruses. The strategy is the latest development in immunotherapy, a revolutionary approach to cancer treatment that uses a series of precision strikes to disintegrate cancer from within the body itself. Joining the trial was risky, since other attempts to activate the immune system hadn’t really worked in the past. Mandy, her husband James and Kaitlyn traveled from their home in Texas to Children’s Hospital of Philadelphia (CHOP), where they stayed in a hotel for eight weeks while Kaitlyn received the therapy and recovered. “The thought crossed my mind that Kaitlyn might not come home again,” says Mandy. “I couldn’t tell you how many times I would be in the bathroom at the hospital, spending an hour in the shower just crying, thinking, What are we going to do if this doesn’t help her?”

But it did. After receiving the therapy in 2015, the cancer cells in Kaitlyn’s body melted away. Test after test, including one that picks up one cancer cell in a million, still can’t detect any malignant cells lurking in Kaitlyn’s blood. What saved Kaitlyn was an infusion of her own immune cells that were genetically modified to destroy her leukemia. “You take someone who essentially has no possibility for a cure–almost every single one of these patients dies–and with [this] therapy, 90% go into remission,” says Dr. David Porter, director of blood and bone-marrow transplantation at the University of Pennsylvania. Such radical immune-based approaches were launched in 2011 with the success of intravenous drugs that loosen the brakes on the immune system so it can see cancer cells and destroy them with the same vigor with which they attack bacteria and viruses. Now, with the genetically engineered immune cells known as chimeric antigen receptor (CAR) T cells that were used in Kaitlyn’s study, doctors are crippling cancer in more precise and targeted ways than surgery, chemotherapy and radiation ever could. While the first cancer immunotherapies were broadly aimed at any cancer, experts are now repurposing the immune system into a personalized precision treatment that can not only recognize but also eliminate the cancer cells unique to each individual patient.

What makes immune-based therapies like CAR T cell therapy so promising–and so powerful–is that they are a living drug churned out by the patients themselves. The treatment isn’t a pill or a liquid that has to be taken regularly, but a one-hit wonder that, when given a single time, trains the body to keep on treating, ideally for a lifetime.

“This therapy is utterly transformative for this kind of leukemia and also lymphoma,” says Stephan Grupp, director of the cancer immunotherapy program at CHOP and one of the lead doctors treating patients in the study in which Kaitlyn participated.

Eager to bring this groundbreaking option to more patients, including those with other types of cancers, an advisory panel for the Food and Drug Administration voted unanimously in July to move the therapy beyond the testing phase, during which several hundred people have been able to take advantage of it, to become a standard therapy for children with certain leukemias if all other treatments have failed. While the FDA isn’t obligated to follow the panel’s advice, it often does, and it is expected to announce its decision in a matter of weeks.

Across the country, doctors are racing to enroll people with other cancers–breast, prostate, pancreatic, ovarian, sarcoma and brain, including the kind diagnosed in Senator John McCain–in hundreds of trials to see if they, too, will benefit from this novel approach. They are even cautiously allowing themselves to entertain the idea that this living drug may even lead to a cure for some of these patients. Curing cancers, rather than treating them, would result in a significant drop in the more than $120 billion currently spent each year on cancer care in the U.S., as well as untold suffering.

Cancer-newest-miracle-cure-06Lon Tweeten for TIME 

This revolutionary therapy, however, almost didn’t happen. While the idea of using the body’s immune cells against cancer has been around for a long time, the practical reality had proved daunting. Unlike infection-causing bacteria and viruses that are distinctly foreign to the body, cancer cells start out as healthy cells that mutate and grow out of control, and the immune system is loath to target its own cells.

“Only a handful of people were doing the research,” says Dr. Carl June, director of the Center for Cellular Immunotherapy at the University of Pennsylvania’s Abramson Cancer Center and the scientist who pioneered the therapy. A graduate of the U.S. Naval Academy, June is all too familiar with the devastating effects of cancer, having lost his first wife to ovarian cancer and battled skin cancer himself. Trial after trial failed as reinfusions of immune cells turned out to be more of a hit-or-miss endeavor than a reliable road to remission.

After spending nearly three decades on the problem, June zeroed in on a malignant fingerprint that could be exploited to stack the deck of a cancer patient’s immune system with the right destructive cells to destroy the cancer.

In the case of leukemias, that marker turned out to be CD19, a protein that all cancerous blood cells sprout on their surface. June repurposed immune cells to carry a protein that would stick to CD19, along with another marker that would activate the immune cells to start attacking the cancer more aggressively once they found their malignant marks. Using a design initially developed by researchers at St. Jude Children’s Research Hospital for such a combination, June and his colleague Bruce Levine perfected a way to genetically modify and grow these cancer-fighting cells in abundance in the lab and to test them in animals with leukemia. The resulting immune platoon of CAR T cells is uniquely equipped to ferret out and destroy cancer cells. But getting them into patients is a complex process. Doctors first remove a patient’s immune cells from the blood, genetically tweak them in the lab to carry June’s cancer-targeting combination and then infuse the modified cells back into the patient using an IV.

Because these repurposed immune cells continue to survive and divide, the therapy continues to work for months, years and, doctors hope, perhaps a lifetime. Similar to the way vaccines prompt the body to produce immune cells that can provide lifelong protection against viruses and bacteria, CAR T cell therapy could be a way to immunize against cancer. “The word vaccination would not be inappropriate,” says Dr. Otis Brawley, chief medical officer of the American Cancer Society.

June’s therapy worked surprisingly well in mice, shrinking tumors and, in some cases, eliminating them altogether. He applied for a grant at the National Cancer Institute at the National Institutes of Health to study the therapy in people from 2010 to 2011. But the idea was still so new that many scientists believed that testing it in people was too risky. In 1999, a teenager died days after receiving an experimental dose of genes to correct an inherited disorder, and anything involving gene therapy was viewed suspiciously. While such deaths aren’t entirely unusual in experimental studies, there were ethical questions about whether the teenager and his family were adequately informed of the risks and concerns that the doctor in charge of the study had a financial conflict of interest in seeing the therapy develop. Officials in charge of the program acknowledged that important questions were raised by the trial and said they took the questions and concerns very seriously. But the entire gene-therapy program was shut down. All of that occurred at the University of Pennsylvania–where June was. His grant application was rejected.

It would take two more years before private funders–the Leukemia and Lymphoma Society and an alumnus of the university who was eager to support new cancer treatments–donated $5 million to give June the chance to bring his therapy to the first human patients.

The date July 31 has always been a milestone for Bill Ludwig, a retired corrections officer in New Jersey. It’s the day that he joined the Marines as an 18-year-old, and the day, 30 years later, that he married his wife Darla.

It was also the day he went to the hospital to become the first person ever to receive the combination gene and CAR T cell therapy, in 2010. For Ludwig, the experimental therapy was his only remaining option. Like many people with leukemia, Ludwig had been living on borrowed time for a decade, counting the days between the chemotherapy treatments that would hold the cancer in his blood cells at bay for a time. Inevitably, like weeds in an untended garden, the leukemia cells would grow and take over his blood system again.

But the periods of reprieve were getting dangerously short. “I was running out of treatments,” says Ludwig. So when his doctor mentioned the trial conducted by June and Porter at the University of Pennsylvania, he didn’t hesitate. “I never thought that the clinical trial was going to cure me,” he says. “I just wanted to live and to continue to fight. If there was something that would put me into the next month, still breathing, then that’s what I was looking for.”

