Scary tomato appears to bleed

A newly discovered species of tomato belongs in a haunted house, not on a sandwich.

Fruit from the bush tomato plant Solanum ossicruentum bears little resemblance to its cultivated cousins. The Australian tomato, about a couple centimeters wide, grows enclosed in a shell of spikes. These burrs probably help the fruit latch on to the fur of passing mammals, which then spread the tomato’s seeds elsewhere, researchers at Bucknell University in Lewisburg, Pa., report May 3 in PhytoKeys.

Slice open the fearsome fruit and within five minutes, its sticky white-green flesh appears to bleed, flushing bright red to dark maroon in response to air exposure. One brave researcher tasted an unripe fruit and deemed it salty. The bush tomato becomes no more appetizing with time: Mature fruits harden into dry, bony nuggets.

The tomato’s gruesome qualities inspired its name, courtesy of a group of Pennsylvanian seventh-grade science students: “Ossicruentum” combines the Latin words for “bone” and “bloody.”

Monitoring online groups offers insight into ISIS attacks

Social media supporters of the Islamic State, or ISIS, form online groups that may provide clues crucial to predicting when terrorist attacks will take place, a new analysis finds.

These virtual communities drive ISIS activity on a Facebook-like site called VKontakte, say physicist Neil Johnson of the University of Miami in Coral Gables, Fla., and colleagues. VKontakte, a social networking service based in Russia with more than 350 million users, allows messaging in many languages and is used worldwide.
In the June 17 Science, Johnson’s team describes a mathematical model that predicts online groups of ISIS supporters will proliferate days before real-world Islamic State attacks. That’s just what happened in September 2014, researchers say. Pro-ISIS groups on VKontakte mushroomed the day before Islamic State forces overran Kobane, a small Syrian town.

The researchers refer to groups of followers of an online page that form spontaneously as aggregates. “Our work suggests that, to manage and monitor online ISIS activity, we need to focus on aggregates rather than individuals,” Johnson says.

Pro-ISIS aggregates on VKontakte exchanged information on issues such as recruiting fighters to Syria and how to survive drone attacks.

The new model suggests that authorities need to shut down online pro-ISIS groups in their early stages. Small-scale aggregates favoring the same cause gradually expand when left alone and eventually merge into a much larger online community that’s more difficult to break up, Johnson’s model forecasts.

“This is the first serious, large-scale, data-driven study that shows how online support develops for terrorist groups such as ISIS,” says computer scientist V.S. Subrahmanian of the University of Maryland in College Park, who builds computational models of terror networks. But it remains to be seen whether the new model can predict when and possibly where future Islamic State attacks will occur, Subrahmanian cautions.
Johnson’s team identified 196 pro-ISIS aggregates, consisting of 108,086 individual followers, which operated between January 1 and August 31, 2015. Intelligence agencies, hackers and website moderators work to shut down these online groups, but to a lesser extent on VKontakte than on Facebook.

Pro-ISIS aggregates rapidly adapted to these survival threats in several ways, the researchers say. Fifteen percent of aggregates changed their online names; 7 percent flipped back and forth between opening their content to any VKontakte user or to current aggregate followers only; and 4 percent engaged in a digital form of reincarnation. Pro-ISIS aggregates under unusually intense attack by hackers and others opted for reincarnation, Johnson says.

Reincarnating aggregates disappeared and then returned, often within weeks, with new names and at least 60 percent of the same followers as before. Aggregates that vanished appear to have reassembled without any direction or urging from one or a few members, Johnson says. New names of reincarnated groups often resembled original names enough to alert former members but not enough to trigger VKontakte’s automated system for identifying names of probable pro-militant groups, he points out.

As with predictions of terror attacks based on the expansion of pro-ISIS aggregates, the new model shows promise in predicting when mass public protests will occur based on sudden jumps in numbers of pro-protest aggregates, the scientists say. There is a difference: Reincarnation did not appear within the last three years among Facebook aggregates consisting of civil protesters in Brazil and several other Latin American countries, the researchers found. Those online groups experienced fewer pressures to shut down than pro-ISIS aggregates on VKontakte did.

Johnson’s analysis moves the study of online militant groups forward, says terrorism analyst J.M. Berger of George Washington University in Washington, D.C. But it’s likely that considerably fewer members of pro-ISIS aggregates than the total studied in the new analysis were actually hard-core Islamic State supporters, Berger contends. Concerted efforts to shut down online terrorist networks have depressed numbers of committed ISIS supporters using social media, in his view. Berger and a colleague have found that English-language Twitter use has declined sharply among ISIS supporters over the last two years, due to suspensions of their accounts by the social media site.