When Ludwig signed the consent form for the treatment, he wasn’t even told what to expect in terms of side effects or adverse reactions. The scientists had no way of predicting what would happen. “They explained that I was the first and that they obviously had no case law, so to speak,” he says. So when he was hit with a severe fever, had difficulty breathing, showed signs of kidney failure and was admitted to the intensive care unit, he assumed that the treatment wasn’t working.

His condition deteriorated so quickly and so intensely that doctors told him to call his family to his bedside, just four days after he received the modified cells. “I told my family I loved them and that I knew why they were there,” he says. “I had already gone and had a cemetery plot, and already paid for my funeral.”

Rather than signaling the end, Ludwig’s severe illness turned out to be evidence that the immune cells he received were furiously at work, eliminating and sweeping away the huge burden of cancer cells choking up his bloodstream. But his doctors did not realize it at the time.

It wasn’t until the second patient, Doug Olson, who received his CAR T cells about six weeks after Ludwig, that Porter had a eureka moment. When he received the call that Olson was also running a high fever, having trouble breathing and showing abnormal lab results, Porter realized that these were signs that the treatment was working. “It happens when you kill huge amounts of cancer cells all at the same time,” Porter says. What threw him off initially is that it’s rare for anything to wipe out that much cancer in people with Ludwig’s and Olson’s disease. June and Porter have since calculated that the T cells obliterated anywhere from 2.5 lb. to 7 lb. of cancer in Ludwig’s and Olson’s bodies. “I couldn’t fathom that this is why they both were so sick,” says Porter. “But I realized this is the cells: they were working, and working rapidly. It was not something we see with chemotherapy or anything else we have to treat this cancer.”

Ludwig has now been in remission for seven years, and his success led to the larger study of CAR T cell therapy in children like Kaitlyn, who no longer respond to existing treatments for their cancer. The only side effect Ludwig has is a weakened immune system; because the treatment wipes out a category of his immune cells–the ones that turned cancerous–he returns to the University of Pennsylvania every seven weeks for an infusion of immunoglobulins to protect him from pneumonia and colds. Olson, too, is still cancer-free.

While the number of people who have received CAR T cell therapy is still small, the majority are in remission. That’s especially encouraging for children, whose lives are permanently disrupted by the repeated cycles of treatments that currently are their only option. “It’s a chance for these kids to have a normal life and a normal childhood that doesn’t involve constant infusions, transfusions, infections and being away from their home, family and school,” says Dr. Gwen Nichols, chief medical officer of the Leukemia and Lymphoma Society.

The hope is that while CAR T cell therapy will at first be reserved for people who have failed to respond to all standard treatments, eventually they won’t have to wait that long. As doctors learn from pioneers like Kaitlyn, Ludwig and Olson, they will have more confidence in pushing the therapy earlier, when patients are stronger and the cancer is less advanced–perhaps as a replacement for or in combination with other treatments.

The severe immune reaction triggered by the therapy remains a big concern. While it can be monitored in the hospital and managed with steroids or antibodies that fight inflammation, there have been deaths in other trials involving CAR T cells. One drug company put one of its studies on hold due to the toxic side effects. “I am excited by CAR T therapy, but I’m also worried that some people might get too excited,” says the American Cancer Society’s Brawley. “It’s important that we proceed slowly and do this meticulously so that we develop this in the right way.”

For now, CAR T cells are expensive–some analysts estimate that each patient’s batch of cells would cost hundreds of thousands of dollars–because they require a bespoke production process. If approved, Novartis, which licensed the technology from the University of Pennsylvania, will provide the therapy in about 35 cancer centers in the U.S. by the end of the year. Other companies are already working toward universal T cells that could be created for off-the-shelf use in any patient with cancer. “This is just the beginning,” says June.

Since Ludwig’s cancer has been in remission, he and his wife have packed their RV and taken the vacations they missed while he was a slave to his cancer and chemotherapy schedule. This year, they’re visiting Mount Rushmore, Grand Teton National Park and Yellowstone National Park before taking their granddaughter to Disney World in the fall. “When they told me I was cancer-free, it was just like someone said, ‘You won the lottery,'” he says. “If somebody else with this disease has the chance to walk in my shoes and live past it, that would be the greatest gift for me.”


SPINY GRASS AND SCRAGGLY PINES creep amid the arts-and-crafts buildings of the Asilomar Conference Grounds, 100 acres of dune where California’s Monterey Peninsula hammerheads into the Pacific. It’s a rugged landscape, designed to inspire people to contemplate their evolving place on Earth. So it was natural that 140 scientists gathered here in 1975 for an unprecedented conference.

They were worried about what people called “recombinant DNA,” the manipulation of the source code of life. It had been just 22 years since James Watson, Francis Crick, and Rosalind Franklin described what DNA was—deoxyribonucleic acid, four different structures called bases stuck to a backbone of sugar and phosphate, in sequences thousands of bases long. DNA is what genes are made of, and genes are the basis of heredity.

Preeminent genetic researchers like David Baltimore, then at MIT, went to Asilomar to grapple with the implications of being able to decrypt and reorder genes. It was a God-like power—to plug genes from one living thing into another. Used wisely, it had the potential to save millions of lives. But the scientists also knew their creations might slip out of their control. They wanted to consider what ought to be off-limits.

By 1975, other fields of science—like physics—were subject to broad restrictions. Hardly anyone was allowed to work on atomic bombs, say. But biology was different. Biologists still let the winding road of research guide their steps. On occasion, regulatory bodies had acted retrospectively—after Nuremberg, Tuskegee, and the human radiation experiments, external enforcement entities had told biologists they weren’t allowed to do that bad thing again. Asilomar, though, was about establishing prospective guidelines, a remarkably open and forward-thinking move.


At the end of the meeting, Baltimore and four other molecular biologists stayed up all night writing a consensus statement. They laid out ways to isolate potentially dangerous experiments and determined that cloning or otherwise messing with dangerous pathogens should be off-limits. A few attendees fretted about the idea of modifications of the human “germ line”—changes that would be passed on from one generation to the next—but most thought that was so far off as to be unrealistic. Engineering microbes was hard enough. The rules the Asilomar scientists hoped biology would follow didn’t look much further ahead than ideas and proposals already on their desks.

Earlier this year, Baltimore joined 17 other researchers for another California conference, this one at the Carneros Inn in Napa Valley. “It was a feeling of déjà vu,” Baltimore says. There he was again, gathered with some of the smartest scientists on earth to talk about the implications of genome engineering.

The stakes, however, have changed. Everyone at the Napa meeting had access to a gene-editing technique called Crispr-Cas9. The first term is an acronym for “clustered regularly interspaced short palindromic repeats,” a description of the genetic basis of the method; Cas9 is the name of a protein that makes it work. Technical details aside, Crispr-Cas9 makes it easy, cheap, and fast to move genes around—any genes, in any living thing, from bacteria to people. “These are monumental moments in the history of biomedical research,” Baltimore says. “They don’t happen every day.”

Using the three-year-old technique, researchers have already reversed mutations that cause blindness, stopped cancer cells from multiplying, and made cells impervious to the virus that causes AIDS. Agronomists have rendered wheat invulnerable to killer fungi like powdery mildew, hinting at engineered staple crops that can feed a population of 9 billion on an ever-warmer planet. Bioengineers have used Crispr to alter the DNA of yeast so that it consumes plant matter and excretes ethanol, promising an end to reliance on petrochemicals. Startups devoted to Crispr have launched. International pharmaceutical and agricultural companies have spun up Crispr R&D. Two of the most powerful universities in the US are engaged in a vicious war over the basic patent. Depending on what kind of person you are, Crispr makes you see a gleaming world of the future, a Nobel medallion, or dollar signs.

The technique is revolutionary, and like all revolutions, it’s perilous. Crispr goes well beyond anything the Asilomar conference discussed. It could at last allow genetics researchers to conjure everything anyone has ever worried they would—designer babies, invasive mutants, species-specific bioweapons, and a dozen other apocalyptic sci-fi tropes. It brings with it all-new rules for the practice of research in the life sciences. But no one knows what the rules are—or who will be the first to break them.

IN A WAY, humans were genetic engineers long before anyone knew what a gene was. They could give living things new traits—sweeter kernels of corn, flatter bulldog faces—through selective breeding. But it took time, and it didn’t always pan out. By the 1930s refining nature got faster. Scientists bombarded seeds and insect eggs with x-rays, causing mutations to scatter through genomes like shrapnel. If one of hundreds of irradiated plants or insects grew up with the traits scientists desired, they bred it and tossed the rest. That’s where red grapefruits came from, and most barley for modern beer.

Genome modification has become less of a crapshoot. In 2002, molecular biologists learned to delete or replace specific genes using enzymes called zinc-finger nucleases; the next-generation technique used enzymes named TALENs.

Yet the procedures were expensive and complicated. They only worked on organisms whose molecular innards had been thoroughly dissected—like mice or fruit flies. Genome engineers went on the hunt for something better.

Scientists have used it to render wheat invulnerable to killer fungi. Such crops could feed billions of people.

As it happened, the people who found it weren’t genome engineers at all. They were basic researchers, trying to unravel the origin of life by sequencing the genomes of ancient bacteria and microbes called Archaea (as in archaic), descendants of the first life on Earth. Deep amid the bases, the As, Ts, Gs, and Cs that made up those DNA sequences, microbiologists noticed recurring segments that were the same back to front and front to back—palindromes. The researchers didn’t know what these segments did, but they knew they were weird. In a branding exercise only scientists could love, they named these clusters of repeating palindromes Crispr.

Then, in 2005, a microbiologist named Rodolphe Barrangou, working at a Danish food company called Danisco, spotted some of those same palindromic repeats in Streptococcus thermophilus, the bacteria that the company uses to make yogurt and cheese. Barrangou and his colleagues discovered that the unidentified stretches of DNA between Crispr’s palindromes matched sequences from viruses that had infected their S. thermophilus colonies. Like most living things, bacteria get attacked by viruses—in this case they’re called bacteriophages, or phages for short. Barrangou’s team went on to show that the segments served an important role in the bacteria’s defense against the phages, a sort of immunological memory. If a phage infected a microbe whose Crispr carried its fingerprint, the bacteria could recognize the phage and fight back. Barrangou and his colleagues realized they could save their company some money by selecting S. thermophilus species with Crispr sequences that resisted common dairy viruses.

As more researchers sequenced more bacteria, they found Crisprs again and again—half of all bacteria had them. Most Archaea did too. And even stranger, some of Crispr’s sequences didn’t encode the eventual manufacture of a protein, as is typical of a gene, but instead led to RNA—single-stranded genetic material. (DNA, of course, is double-stranded.)

That pointed to a new hypothesis. Most present-day animals and plants defend themselves against viruses with structures made out of RNA. So a few researchers started to wonder if Crispr was a primordial immune system. Among the people working on that idea was Jill Banfield, a geomicrobiologist at UC Berkeley, who had found Crispr sequences in microbes she collected from acidic, 110-degree water from the defunct Iron Mountain Mine in Shasta County, California. But to figure out if she was right, she needed help.

Luckily, one of the country’s best-known RNA experts, a biochemist named Jennifer Doudna, worked on the other side of campus in an office with a view of the Bay and San Francisco’s skyline. It certainly wasn’t what Doudna had imagined for herself as a girl growing up on the Big Island of Hawaii. She simply liked math and chemistry—an affinity that took her to Harvard and then to a postdoc at the University of Colorado. That’s where she made her initial important discoveries, revealing the three-dimensional structure of complex RNA molecules that could, like enzymes, catalyze chemical reactions.

The mine bacteria piqued Doudna’s curiosity, but when Doudna pried Crispr apart, she didn’t see anything to suggest the bacterial immune system was related to the one plants and animals use. Still, she thought the system might be adapted for diagnostic tests.

Banfield wasn’t the only person to ask Doudna for help with a Crispr project. In 2011, Doudna was at an American Society for Microbiology meeting in San Juan, Puerto Rico, when an intense, dark-haired French scientist asked her if she wouldn’t mind stepping outside the conference hall for a chat. This was Emmanuelle Charpentier, a microbiologist at Ume˚a University in Sweden.

As they wandered through the alleyways of old San Juan, Charpentier explained that one of Crispr’s associated proteins, named Csn1, appeared to be extraordinary. It seemed to search for specific DNA sequences in viruses and cut them apart like a microscopic multitool. Charpentier asked Doudna to help her figure out how it worked. “Somehow the way she said it, I literally—I can almost feel it now—I had this chill down my back,” Doudna says. “When she said ‘the mysterious Csn1’ I just had this feeling, there is going to be something good here.”

Back in Sweden, Charpentier kept a colony of Streptococcus pyogenesin a biohazard chamber. Few people want S. pyogenes anywhere near them. It can cause strep throat and necrotizing fasciitis—flesh-eating disease. But it was the bug Charpentier worked with, and it was in S. pyogenes that she had found that mysterious yet mighty protein, now renamed Cas9. Charpentier swabbed her colony, purified its DNA, and FedExed a sample to Doudna.

Working together, Charpentier’s and Doudna’s teams found that Crispr made two short strands of RNA and that Cas9 latched onto them. The sequence of the RNA strands corresponded to stretches of viral DNA and could home in on those segments like a genetic GPS. And when the Crispr-Cas9 complex arrives at its destination, Cas9 does something almost magical: It changes shape, grasping the DNA and slicing it with a precise molecular scalpel.

Here’s what’s important: Once they’d taken that mechanism apart, Doudna’s postdoc, Martin Jinek, combined the two strands of RNA into one fragment—“guide RNA”—that Jinek could program. He could make guide RNA with whatever genetic letters he wanted; not just from viruses but from, as far as they could tell, anything. In test tubes, the combination of Jinek’s guide RNA and the Cas9 protein proved to be a programmable machine for DNA cutting. Compared to TALENs and zinc-finger nucleases, this was like trading in rusty scissors for a computer-controlled laser cutter. “I remember running into a few of my colleagues at Berkeley and saying we have this fantastic result, and I think it’s going to be really exciting for genome engineering. But I don’t think they quite got it,” Doudna says. “They kind of humored me, saying, ‘Oh, yeah, that’s nice.’”

On June 28, 2012, Doudna’s team published its results in Science. In the paper and in an earlier corresponding patent application, they suggest their technology could be a tool for genome engineering. It was elegant and cheap. A grad student could do it.