Johnson suspects most pro-ISIS aggregate members were staunch supporters, since aggregates aggressively weed out those deemed unserious or hostile.

Terrorists use chains of social and messaging sites online to achieve their ends, Subrahmanian says. How that works, and whether the same aggregate operates under different guises from one site to the next, has yet to be studied.

Tight spaces cause spreading cancer cells to divide improperly

Scientists have found a new way to study how cancer cells divide and thrive in difficult-to-reach crannies of the body.

Transparent artificial membranes — just nanometers thick — can be rolled into tubes to mimic capillaries that host spreading cancer cells, researchers report in the June ACS Nano. Cells squished inside such tubes didn’t organize their internal components the way they normally do before splitting. As a result, the cells divided unevenly, potentially introducing new mutations.
Inside the body, cancer cells fight for space. Sometimes they’ll spread, or metastasize, to other organs via tight blood vessels. Although cancer cells are more likely to kill once they spread, scientists still don’t understand how the abnormal cells divide inside such tiny tubes. These cells are difficult to study in the body because they’re tucked away in hard-to-reach places. They’re challenging to study in the lab, too, because they behave differently in a petri dish than in their natural environment.

By replicating that environment more closely, this experiment gives “an appreciation for what it’s like to be a cell in a body,” says Buzz Baum, a biologist at University College London who was not involved in the work.

Other researchers have looked at cells constrained in other ways. But the nanotubes are round, just like blood vessels. They’re transparent, making it easier to visualize what’s happening inside. And it’s possible to study many cells at once by growing nearly a thousand tubes, all exactly the same size, on a chip slightly larger than a postage stamp.

When watching single human cancer cells inside the tubes through high-powered microscopes, the team noticed that the squished cells didn’t divide symmetrically. Instead of sending half the chromosomes to each new cell, some of those cells got extra genetic instructions, while others were shortchanged.

The squished cells also took longer to divide. And the protein structures that help guide the chromosomes and organelles into place before division didn’t develop correctly, says study coauthor Wang Xi, a materials scientist who did the work at the Leibniz Institute for Solid State and Materials Research Dresden in Germany.
Cancer cells succeed by mutating enough that they can evade capture, without becoming too mutated to keep replicating.

“A cell that makes too many mistakes will just die,” says Baum.

When cells are trapped inside blood vessels, “they become misshapen but they’re still able to divide,” says Xi, now at the National University of Singapore.

Xi and his colleagues think that a bulging of the cell membrane in response to pressure, called blebbing, might help the trapped cells divide in a slightly less distorted way. When the researchers prevented the cells from blebbing, the division was even more uneven. But because the team can’t yet explain why this would be the case, it’s too soon to say whether blebbing itself is responsible for the improved division.

Baum says he has shown similar deformities in cancer cells dividing under other types of constraints. But, he adds, it’s important to have systems that more closely replicate the body’s internal environment. Otherwise, it can be a big jump between doing tests in a petri dish and in a live animal.

Study coauthor Christine Schmidt, a biochemist at the University of Cambridge, says understanding how cancer cells manage to divide in tight quarters could eventually inspire ways to kill spreading cancer cells without hurting healthy cells.

Mini ‘wind farm’ could capture energy from microbes in motion

Fluid filled with lively, churning bacteria could one day become a small-scale power source.

New computer simulations indicate that a miniature wind farm‒like device could harvest the energy of chaotically swirling bacteria. That energy could be used to power micromachines or pump fluids through tiny channels. In the simulations, bacteria tended to spontaneously swim in an orderly fashion around an array of cylindrical turbines. These turbines then rotated steadily like windmills in a breeze, scientists report July 8 in Science Advances.
Previous research has harnessed the energy of the motion in such chaotic fluids using tiny, asymmetric gears, which spin as bacteria bump into their teeth. But the new result shows that a very simple system can serve the same purpose — a result that could make such devices easier to construct. “You don’t have to muck around with getting the teeth right; you just have a nice smooth cylinder,” says biophysicist and study coauthor Tyler Shendruk of the University of Oxford. The technique would sidestep the need to manufacture complicated microscopic gears.

“I think it’s quite surprising because previous work showed that you need to have a certain nonsymmetry in the system” to generate rotation, says physicist Igor Aronson of Argonne National Laboratory in Illinois, who was not involved with the new work.

The researchers studied simulations of a liquid filled with many self-propelled particles, called a dense active fluid. These fluids can be made up of swimming bacteria or biological motors found inside cells — for instance, the proteins myosin and actin, which cause muscles to contract. Such fluids are normally turbulent, with swarms of particles generating rapidly and unpredictably changing flows. That makes it a challenge to harvest energy from the fluid. “It’s chaotic, so you can’t use it to do anything useful because it’s a random flow,” Shendruk says.
But when Shendruk and colleagues added a grid of cylindrical rotors, each a few hundredths of a millimeter in diameter, into their simulated fluid, they found that bacteria would spontaneously organize, like sailors all rowing in the same direction. The swimming bacteria produced a circular fluid flow that spun the rotors. That rotation could be used to generate electrical power in the same manner as windmills do, but in much smaller amounts that might be used to power tiny electronics. Each rotor might produce around a quadrillionth of a watt of electrical power, Shendruk estimates.
A single rotor on its own didn’t work as well: Its spin changed direction periodically as the chaotic fluid swirled around it. But with an array of rotors close together, the bacteria became steady synchronized swimmers squeezing through gaps between the rotors — and making each rotor consistently spin in the direction opposite to that of its neighbors.

The system should translate well from simulation to the real world, says Shendruk, and the researchers are already discussing the possibilities for constructing it. But, says applied mathematician Jörn Dunkel of MIT, the details of the real world are important. Whether the rotors would behave the same way in a real-life system where the rotors experience friction is uncertain. “The effect is there — I don’t doubt that. The question is how strong.”

Scientists throw a curve at knuckleball explanation

Knuckleballs baffle baseball players with their unpredictable swerves. A new study suggests a possible cause of the pitch’s erratic flight — sudden changes in the drag force on a ball, due to a phenomenon called a drag crisis.

The result is at odds with previous research that attributed the zigzags to the effect of airflow over the baseball’s seams. Scientists report the finding July 13 in the New Journal of Physics.

Knuckleballs are well known in baseball, but similar phenomena also confound players in soccer and volleyball. Knuckleballs occur when balls sail through the air with very little spin, producing unstable flight.
In drag crisis, the thin layer of air surrounding the ball flips between turbulent and smooth flow, abruptly changing the drag forces on the ball. If the transition occurs asymmetrically, it can push the ball to one side. “This phenomenon is intermittent” and hard to predict, says study coauthor Caroline Cohen, a physicist at École Polytechnique in Palaiseau, France. “We can’t know in advance [to] which side it will go.” Balls must move at a certain speed to experience a drag crisis, which may be why knuckleballs tend to be thrown slower than other pitches, the researchers suggest. While the fastest pitches can top 100 miles per hour, knuckleballs are usually closer to 60 or 70 miles per hour.

The scientists built a knuckleball machine, designed to launch a beach ball without any spin, and measured how much the ball veered off course. Then they calculated the ball’s expected motion based on the physics of the drag crisis and found that the predicted trajectories matched the experiments. The scientists’ calculations also correctly predict knuckleball-like phenomena in soccer, volleyball, cricket and baseball — but not in sports like tennis or basketball, where knuckleballs aren’t seen due to the properties of the ball, including texture, typical speed and how far it flies.

“It’s a fine piece of work,” says Alan Nathan of the University of Illinois at Urbana-Champaign, who studies the physics of baseball (SN: 3/23/13, p.32). But he is not entirely convinced by the explanation of knuckleballs. “Wind tunnel experiments seem to strongly suggest that it’s associated with the seams on the ball,” Nathan says, which can create turbulence that causes the ball to swerve.

So knuckleballs may remain as much of a challenge to explain as it is to hit them.

Jupiter’s Great Red Spot is hot

On Jupiter, the Great Red Spot is the hottest thing going. Temperatures over the ruddy oval, a storm that could engulf Earth, are hundreds of degrees warmer than neighboring parcels of air and higher than anywhere else on the planet, researchers report online July 27 in Nature. Heat from the storm might help explain why Jupiter is unusually toasty given its distance from the sun.

Astronomers have known for over 40 years that Jupiter’s upper atmosphere is surprisingly hot. Mid-latitude temperatures are about 530° Celsius, roughly 600 degrees warmer than they would be if the sun was the only source of heat. Warmth must come from inside the planet, but until now, researchers had not come up with a satisfactory explanation for how.
Active storms all around Jupiter could be injecting heat into the atmosphere, suggest James O’Donoghue, an astrophysicist at Boston University, and colleagues. Using observations from NASA’s Infrared Telescope Facility in Hawaii, the researchers found that the temperature over the Great Red Spot is about 1,300° Celsius. Sound waves generated by turbulence might be heating the air above the storm, the researchers suggest. Similar heating (on a much smaller scale) has been seen on Earth, as air ripples over the Andes Mountains in South America.