The finding got noticed. In the 10 years preceding 2012, 200 papers mentioned Crispr. By 2014 that number had more than tripled. Doudna and Charpentier were each recently awarded the $3 million 2015 Breakthrough Prize. Time magazine listed the duo among the 100 most influential people in the world. Nobody was just humoring Doudna anymore.

MOST WEDNESDAY AFTERNOONS, Feng Zhang, a molecular biologist at the Broad Institute of MIT and Harvard, scans the contents of Scienceas soon as they are posted online. In 2012, he was working with Crispr-Cas9 too. So when he saw Doudna and Charpentier’s paper, did he think he’d been scooped? Not at all. “I didn’t feel anything,” Zhang says. “Our goal was to do genome editing, and this paper didn’t do it.” Doudna’s team had cut DNA floating in a test tube, but to Zhang, if you weren’t working with human cells, you were just screwing around.

That kind of seriousness is typical for Zhang. At 11, he moved from China to Des Moines, Iowa, with his parents, who are engineers—one computer, one electrical. When he was 16, he got an internship at the gene therapy research institute at Iowa Methodist hospital. By the time he graduated high school he’d won multiple science awards, including third place in the Intel Science Talent Search.

When Doudna talks about her career, she dwells on her mentors; Zhang lists his personal accomplishments, starting with those high school prizes. Doudna seems intuitive and has a hands-off management style. Zhang … pushes. We scheduled a video chat at 9:15 pm, and he warned me that we’d be talking data for a couple of hours. “Power-nap first,” he said.

If new genes that wipe out malaria also make mosquitoes go extinct, what will bats eat?

Zhang got his job at the Broad in 2011, when he was 29. Soon after starting there, he heard a speaker at a scientific advisory board meeting mention Crispr. “I was bored,” Zhang says, “so as the researcher spoke, I just Googled it.” Then he went to Miami for an epigenetics conference, but he hardly left his hotel room. Instead Zhang spent his time reading papers on Crispr and filling his notebook with sketches on ways to get Crispr and Cas9 into the human genome. “That was an extremely exciting weekend,” he says, smiling.

Just before Doudna’s team published its discovery in Science, Zhang applied for a federal grant to study Crispr-Cas9 as a tool for genome editing. Doudna’s publication shifted him into hyperspeed. He knew it would prompt others to test Crispr on genomes. And Zhang wanted to be first.

Even Doudna, for all of her equanimity, had rushed to report her finding, though she hadn’t shown the system working in human cells. “Frankly, when you have a result that is exciting,” she says, “one does not wait to publish it.”

In January 2013, Zhang’s team published a paper in Science showing how Crispr-Cas9 edits genes in human and mouse cells. In the same issue, Harvard geneticist George Church edited human cells with Crispr too. Doudna’s team reported success in human cells that month as well, though Zhang is quick to assert that his approach cuts and repairs DNA better.

That detail matters because Zhang had asked the Broad Institute and MIT, where he holds a joint appointment, to file for a patent on his behalf. Doudna had filed her patent application—which was public information—seven months earlier. But the attorney filing for Zhang checked a box on the application marked “accelerate” and paid a fee, usually somewhere between $2,000 and $4,000. A series of emails followed between agents at the US Patent and Trademark Office and the Broad’s patent attorneys, who argued that their claim was distinct.

A little more than a year after those human-cell papers came out, Doudna was on her way to work when she got an email telling her that Zhang, the Broad Institute, and MIT had indeed been awarded the patent on Crispr-Cas9 as a method to edit genomes. “I was quite surprised,” she says, “because we had filed our paperwork several months before he had.”

The Broad win started a firefight. The University of California amended Doudna’s original claim to overlap Zhang’s and sent the patent office a 114-page application for an interference proceeding—a hearing to determine who owns Crispr—this past April. In Europe, several parties are contesting Zhang’s patent on the grounds that it lacks novelty. Zhang points to his grant application as proof that he independently came across the idea. He says he could have done what Doudna’s team did in 2012, but he wanted to prove that Crispr worked within human cells. The USPTO may make its decision as soon as the end of the year.

The stakes here are high. Any company that wants to work with anything other than microbes will have to license Zhang’s patent; royalties could be worth billions of dollars, and the resulting products could be worth billions more. Just by way of example: In 1983 Columbia University scientists patented a method for introducing foreign DNA into cells, called cotransformation. By the time the patents expired in 2000, they had brought in $790 million in revenue.

It’s a testament to Crispr’s value that despite the uncertainty over ownership, companies based on the technique keep launching. In 2011 Doudna and a student founded a company, Caribou, based on earlier Crispr patents; the University of California offered Caribou an exclusive license on the patent Doudna expected to get. Caribou uses Crispr to create industrial and research materials, potentially enzymes in laundry detergent and laboratory reagents. To focus on disease—where the long-term financial gain of Crispr-Cas9 will undoubtedly lie—Caribou spun off another biotech company called Intellia Therapeutics and sublicensed the Crispr-Cas9 rights. Pharma giant Novartis has invested in both startups. In Switzerland, Charpentier cofounded Crispr Therapeutics. And in Cambridge, Massachusetts, Zhang, George Church, and several others founded Editas Medicine, based on licenses on the patent Zhang eventually received.

Thus far the four companies have raised at least $158 million in venture capital.

ANY GENE TYPICALLY has just a 50–50 chance of getting passed on. Either the offspring gets a copy from Mom or a copy from Dad. But in 1957 biologists found exceptions to that rule, genes that literally manipulated cell division and forced themselves into a larger number of offspring than chance alone would have allowed.

A decade ago, an evolutionary geneticist named Austin Burt proposed a sneaky way to use these “selfish genes.” He suggested tethering one to a separate gene—one that you wanted to propagate through an entire population. If it worked, you’d be able to drive the gene into every individual in a given area. Your gene of interest graduates from public transit to a limousine in a motorcade, speeding through a population in flagrant disregard of heredity’s traffic laws. Burt suggested using this “gene drive” to alter mosquitoes that spread malaria, which kills around a million people every year. It’s a good idea. In fact, other researchers are already using other methods to modify mosquitoes to resist the Plasmodium parasite that causes malaria and to be less fertile, reducing their numbers in the wild. But engineered mosquitoes are expensive. If researchers don’t keep topping up the mutants, the normals soon recapture control of the ecosystem.

Push those modifications through with a gene drive and the normal mosquitoes wouldn’t stand a chance. The problem is, inserting the gene drive into the mosquitoes was impossible. Until Crispr-Cas9 came along.

Today, behind a set of four locked and sealed doors in a lab at the Harvard School of Public Health, a special set of mosquito larvae of the African species Anopheles gambiae wriggle near the surface of shallow tubs of water. These aren’t normal Anopheles, though. The lab is working on using Crispr to insert malaria-resistant gene drives into their genomes. It hasn’t worked yet, but if it does … well, consider this from the mosquitoes’ point of view. This project isn’t about reengineering one of them. It’s about reengineering them all.

Kevin Esvelt, the evolutionary engineer who initiated the project, knows how serious this work is. The basic process could wipe out any species. Scientists will have to study the mosquitoes for years to make sure that the gene drives can’t be passed on to other species of mosquitoes. And they want to know what happens to bats and other insect-eating predators if the drives make mosquitoes extinct. “I am responsible for opening a can of worms when it comes to gene drives,” Esvelt says, “and that is why I try to ensure that scientists are taking precautions and showing themselves to be worthy of the public’s trust—maybe we’re not, but I want to do my damnedest to try.”