New fossil suggests echolocation evolved early in whales

A roughly 27-million-year-old fossilized skull echoes growing evidence that ancient whales could navigate using high-frequency sound.

Discovered over a decade ago in a drainage ditch by an amateur fossil hunter on the South Carolina coast, the skull belongs to an early toothed whale. The fossil is so well-preserved that it includes rare inner ear bones similar to those found in modern whales and dolphins. Inspired by the Latin for “echo hunter,” scientists have now named the ancient whale Echovenator sandersi.
“It suggests that the earliest toothed whales could hear high-frequency sounds,” which is essential for echolocation, says Morgan Churchill, an anatomist at the New York Institute of Technology in Old Westbury. Churchill and his colleagues describe the specimen online August 4 in Current Biology.

Modern whales are divided on the sound spectrum. Toothed whales, such as orcas and porpoises, use high-frequency clicking sounds to sense predators and prey.

Filter-feeding baleen whales, on the other hand, use low-frequency sound for long-distance communication. Around 35 million years ago, the two groups split, and E. sandersi emerged soon after.

CT scans show that E. sandersi had a few features indicative of ultrasonic hearing in modern whales and dolphins. Most importantly, it had a spiraling inner ear bone with wide curves and a long bony support structure, both of which allow a greater sensitivity to higher-frequency sound. A small nerve canal probably transmitted sound signals to the brain.
“Scientists have long suspected that early toothed whales could produce the high-frequency sounds needed for echolocation based on features on their skulls,” says Travis Park of Monash University in Melbourne, Australia. Previous work points to early toothed whales sensing those high frequencies. Park and his colleagues reported in April in Biology Letters the discovery of a 26-million-year-old lone ear bone showing signs of high-frequency hearing. But it wasn’t connected to a skull and, thus, couldn’t be tied to a specific whale species.

Tracing inner ear features in CT scans of 24 ancient and modern whales, including E. sandersi, plus two hippos, whales’ closest living relatives, Churchill’s team ups the ante. Because primitive versions of the bony spiral and nerve canal appeared before the first known toothed whale, the researchers suggest that rudimentary high-frequency hearing might have emerged in the common ancestor of toothed and baleen whales at least 43 million years ago. If so, baleen whales lost their high-frequency hearing at some point. Determining whether that’s truly the case requires more analysis and a wider array of fossil data, says Park, who’s unconvinced.

But there is growing consensus that the first toothed whales could hear and produce sounds at high-frequency ranges. The skull of a 28-million-year-old toothed whale also suggests that such animals could make high-frequency calls (SN: 4/19/14, p. 6). “The next step is to look at when their brains got big enough to process echolocation signals,” says Nicholas Pyenson, a paleobiologist at the Smithsonian’s National Museum of Natural History who was not affiliated with the study. “This is great, but there’s more to be done.”

Capybaras may be poised to be Florida’s next invasive rodent

Capybaras, giant rodents native to South America, could become Florida’s next big invasive species, a biologist warned August 3 in Columbia, Mo., at the 53rd Annual Conference of the Animal Behavior Society.

“Capybaras have been introduced to northern Florida,” said Elizabeth Congdon of Bethune-Cookman University in Daytona Beach, Fla. And there are enough similarities to nutria — large invasive rodents that have caused havoc in many states — to warrant a closer look at the South American newcomers.

There are currently about 50 capybara loose in northern Florida. Now, that may not seem like an invasion, and it’s not — yet. But these animals are the world’s largest rodent, growing to 50 kilograms or more. In the wild, the semiaquatic animals live in social groups in forests where they can be near bodies of water, such as rivers, lakes or swamps. They are herbivores that can subsist on a wide variety of vegetation, from grass to tree bark. And they reproduce at a fair pace, producing an average of four, and up to eight, pups per litter.

Most people wouldn’t look at those characteristics and think “I want to own one of those animals,” but some have. Capybaras are one of the many exotic creatures that people have tried to turn into pets. (Owning one is legal in some states.) But the animals can get loose, or people may purposely release them when they no longer want to own a giant rodent.

A capybara (or 50) loose in the countryside or city is not automatically an invasive species. The difference between an invasive and a nonnative exotic is whether an organism is causing environmental or economic harm, or harm to human health.

Congdon and her undergraduate students have been studying the potential for capybaras to make that transition from exotic to invasive, and they have been looking for similarities to nutria. Those large rodents were first imported to the United States in the early 1900s; the animals were farmed for their fur in Louisiana. But they escaped — some were also purposely released as weed mitigators — and quickly established themselves in Louisiana’s many swamps. Efforts to control the animals, such as hunting, have largely failed.