Esvelt talked all this over with his adviser—Church, who also worked with Zhang. Together they decided to publish their gene-drive idea before it was actually successful. They wanted to lay out their precautionary measures, way beyond five nested doors. Gene drive research, they wrote, should take place in locations where the species of study isn’t native, making it less likely that escapees would take root. And they also proposed a way to turn the gene drive off when an engineered individual mated with a wild counterpart—a genetic sunset clause. Esvelt filed for a patent on Crispr gene drives, partly, he says, to block companies that might not take the same precautions.

Within a year, and without seeing Esvelt’s papers, biologists at UC San Diego had used Crispr to insert gene drives into fruit flies—they called them “mutagenic chain reactions.” They had done their research in a chamber behind five doors, but the other precautions weren’t there.Church said the San Diego researchers had gone “a step too far”—big talk from a scientist who says he plans to use Crispr to bring back an extinct woolly mammoth by deriving genes from frozen corpses and injecting them into elephant embryos. (Church says tinkering with one woolly mammoth is way less scary than messing with whole populations of rapidly reproducing insects. “I’m afraid of everything,” he says. “I encourage people to be as creative in thinking about the unintended consequences of their work as the intended.”)

Ethan Bier, who worked on the San Diego fly study, agrees that gene drives come with risks. But he points out that Esvelt’s mosquitoes don’t have the genetic barrier Esvelt himself advocates. (To be fair, that would defeat the purpose of a gene drive.) And the ecological barrier, he says, is nonsense. “In Boston you have hot and humid summers, so sure, tropical mosquitoes may not be native, but they can certainly survive,” Bier says. “If a pregnant female got out, she and her progeny could reproduce in a puddle, fly to ships in the Boston Harbor, and get on a boat to Brazil.”

These problems don’t end with mosquitoes. One of Crispr’s strengths is that it works on every living thing. That kind of power makes Doudna feel like she opened Pandora’s box. Use Crispr to treat, say, Huntington’s disease—a debilitating neurological disorder—in the womb, when an embryo is just a ball of cells? Perhaps. But the same method could also possibly alter less medically relevant genes, like the ones that make skin wrinkle. “We haven’t had the time, as a community, to discuss the ethics and safety,” Doudna says, “and, frankly, whether there is any real clinical benefit of this versus other ways of dealing with genetic disease.”

Researchers in China announced they had used Crispr to edit human embryos.

That’s why she convened the meeting in Napa. All the same problems of recombinant DNA that the Asilomar attendees tried to grapple with are still there—more pressing now than ever. And if the scientists don’t figure out how to handle them, some other regulatory body might. Few researchers, Baltimore included, want to see Congress making laws about science. “Legislation is unforgiving,” he says. “Once you pass it, it is very hard to undo.”

In other words, if biologists don’t start thinking about ethics, the taxpayers who fund their research might do the thinking for them.

All of that only matters if every scientist is on board. A month after the Napa conference, researchers at Sun Yat-sen University in Guangzhou, China, announced they had used Crispr to edit human embryos. Specifically they were looking to correct mutations in the gene that causes beta thalassemia, a disorder that interferes with a person’s ability to make healthy red blood cells.

The work wasn’t successful—Crispr, it turns out, didn’t target genes as well in embryos as it does in isolated cells. The Chinese researchers tried to skirt the ethical implications of their work by using nonviable embryos, which is to say they could never have been brought to term. But the work attracted attention. A month later, the US National Academy of Sciences announced that it would create a set of recommendations for scientists, policymakers, and regulatory agencies on when, if ever, embryonic engineering might be permissible. Another National Academy report will focus on gene drives. Though those recommendations don’t carry the weight of law, federal funding in part determines what science gets done, and agencies that fund research around the world often abide by the academy’s guidelines.

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

crispr new tools

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).

Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).

Figure 1. Cas9 in vivo: Bacterial Adaptive Immunity

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).

Cas9 and CRISPR as a New Tool in Molecular Biology

The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).

To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about “paired nickases” in Figure 2B).

The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 23–26). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).

Figure 2. CRISPR/Cas9 System Applications

A. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.
B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.
C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.

Targeting Efficiency and Off-target Mutations

Targeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (29–31). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 2–5% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).

A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).

Figure 3. T7 Endonuclease I Targeting Efficiency Assay

Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding 3 possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency.

Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.

Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5´ end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of “paired nickases” (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.

By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).

Applications as a Genome-editing and Genome Targeting Tool

Following its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila(45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.

The future of CRISPR/Cas9

The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9’s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.


Jakarta detik – Ilmuwan Amerika Serikat (AS) kini telah berhasil membikin genom (kumpulan gen) terkecil yang bisa hidup. Genom ini terdiri dari gen-gen dasar yang diperlukan bagi organisme untuk berfungsi dan bereproduksi sendiri.

Sebagaimana dilansir AFP, Sabtu (26/3/2016), ini adalah langkah besar menyibak misteri bagaimana kehidupan diciptakan. Genom sintetis dari bakteria ini punya julukan JCVI-syn3.0. Bakteri bikinan manusia ini hanya terdiri dari 473 gen, bandingkan dengan yang dipunya manusia, yakni terdiri dari 20.000 gen.

Pimpinan penelitian ini, Craig Venter, adalah orang pertama yang bisa menguraikan kode genom manusia. Selain Craig Venter, ada pula Clyde Hutchinson yang memimpin penelitian ini.

Meski begitu, tim ilmuwan ini belum menentukan fungsi dari 149 gen alias sepertiga dari total gen-gen yang mereka gabungkan itu. “Tugas pertama para investigator adalah menyelidiki aturan main gen-gen ini. Tugas ini menjanjikan pemahaman baru atas dasar kehidupan biologis,” kata Chris Voigt, ahli biologi sintetis dari Massachusetts Institute of Technology yang tak berpartisipasi dalam penelitian itu.

Sejumlah gen potensial yang berkesesuaian telah ditemukan di organisme lain. Ini mendorong mereka untuk mengkode protein universal dengan fungsi yang sampai sekarang masih belum bisa dipastikan.

Para peneliti itu menggunakan proses tes rancangan untuk mengidentifikasi gen quasi-esensial (gen yang sebenarnya tidak punya peran penting). Gen quasi-esensial itu dibutuhkan untuk pertumbuhan yang sehat, namun bukan untuk kehidupan.

Studi ini dipublikasikan pada jurnal ‘Science’ edisi Kamis (24/3). Melalui serangkaian eksperimen, mereka mencapai genom sintetis yang tereduksi, dibikin sekecil mungkin untuk menjamin tak ada lagi gen yang bisa mengacaukan genom ini.

Langkah Penting

“Satu-satunya  cara untuk menjawab pertanyaan mendasar tentang hidup adalah dengan cara mendapatkan genom paling minimal,” kata Craig Venter melalui telekonferensi.

“Mungkin satu-satunya jalan untuk melakukan itu adalah mencoba mensintesiskan (membikin -red) sebuah genom,” imbuh Venter.

24-Mar-2016                                        kehidupan sintetis

First Minimal Synthetic Bacterial Cell Designed and Constructed by Scientists at Venter Institute and Synthetic Genomics, Inc.

Cell, JCVI-syn3.0, was minimized to just 473 genes

(LA JOLLA, CA)—March 24, 2016—Researchers from the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) announced today the design and construction of the first minimal synthetic bacterial cell, JCVI-syn3.0.