Nutria, which are smaller than capybaras, reproduce at about the same rate as the giant rodents. But one of the things that have made nutria such a menace — their propensity to dig into riverbanks, levees and other places that can cause problems when the ground disintegrates — appears to be a trait they don’t share with capybaras.
While coyotes and dogs are know to hunt nutria, it appears that nothing in the United States, other than a human, is big enough to kill a capybara. No animals here are equivalent to the capybara’s natural South American predators, which include anacondas, puma and jaguar, Congdon noted.

The state of Florida says only that a breeding population of capybaras “may exist,” but Congdon is pretty sure that there is one. In 1995, five animals escaped from a wildlife facility near Gainesville, and they are probably at least part of the source of those 50 capybaras now living in Florida. “Several sightings suggest they have been breeding,” Congdon said, including the finding of a juvenile capybara. Given the similarities to the nutria, and the ability of capybaras to adapt to a variety of habitats, including cities, “they might be able to make a go of it in the United States,” Congdon concluded.

But Congdon isn’t advocating that wildlife managers kill all the capybaras in Florida. The animals represent “an opportunity to study the process of invasion,” she said. Plus, a population in Florida would be a lot easier for her to access than the one she studied in Venezuela as a gradate student. “We want to keep them from spreading,” she said, “but can we please not kill them all so I can study them?”

How to get Ötzi’s look

Ötzi had Copper Age style.

The 5,300-year-old Tyrolean Iceman, whose body was found poking out of a glacier in the Italian Alps in 1991, incorporated hides from at least five domesticated and wild animal species into his apparel, a new genetic study finds. Comparing mitochondrial DNA extracted from nine ancient leather fragments with DNA of living animals revealed the makeup of Ötzi’s clothes and a key accessory, says a team led by paleogeneticist Niall O’Sullivan. Mitochondrial DNA typically gets passed from mothers to their offspring.
Little is known about what people wore during Ötzi’s time. The findings provide a glimpse into how ancient European populations exploited domesticated animals to make clothes and other items.

Ötzi’s coat consisted of hides from at least three goats and one sheep, the scientists report August 18 in Scientific Reports. This garment may have been periodically patched with leather from whatever animals were available, the team suggests.

Goats also provided skin for the Iceman’s leggings, the new analysis indicates.

A sheepskin loincloth and a shoelace derived from European cattle round out Ötzi’s attire made from domesticated animals.

As for wild animals, Ötzi wore a brown-bear cap and toted a quiver made from roe deer. It’s impossible to know if the ancient man attached any special meaning to brown bears, “but he may have been an opportunistic hunter or a scavenger,” says O’Sullivan, of University College Dublin and EURAC Research in Bolzano, Italy.
A 2012 analysis of proteins from fur samples taken from Ötzi’s clothing identified sheep and a goatlike animal called a chamois as sources for the Iceman’s coat. A team led by biochemist Klaus Hollemeyer of Saarland University in Saarbrücken, Germany, also pegged goats and dogs or wolves as sources of skin for Ötzi’s leggings.

Disparities between Hollemeyer’s and O’Sullivan’s studies may stem from the two groups having sampled different parts of patchwork garments. In addition, the new report used advanced techniques for extracting and analyzing ancient DNA. That enabled O’Sullivan’s team to retrieve six complete mitochondrial genomes from Ötzi’s leather belongings.

O’Sullivan’s investigation “opens a new field of potential identification procedures for mammalian species in ancient leathers and furs,” Hollemeyer says.

A roughly 4,200-year-old legging found in the Swiss Alps in 2004 also features goat hide. Mitochondrial DNA extracted from that garment came from an ancient line of European goats that has largely been replaced by a genetically distinct goat population, a team led by archaeologist Angela Schlumbaum of the University of Basel in Switzerland reported in 2010.

The Swiss legging was found with pieces of bows and arrows, woolen clothes and many other artifacts where an ice patch in a mountain pass had partly melted. No human bodies have been found there.

“Possibly, goat leather was most comfortable” as legging material, says University of Bern archaeologist Albert Hafner, a coauthor of the Swiss legging study. “Modern leather trousers often use goat as well.”

CRISPR inspires new tricks to edit genes

Scientists usually shy away from using the word miracle — unless they’re talking about the gene-editing tool called CRISPR/Cas9. “You can do anything with CRISPR,” some say. Others just call it amazing.

CRISPR can quickly and efficiently manipulate virtually any gene in any plant or animal. In the four years since CRISPR has been around, researchers have used it to fix genetic diseases in animals, combat viruses, sterilize mosquitoes and prepare pig organs for human transplants. Most experts think that’s just the beginning. CRISPR’s powerful possibilities — even the controversial notions of creating “designer babies” and eradicating entire species — are stunning and sometimes frightening.