Using the first synthetic cell, Mycoplasma mycoides JCVI-syn1.0 (created by this same team in 2010), JCVI-syn3.0 was developed through a design, build, and test process using genes from JCVI-syn1.0. The new minimal synthetic cell contains 531,560 base pairs and just 473 genes, making it the smallest genome of any organism that can be grown in laboratory media. Of these genes 149 are of unknown biological function. By comparison the first synthetic cell, M. mycoides JCVI-syn1.0 has 1.08 million base pairs and 901 genes.

A paper describing this research is being published in the March 25th print version of the journal Science by lead authors Clyde A. Hutchison, III, Ph.D. and Ray-Yuan Chuang, Ph.D., senior author J. Craig Venter, Ph.D., and senior team of Hamilton O. Smith, MD, Daniel G. Gibson, Ph.D., and John I. Glass, Ph.D.

“Our attempt to design and create a new species, while ultimately successful, revealed that 32% of the genes essential for life in this cell are of unknown function, and showed that many are highly conserved in numerous species. All the bioinformatics studies over the past 20 years have underestimated the number of essential genes by focusing only on the known world. This is an important observation that we are carrying forward into the study of the human genome,” said Dr. Venter, Founder, Executive Chairman, and CEO, JCVI.

The research to construct the first minimal synthetic cell at JCVI was the culmination of 20 years of research that began in 1995 after the genome sequencing of the first free-living organism, Haemophilus influenza, followed by the sequencing of Mycoplasma genitalium. A comparison of these two genomes revealed a common set of 256 genes which the team thought could be a minimal set of genes needed for viability. In 1999 Dr. Hutchison led a team who published a paper describing the use of global transposon mutagenesis techniques to identify the nonessential genes in M. genitalium.

Over the last 50 years more than 2,000 publications have contemplated minimal cells and their use in elucidating first principals of biology. From the start, the goal of the JCVI team was similar—build a minimal operating system of a cell to understand biology but to also have a desirable chassis for use in industrial applications. The creation of the first synthetic cell in 2010 did not inform new genome design principles since the M. mycoides genome was mostly recapitulated as in nature.  Rather, it established a work flow for building and testing whole genome designs, including a minimal cell, from the bottom up starting from a genome sequence.

To create JCVI-syn3.0, the team used an approach of whole genome design and chemical synthesis followed by genome transplantation to test if the cell was viable. Their first attempt to minimize the genome began with a simple approach using information in the biochemical literature and some limited transposon mutagenesis work, but this did not result in a viable genome. After improving transposon methods, they discovered a set of quasi-essential genes that are necessary for robust growth which explained the failure of their first attempt.

To facilitate debugging of non-functional reduced genome segments, the team built the genome in eight segments at a time so that each could be tested separately before combining them to generate a minimal genome. The team also explored gene order and how that affects cell growth and viability, noting that gene content was more critical to cell viability than gene order. They went through three cycles of designing, building, and testing ensuring that the quasi-essential genes remained, which in the end resulted in a viable, self-replicating minimal synthetic cell that contained just 473 genes, 35 of which are RNA-coding. In addition, the cell contains a unique 16S gene sequence.

The team was able to assign biological function to the majority of the genes with 41% of them responsible for genome expression information, 18% related to cell membrane structure and function, 17% related to cytosolic metabolism, and 7% preservation of genome information. However, a surprising 149 genes could not be assigned a specific biological function despite intensive study. This remains an area of continued work for the researchers.

“This paper represents more than five years of work by an amazingly talented group of people. Our goal is to have a cell for which the precise biological function of every gene is known,” said Dr. Hutchison, Distinguished Professor, JCVI.

The team concludes that a major outcome of this minimal cell program are new tools and semi-automated processes for whole genome synthesis.  Many of these synthetic biology tools and services are commercially available through SGI and SGI-DNA including a synthetic DNA construction service specializing in building large and complex DNA fragments including combinatorial gene libraries, Archetype® genomics software, Gibson Assembly® kits, and the BioXp™, which is a benchtop instrument for producing accurate synthetic DNA fragments.

“This paper signifies a major step toward our ability to design and build synthetic organisms from the bottom up with predictable outcomes.  The tools and knowledge gained from this work will be essential to producing next generation production platforms for a wide range of disciplines,” said Dr. Gibson, Vice President, DNA Technologies, SGI; Associate Professor, JCVI.

The other researchers on this paper have been integral to this work for much of the last decade. Current and former JCVI and SGI scientists are: Chuck Merryman, Ph.D., Ray-Yuan Chuang, Ph.D., Vladimir Noskov, Ph.D., Nacyra Assad-Garcia, John Gill, Krishna Kannan, Ph.D., Bogumil Karas, Ph.D., Li Ma, Zhi-Qing Qi, Ph.D., R. Alexander Richter, Ph.D., Lijie Sun, Ph.D., Yo Suzuki, Ph.D., Billyana Tsvetanova, Ph.D. and Kim Wise, Ph.D.

Other authors on the paper are: Thomas J. Deerinck and Mark H. Ellisman, Ph.D., University of California, San Diego National Center for Microscopy and Imaging Research; James F. Pelletier, Center for Bits and Atoms and Department of Physics, Massachusetts Institute of Technology; Elizabeth A. Strychalski, National Institute of Standards and Technology.

This work was funded by SGI, the JCVI endowment and the Defense Advanced Research Projects Agency’s Living Foundries program, HR0011-12-C-0063.

About J. Craig Venter Institute

The JCVI is a not-for-profit research institute in Rockville, MD and La Jolla, CA dedicated to the advancement of the science of genomics; the understanding of its implications for society; and communication of those results to the scientific community, the public, and policymakers. Founded by J. Craig Venter, Ph.D., the JCVI is home to approximately 200 scientists and staff with expertise in human and evolutionary biology, genetics, bioinformatics/informatics, information technology, high-throughput DNA sequencing, genomic and environmental policy research, and public education in science and science policy. The JCVI is a 501 (c)(3) organization. For additional information, please visit http://www.JCVI.org.

JCVI Media Contact

Heather Kowalski, hkowalski(AT)jcvi.org

– See more at: http://www.jcvi.org/cms/press/press-releases/full-text/article/first-minimal-synthetic-bacterial-cell-designed-and-constructed-by-scientists-at-venter-institute-an/#sthash.kTm3YrGS.dpuf

rose KECIL

DNA Robots Programmed to Kill Cancer Cells, Harvard Study Shows

Usaha ini mengantarkan ilmuwan kepada Mycoplasma, yakni bakteri dengan sel genom terkecil. Mycoplasma dapat mereplikasi (menggandakan) dirinya sendiri secara otonom.

“Bila Anda tak tahu apapun soal pesawat terbang dan Anda sedang melihat Boeing 777, dan Anda cuma mencari tahu fungsi bagian-bagian pesawat itu dengan cara mengurainya, dan anda mempreteli mesinnya dari sayap kanan, pesawat itu masih bisa terbang dan mendarat,” kata Venter mencoba menjelaskan dengan analogi.

“Jadi mungkin Anda mengatakan ini adalah komponen yang tidak penting, dan Anda tak benar-benar menemukan hal yang paling esensial sampai anda mempreteli (mesin pesawat) yang satunya lagi. Dan itulah yang terjadi, lagi dan lagi, pada biologi saat kami akan mendapati komponen yang non-esensial, sampai pada saat kita mempreteli pasangannya,” kata Venter.