So far CRISPR’s biggest impact has been felt in basic biology labs around the world. The inexpensive, easy-to-use gene editor has made it possible for researchers to delve into fundamental mysteries of life in ways that had been difficult or impossible. Developmental biologist Robert Reed likens CRISPR to a computer mouse. “You can just point it at a place in the genome and you can do anything you want at that spot.”

Anything, that is, as long as it involves cutting DNA. CRISPR/Cas9 in its original incarnation is a homing device (the CRISPR part) that guides molecular scissors (the Cas9 enzyme) to a target section of DNA. Together, they work as a genetic-engineering cruise missile that disables or repairs a gene, or inserts something new where it cuts.

Even with all the genetic feats the CRISPR/Cas9 system can do, “there were shortcomings. There were things we wanted to do better,” says MIT molecular biologist Feng Zhang, one of the first scientists to wield the molecular scissors. From his earliest report in 2013 of using CRISPR/Cas9 to cut genes in human and mouse cells, Zhang has described ways to make the system work more precisely and efficiently.

He isn’t alone. A flurry of papers in the last three years have detailed improvements to the editor. Going even further, a bevy of scientists, including Zhang, have dreamed up ways to make CRISPR do a toolbox’s worth of jobs.

Turning CRISPR into a multitasker often starts with dulling the cutting-edge technology’s cutting edge. In many of its new adaptations, the “dead” Cas9 scissors can’t snip DNA. Broken scissors may sound useless, but scientists have upcycled them into chromosome painters, typo-correctors, gene activity stimulators and inhibitors and general genome tinkerers.

“The original Cas9 is like a Swiss army knife with only one application: It’s a knife,” says Gene Yeo, an RNA biologist at the University of California, San Diego. But Yeo and other researchers have bolted other proteins and chemicals to the dulled blades and transformed the knife into a multifunctional tool.

Zhang and colleagues are also exploring trading the Cas9 part of the system for other enzymes that might expand the types of manipulations scientists can perform on DNA and other molecules. With the expanded toolbox, researchers may have the power to pry open secrets of cancer and other diseases and answer new questions about biology.
Many enzymes can cut DNA; the first were discovered in the 1970s and helped to launch the whole field of genetic engineering. What makes CRISPR/Cas9 special is its precision. Scientists can make surgical slices in one selected spot, as opposed to the more scattershot approach of early tools. A few recent gene-editing technologies, such as zinc finger nucleases and TALENs, could also lock on to a single target. But those gene editors are hard to redirect. A scientist who wants to snip a new spot in the genome has to build a new editor. That’s like having to assemble a unique guided missile for every possible target on a map. With CRISPR/Cas9, that’s not necessary.

The secret to CRISPR’s flexibility is its guidance system. A short piece of RNA shepherds the Cas9 cutting enzyme to its DNA target. The “guide RNA” can home in on any place a researcher selects by chemically pairing with DNA’s information-containing building blocks, or bases (denoted by the letters A, T, C and G). Making a new guide RNA is easy; researchers often simply order one online by typing in the desired sequence of bases.

That guidance system is taking genetic engineers to places they’ve never been. “With CRISPR, literally overnight what had been the biggest frustration of my career turned into an undergraduate side project,” says Reed, of Cornell University. “It was incredible.”
Reed studies how patterns are painted on butterfly and moth wings. Color patterning is one of the fundamental questions evolutionary and developmental biologists have been trying to answer for decades. In 1994, Sean B. Carroll and colleagues discovered that a gene called Distal-less is turned on in butter­fly wings in places where eyespots later form. The gene appeared to be needed for eyespot formation, but the evidence was only circumstantial. That’s where researchers have been stuck for 20 years, Reed says. They had no way to manipulate genes in butter­fly wings to get more direct proof of the role of different genes in painting wing patterns.

With CRISPR/Cas9, Reed and Cornell colleague Linlin Zhang cut and disabled the Distal-less gene at an early stage of wing development and got an unexpected result: Rather than cause eyespots, Distal-less limits them. When CRISPR/Cas9 knocks out Distal-less, more and bigger eyespots appear, the researchers reported in June in Nature Communications. Reed and colleagues have snipped genes in not just one, but six different butterfly species using CRISPR, he says.