Pada satu penemuan penting, tim peneliti belajar bahwa beberapa gen digolongkan sebagai ‘non-esensial (tidak penting)’. Artinya, gen satunya lagi adalah gen yang wajib diperlukan, alias gen yang ‘esensial’, atau gen minimal yang dibutuhkan.

Genom minimal tak punya gen yang bisa memodifikasi dan membatasi DNA. Genom minimal juga kurang akan gen yang mengkode lipoprotein.

Bagaimanapun juga, ‘genom minimalis’ ini mengandung semua gen yang diperlukan dalam pembacaan dan mengekspresikan informasi genetik dalam genom, sebagaimana dalam hal menjaga informasi genetik dari generasi ke generasi.



By Elizabeth Lopatto – Feb 16, 2012

Scientists have created a robot made entirely from DNA that can be instructed to find diseased cells in the body and deliver a payload to kill or reprogram them, according to a study from Harvard University.

The robot was constructed by folding DNA strands into a shape that looks roughly like a clamshell. The researchers programmed the nano-sized device to open in the presence of leukemia and lymphoma cells in a laboratory dish, where they delivered immune system antibodies that caused the cells to self-destruct, according to a report in the journal Science.

The next step will be to test the system in animals, tweaking the robot so that it can circulate longer in the blood to locate all cancer cells. The technology isn’t yet ready for commercial use, said Shawn Douglas, an author of the study.

“In diseases such as cancer we know if we can find every single last cell and kill or reprogram it, we can cure that disease,” said Douglas, a researcher at the Wyss Institute for Biologically Inspired Engineering at Harvard, in Boston. “A lot of our current therapies fall short.”

The idea is based on the behavior of the body’s immune cells, which recognize viruses or other invaders and attack them, Douglas said. The DNA nano-robots, with similar capabilities, may potentially lead to the development of new types of targeted cancer treatments that kill only abnormal cells, he said.

The robots don’t reproduce. They have to be constructed in a process that has gained traction since the idea of DNA nanotechnology was first suggested in 1982.
Genetic Information

DNA is a material, shaped in the form of a revolving ladder, which carries the genetic information in our cells.

The double-sided strands have so-called sticky ends that allow them to be joined together with other DNA. Scientists, led by Nadrian Seeman, now head of the Department of Chemistry at New York University, have used those sticky ends to form DNA into lattices that can be shaped in various ways.

The latest research created a robot in a clamshell shape that’s held together with a “zipper” constructed of a special sequence of DNA, the report said. The zipper was programmed to release its grip when it recognized specific targets on a cell, allowing the robot to release its payload.

In the experiment, Douglas and his fellow scientists used the robot they constructed to deliver instructions encoded in antibodies to the cancer cells.

“It’s an important step forward in specific targeting,” said Milan Stojanovic, an assistant professor of experimental therapeutics at Columbia University in New York who wasn’t involved in the research, in an e-mail. “It looks very exciting.”

Besides cancer, the robots may also benefit people with autoimmune disease, Douglas said. One day, the robots might be used to find immune cells wrongly attacking the body and reprogram them, he said.


Agustus 15, 2017

terlanjur cinta: berbahagialah ORANG YANG BERBAHAGIA…

Filed under: GLOBAL ECONOMY — bumi2009fans @ 2:09 am

Kabar24.com, JAKARTA–Survei Pengukuran Tingkat Kebahagiaan (SPTK) yang dilakukan oleh Badan Pusat Statistik (BPS) menunjukkan Provinsi Maluku Utara sebagai wilayah yang memiliki tingkat kebahagiaan tertinggi.

Dari survei BPS, indeks kebahagiaan penduduk provinsi Maluku Utara mencapai 75,68, lebih tinggi dari indeks kebahagiaan nasional yang hanya 70,69.

Kepala BPS Suhariyanto mengatakan posisi kedua ditempati oleh Maluku dengan indeks kebahagiaan penduduknya sebesar 73,77 dan Sulawesi Utara sebesar 73,69.

Sementara itu, tiga provinsi dengan indeks kebahagiaan terendah adalah Papua 67,52, Sumatera Utara 68,41 dan NTT 68,98.

Menurut Kepala BPS, Maluku Utara menjadi provinsi dengan tingkat kebahagiaan yang tinggi dilihat dimensi kepuasan hidup mencapai 77,09, dimensi perasaan 70,48, dan dimensi makna hidup 79,00.

“Sangat berbeda ketika kita pergi ke Papua yang kebahagiaannya paling rendah, bisa dipahami di sana per sub dimensinya personal, karena berkaitan dengan pendidikan, pendapatan dan kondisi rumah,” kata Suhariyanto.

Semua aspek di atas, lanjutnya, sangat mempengaruhi tingkat kebahagiaan penduduk.

Jika dibandingkan dengan survei kebahagiaan 2014, BPS mencatat ada 6 provinsi yang mengalami peningkatan kebahagiaannya pada tahun ini. Provinsi tersebut a.l. Maluku Utara, Gorontalo, Papua, Kepulauan Bangka Belitung, Sumatera Barat, dan Aceh.

Di sisi lain, provinsi yang indeks kebahagiaannya turun drastis pada tahun ini a.l. Kalimantan Selatan, Kepulauan Riau, Sumatera Utara, Kalimantan Tengah, dan Lampung.

Deputi Bidang Statistik Sosial BPS Sairi Hasbullah menjelaskan mengapa indeks kebahagiaan Maluku Utara dan Maluku lebih tinggi dibandingkan Jakarta yang hanya mencapai 71,33.

Menurutnya, masyarakat yang tinggal di desa memiliki keunggulan dari sisi hubungan sosial yang lebih baik dan nyaman.

“Itu yang menyebabkan indeks kebahagiaan Maluku tinggi,” tegasnya.

Sementara itu, dimensi kebahagiaan di perkotaan seperti Jakarta hanya mencakup dimensi personal seperti kesehatan, pendidikan, lapangan pekerjaan dan lain sebagainya.

statistik kebahagiaan

Kabar24.com, JAKARTA – Penduduk Indonesia yang masih bujangan ternyata lebih bahagia dibandingkan dengan penduduk dengan status perkawinan lainnya.

Hal ini terekam dari indeks kebahagiaan Indonesia 2017 yang baru saja dirilis Badan Pusat Statistik (BPS) pada hari ini, Selasa (15/8/2017). Indeks kebahagian penduduk yang belum menikah mencapai 71,53 karena terdongkrak indeks dimensi makna hidup (Eudaimonia).

Setelah penduduk yang belum menikah, tingkat kebahagian secara berurutan dimiliki penduduk menikah (71,09) dan penduduk cerai mati (68,37). Sementara, penduduk dengan status perkawinan cerai hidup memiliki tingkat kebahagian terendah dengan indeks 67,83.

Kendati penduduk yang belum menikah memiliki tingkat kebahagian tertinggi, kepuasan hidup tertinggi dimilikipenduduk yang menikah. Kepuasan ini terutama terlihat dari subdimensi kepuasan hidup sosial.

Sekadar informasi, subdimensi kepuasan hidup sosial mencakup indikator keharmonisan keluarga; ketersediaan waktu luang; hubungan sosial; keadaan lingkungan; serta kondisi keamanan.