CRISPR cuts genes very well, maybe too well, says neuro­scientist Marc Tessier-Lavigne of Rockefeller University in New York City. “The Cas9 enzyme is just so prolific. It cuts and recuts and recuts,” he says. That constant snipping can result in unwanted mutations in genes that researchers are editing or in genes that they never intended to touch. Tessier-Lavigne and colleagues figured out how to tame the overeager enzyme and keep it from julienning the genes of human stem cells grown in lab dishes. With better control, the researchers could make one or two mutations in two genes involved in early-onset Alzheimer’s disease, they reported in the May 5 Nature. Growing the mutated stem cells into brain cells showed that increasing the number of mutated copies of the genes also boosts production of the amyloid-beta peptide that forms plaques in Alzheimer’s-afflicted brains. The technology could make stem cells better mimics of human diseases.
While Tessier-Lavigne and others are working to improve the CRISPR/Cas9 system, building better guide RNAs and increasing the specificity of its cuts, some researchers are turning away from snippy Cas9 altogether.

Nuanced edits
Cas9 isn’t entirely to blame for the mess created when it causes a double-stranded break by slicing through both rails of the DNA ladder. “The cell’s response to double-stranded breaks is the source of a lot of problems,” says David Liu, a chemical biologist at Harvard University. A cell’s go-to method for fixing a DNA breach is to glue the cut ends back together. But often a few bases are missing or bits get stuck where they don’t belong. The result is more genome “vandalism than editing,” Liu says, quoting Harvard colleague George Church.

Liu wanted a gene editor that wouldn’t cause any destructive breaches: One that could A) go to a specific site in a gene and B) change a particular DNA base there, all without cutting DNA. The tool didn’t exist, but in Cas9, Liu and colleagues saw the makings of one, if they could tweak it just a bit.

They started by dulling Cas9’s cutting edge, effectively killing the enzyme. The “dead” Cas9 could still grip the guide RNA and ride it to its destination, but it couldn’t slice through DNA’s double strands. Liu and colleagues then attached a hitch­hiking enzyme, whose job is to initiate a series of steps to change the DNA base C into a T, or a G to an A. The researchers had to tinker with the system in other ways to get the change to stick. Once they worked out the kinks, they could make permanent single base-pair changes in 15 to 75 percent of the DNA they targeted without introducing insertions and deletions the way traditional CRISPR editing often does. Liu and collaborators reported the accomplishment in Nature in May.  A similar base editor, reported in Science in August by researchers in Japan, may be useful for editing DNA in bacteria and other organisms that can’t tolerate having their DNA cut.

There are 12 possible combinations of DNA base swaps. The hitchhiking enzyme that Liu used, cytidine deaminase, can make two of the swaps. Liu and others are working to fuse enzymes to Cas9 that can do the 10 others. Other enzyme hitchhikers may make it possible to edit single DNA bases at will, Liu says. Such a base editor could be used to fix single mutations that cause genetic diseases such as cystic fibrosis or muscular dystrophy. It might even correct the mutations that lead to inherited breast cancer.

Rewriting the score
Dead Cas9 is already helping researchers tinker with DNA in ways they couldn’t before. Variations on the dull blade may help scientists solve one of the great mysteries of biology: How does the same set of 20,000 genes give rise to so many different types of cells in the body?

The genome is like a piano, says Jonathan Weissman, a biochemist at the University of California, San Francisco. “You can play a huge variety of different music with only 88 keys by how hard you hit the keys, what keys you mix up and the timing.” By dialing down or turning up the activity of combinations of genes at precise times during development, cells are coaxed into becoming hundreds of different types of body cells.

For the last 20 years, researchers have been learning more about that process by watching when certain genes turn on and off in different cells. Gene activity is controlled by a dizzying variety of proteins known as transcription factors. When and where a transcription factor acts is at least partly determined by chemical tags on DNA and the histone proteins that package it. Those tags are known collectively as epigenetic marks. They work something like the musical score for an orchestra, telling the transcription factor “musicians” which notes to hit and how loudly or softly to play. So far, scientists have only been able to listen to the music. With dead Cas9, researchers can create molecules that will change epigenetic notes at any place in the score, Weissman says, allowing researchers to arrange their own music.

Epigenetic marks are alleged to be involved in addiction, cancer, mental illness, obesity, diabetes and heart disease. Scientists haven’t been able to prove that epigenetic marks are really behind these and other ailments, because they could never go into a cell and change just one mark on one gene to see if it really produced a sour note.

One such epigenetic mark, the attachment of a chemical called an acetyl group to a particular amino acid in a histone protein, is often associated with active genes. But no one could say for sure that the mark was responsible for making those genes active. Charles Gersbach of Duke University and colleagues reported last year in Nature Biotechnology that they had fused dead Cas9 to an enzyme that could make that epigenetic mark. When the researchers placed the epigenetic mark on certain genes, activity of those genes shot up, evidence that the mark really does boost gene activity. With such CRISPR epigenetic editors in hand, researchers may eventually be able to correct errant marks to restore harmony and health.