The World’s Happiest Countries

A British researcher merged dozens of statistical metrics to rank nations on the elusive notion of contentment

By Marina Kamenev businessweek

Feeling blue? Perhaps you live in the wrong country. A recent study from Britain’s University of Leicester examined a range of statistical data to devise a ranking of the world’s happiest nations. Heading up the list: Denmark, which rose to the top thanks to its wealth, natural beauty, small size, quality education, and good health care. At the bottom were Zimbabwe and Burundi. But there were a few surprises along the way, too. Asian countries scored worse than researcher Adrian White expected. Capitalism — sometimes criticized for its heartlessness — was far from a source of discontent, though the top-scoring capitalist countries also tended to have strong social services. And the U.S. ranked only 23rd, due to nagging poverty and spotty health care. Read on to learn about the world’s 12 happiest countries — by the numbers, at least.


No. 1: Denmark
Population: 5.5 million
Life Expectancy: 77.8 years
GDP Per Capita: $34,600

With a high standard of living, negligible poverty, and a broad range of public and social services, it’s easy to see why Denmark tops the happiness map. There’s a high level of education; public schools are top-quality and private ones are affordable. The low population gives the nation a strong sense of identity. And Denmark’s physical beauty forms a great backdrop to daily life. The weather is a bit tough, though.

No. 2: Switzerland
Population: 7.5 million
Life Expectancy: 80.5 years
GDP Per Capita: $32,300

Smack in the middle of Europe and surrounded by picture-postcard scenery, Switzerland ranks second among the world’s happiest countries. It has a low crime rate, good infrastructure, and a wealth of outdoor activities, from skiing in the Alps to boating on Lake Geneva. Home to the International Red Cross, the World Health Organization, and parts of the U.N., it’s not surprising that the Swiss devote a large portion of private and public money to health care — spending an average of $3,445 per person. It’s pretty peaceful, too: years of political neutrality have sheltered the Swiss from the conflicts of their neighbors.
No. 3: Austria
Population: 8.2 million
Life Expectancy: 79 years
GDP Per Capita: $32,700

Another Alpine hotbed of happiness, Austria also boasts beautiful scenery and a surprisingly rich cultural scene. Like many of the world’s happiest countries, it boasts a strong health-care system, as evidenced by the long average life expectancy of its citizens. Strict environmental regulations are starting to pay dividends, says Oskar Hinteregger, of the Austrian National Tourist Office. He credits the country’s happy mood to its relaxed atmosphere, efficient public transport system, and general cleanliness. Austria does have some poverty, though: nearly 6%
No. 4: Iceland
Population: 300,000
Life Expectancy: 80 years
GDP Per Capita: $35,600

There’s more to Iceland than hot springs and Björk. The tiny country’s extensive welfare system plays a big part in its citizens’ happiness. The Icelandic government offers a broad range of services, such as generous housing subsidies, and with very little poverty, wealth is evenly distributed among Icelandic society. Literacy is high and unemployment, at 2.1%, is low.
No. 5: Bahamas
Population: 303,800
Life Expectancy: 65.6 years
GDP Per Capita: $20,200

Bahamanians know how to enjoy life. “Maybe it’s our ‘Bahama Mamas,’ our sweet sea breeze, our conch salad, and fun loving people,” suggests Kendenique Campbell-Moss, a senior executive at the Bahamas Tourism Ministry. Although the poverty rate, at 9.3%, is relatively high, the beautiful weather and laid-back lifestyle keep Bahamas’ citizens smiling. Campbell-Moss also reckons the fusion of African and European cultures, strong family values, and Christianity contribute to the happy vibe in the Caribbean country.

No. 6: Finland
Population: 5.2 million
Life Expectancy: 78.5 years
GDP Per Capita: $30,900

It’s dark and cold in the winter and has some of the highest taxes in Europe. But that doesn’t get in the way of Finns’ overall happiness. High quality medical care — at little to no cost — contributes to the country’s high average life expectancy. The country’s free educational system is one of the best, resulting in a 100% literacy rate. Poverty is rare; so too, is extreme wealth. “Our beloved government makes sure that taxes are high enough to prevent easy ways to riches,” says Jaakko Lehtonen, director-general of the Finnish Tourism Board. “Finns think a good salary is two cents higher than your neighbor’s; it’s enough to make you feel wealthy and subsequently, happy,” he says.
No. 7: Sweden
Population: 9 million
Life Expectancy: 80.50 years
GDP Per Capita: $29,800

Taxes are high and the winter is trying. But social equality, one of the best welfare systems in Europe, and a great work/life balance keep Swedes smiling. Parents get extensive maternity and paternity leave, and child care is heavily subsidized and available to all. Sweden also has unusually transparent government and a strong emphasis on ensuring the freedom and equality of its people. “Ordinary citizens in Sweden have the right to see the prime minister’s official mail, and they often exercise that right,” notes Susanna Wallgren, of the Swedish Tourism Board.
No. 8: Bhutan
Population: 2.3 million
Life Expectancy: 55 years
GDP Per Capita: $1,400

Here’s a surprise: The small Asian nation of Bhutan ranks eighth in the world, despite relatively low life expectancy, a literacy rate of just 47%, and a very low GDP per capita. Why? Researchers credit an unusually strong sense of national identity. Plus, the country has beautiful scenery and a largely unspoiled culture, thanks to strict governmental limits on tourism, development, and immigration. Pretty counterintuitive, but Bhutan seems to have found a recipe for happiness.
No. 9: Brunei
Population: 380,000
Life Expectancy: 75
GDP Per Capita: $23,600

It helps to have oil. Wealthy and politically stable, Brunei’s government plays a major role in its citizens’ happiness. The same family has ruled the Southeast Asian nation for more than six centuries, providing free medical services and education. Even university-level education is paid for by the government, which also subsidizes rice and housing. That ensures virtually nonexistent poverty.
No. 10: Canada
Population: 33 million
Life Expectancy: 80 years
GDP Per Capita: $34,000

Canada may sometimes feel overshadowed by its giant neighbor to the south, but a strong sense of national identity and abundant natural beauty help make the sprawling and sparsely populated country one of the world’s happiest. Canada also punches above its weight economically, with a huge $1.1 trillion GDP and per-capita that ranks among the world’s highest. It also has strong health care and a low crime rate.
No. 11: Ireland
Population: 4 million
Life Expectancy: 77.7 years
GDP Per Capita: $41,000

Once so poor that its citizens fled by the millions, the Celtic Tiger has enjoyed unprecedented economic growth over the past dozen years. Credit membership in the European Union and a can-do attitude has raised standards of living and even lured former immigrants back home. The excellent education system, open economy, and relaxed pace of life all contribute to the overall happiness of the Irish.
No. 12: Luxembourg
Population: 474,500
Life Expectancy: 79 years
GDP Per Capita: $55,600

Luxembourg’s position proves that sometimes money can buy happiness. It has the highest GDP per capita in the world. And with great access to education, 100% of the population is literate. The people of Luxembourg should find comfort in their surroundings, too. Mercer Resource Human Consulting ranked the city-state as the safest in the world in 2005.

… yang laen jelas abis, duit ada hubungannya dengan kebahagiaan, tapi YANG INI BEDA BO: No. 8: Bhutan
Population: 2.3 million
Life Expectancy: 55 years
GDP Per Capita: $1,400 …

indon mesti belajar dari mereka… HARUS, kalo bener emang tujuan hidup ini adalah untuk berbahagia …


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