Weissman’s lab group was one of the first to turn dead Cas9 into a conductor of gene activity. Parking dead Cas9 on a gene is enough to nudge down the volume of some genes’ activity by blocking the proteins that copy DNA into RNA, the researchers found. Fusing a protein that silences genes to dead Cas9 led to even better noise-dampening of targeted genes. The researchers reported in Cell in 2014 that they could reduce gene activity by 90 to 99 percent for some genes using the silencer (which Weissman and colleagues call CRISPRi, for interference). A similar tool, created by fusing proteins that turn on, or activate, genes to dead Cas9 (called CRISPRa, for activator) lets researchers crank up the volume of activity from certain genes. In a separate study, published in July in the Proceedings of the National Academy of Sciences, Weissman and colleagues used their activation scheme to find new genes that make cancer cells resistant to chemo­therapy drugs.

RNA revolution
New, refitted Cas9s won’t just make manipulating DNA easier. They also could revolutionize RNA biology. There are already multiple molecular tools for grabbing and cutting RNA, Yeo says. So for his purposes, scissors weren’t necessary or even desirable. The homing ability of CRISPR/Cas9 is what Yeo found appealing.

He started simple, by using a tweaked CRISPR/Cas9 to tag RNAs to see where they go in the cell. Luckily, in 2014, Jennifer Doudna at the University of California, Berkeley — one of the researchers who in 2012 introduced CRISPR/Cas9 — and colleagues reported that Cas9 could latch on to messenger RNA molecules, or mRNAs (copies of the protein-building instructions contained in DNA). In a study published in April in Cell, Doudna, Yeo and colleagues strapped fluorescent proteins to the back of a dead Cas9 and pointed it toward mRNAs from various genes.
With the glowing Cas9, the researchers tracked mRNAs produced from several different genes in living cells. (Previous methods for pinpointing RNA’s location in a cell killed the cell.) In May, Zhang of MIT and colleagues described a two-color RNA-tracking system in Scientific Reports. Yet another group of researchers described a CRISPR rainbow for giving DNA a multicolored glow, also in living cells. That glow allowed the team to pinpoint the locations of up to six genes and see how the three-dimensional structure of chromosomes in the nucleus changes over time, the researchers reported in the May Nature Biotechnology. A team from UC San Francisco reported in January in Nucleic Acids Research that it had tracked multiple genes using combinations of two color tags.

But Yeo wants to do more than watch RNA move around. He envisions bolting a variety of different proteins to Cas9 to manipulate and study the many steps an mRNA goes through between being copied from DNA and having its instructions read to make a protein. Learning more about that multistep process and what other RNAs do in a cell could help researchers understand what goes wrong in some diseases, and maybe learn how to fix the problems.

Zhang wants to improve Cas9, but he would also like other versatile tools. He and colleagues are looking for such tools in bacteria.

CRISPR/Cas9 was first discovered in bacteria as a rudimentary immune system for fighting off viruses (SN: 12/12/15, p. 16). It zeroes in on and then shreds the viral DNA. Researchers most often use the Cas9 cutting enzyme from Streptococcus pyogenes bacteria.

But almost half of all bacteria have CRISPR immune systems, scientists now know, and many use enzymes other than Cas9. In the bacterium Francisella novicida U112, Zhang and colleagues found a gene-editing enzyme, Cpf1, which does things a little differently than Cas9 does. It has a different “cut here” signal that could make it more suitable than Cas9 for cutting DNA in some cases, the team reported last October in Cell. Cpf1 can also chop one long guide RNA into multiple guides, so researchers may be able to edit several genes at once. And Cpf1 cuts DNA so that one strand of the DNA is slightly longer than the other. That could make it easier to insert new genes into DNA.

Zhang more recently found an enzyme in the bacterium Leptotrichia shahii that could tinker with RNA. The RNA­ cutting enzyme is called C2c2, he and colleagues reported August 5 in Science. Like Cas9, C2c2 uses a guide RNA to lead the way, but instead of slicing DNA, it chops RNA.

Zhang’s team is exploring other CRISPR/Cas9-style enzymes that could help them “edit or modulate or interact with a genome more efficiently or more effectively,” he says. “Our search is not done yet.”

The explosion of new ways to use CRISPR hasn’t ended. “The field is advancing so rapidly,” says Zhang. “Just looking at how far we have come in the last three and a half years, I think what we’ll see coming in the next few years will just be amazing.”