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  • richardmitnick 11:56 am on May 25, 2017 Permalink | Reply
    Tags: , , Chiral nonlinear spectroscopy, , DNA studies,   

    From Cornell: “Water forms ‘spine of hydration’ around DNA, group finds” 

    Cornell Bloc

    Cornell University

    May 24, 2017
    Tom Fleischman
    tjf85@cornell.edu

    Story Contacts
    Cornell Chronicle
    Tom Fleischman
    607-255-9735
    tjf85@cornell.edu

    Media Contact
    Daryl Lovell
    607-254-4799
    dal296@cornell.edu

    1
    An illustration of what chiral nonlinear spectroscopy reveals: that DNA is surrounded by a chiral water super-structure, forming a “spine of hydration.” Poul Petersen/Provided

    Water is the Earth’s most abundant natural resource, but it’s also something of a mystery due to its unique solvation characteristics – that is, how things dissolve in it.

    “It’s uniquely adapted to biology, and vice versa,” said Poul Petersen, assistant professor of chemistry and chemical biology. “It’s super-flexible. It dissipates energy and mediates interactions, and that’s becoming more recognized in biological systems.”

    How water relates to and interacts with those systems – like DNA, the building block of all living things – is of critical importance, and Petersen’s group has used a relatively new form of spectroscopy to observe a previously unknown characteristic of water.

    “DNA’s chiral spine of hydration,” published May 24 in the American Chemical Society journal Central Science, reports the first observation of a chiral water superstructure surrounding a biomolecule. In this case, the water structure follows the iconic helical structure of DNA, which itself is chiral, meaning it is not superimposable on its mirror image. Chirality is a key factor in biology, because most biomolecules and pharmaceuticals are chiral.

    “If you want to understand reactivity and biology, then it’s not just water on its own,” Petersen said. “You want to understand water around stuff, and how it interacts with the stuff. And particularly with biology, you want to understand how it behaves around biological material – like protein and DNA.”

    Water plays a major role in DNA’s structure and function, and its hydration shell has been the subject of much study. Molecular dynamics simulations have shown a broad range of behaviors of the water structure in DNA’s minor groove, the area where the backbones of the helical strand are close together.

    The group’s work employed chiral sum frequency generation spectroscopy (SFG), a technique Petersen detailed in a 2015 paper in the Journal of Physical Chemistry. SFG is a nonlinear optical method in which two photon beams – one infrared and one visible – interact with the sample, producing an SFG beam containing the sum of the two beams’ frequencies, or energies. In this case, the sample was a strand of DNA linked to a silicon-coated prism.

    More manipulation of the beams and calculation proved the existence of a chiral water superstructure surrounding DNA.

    In addition to the novelty of observing a chiral water structure template by a biomolecule, chiral SFG provides a direct way to examine water in biology.

    “The techniques we have developed provide a new avenue to study DNA hydration, as well as other supramolecular chiral structures,” Petersen said.

    The group admits that their finding’s biological relevance is unclear, but Petersen thinks the ability to directly examine water and its behavior within biological systems is important.

    “Certainly, chemical engineers who are designing biomimetic systems and looking at biology and trying to find applications such as water filtration would care about this,” he said.

    Another application, Petersen said, could be in creating better anti-biofouling materials, which are resistant to the accumulation of microorganisms, algae and the like on wetted surfaces.

    Collaborators included M. Luke McDermott, Ph.D. ’17; Heather Vanselous, a doctoral student in chemistry and chemical biology and a member of the Petersen Group; and Steven Corcelli, professor of chemistry and biochemistry at the University of Notre Dame.

    This work was supported by grants from the National Science Foundation and the Arnold and Mable Beckman Foundation, and made use of the Cornell Center for Materials Research, an NSF Materials Research Science and Engineering Center.

    See the full article here .

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    Once called “the first American university” by educational historian Frederick Rudolph, Cornell University represents a distinctive mix of eminent scholarship and democratic ideals. Adding practical subjects to the classics and admitting qualified students regardless of nationality, race, social circumstance, gender, or religion was quite a departure when Cornell was founded in 1865.

    Today’s Cornell reflects this heritage of egalitarian excellence. It is home to the nation’s first colleges devoted to hotel administration, industrial and labor relations, and veterinary medicine. Both a private university and the land-grant institution of New York State, Cornell University is the most educationally diverse member of the Ivy League.

    On the Ithaca campus alone nearly 20,000 students representing every state and 120 countries choose from among 4,000 courses in 11 undergraduate, graduate, and professional schools. Many undergraduates participate in a wide range of interdisciplinary programs, play meaningful roles in original research, and study in Cornell programs in Washington, New York City, and the world over.

     
  • richardmitnick 3:14 pm on January 23, 2017 Permalink | Reply
    Tags: , , DNA studies, FRET, Histone proteins, Nucleosomes   

    From Cornell: “Slo-mo unwrapping of nucleosomal DNA probes protein’s role” 

    Cornell Bloc

    Cornell University

    Jan. 11, 2017
    Tom Fleischman

    1
    Using X-rays to visualize DNA (dark gray) and fluorescence to monitor the histone proteins (yellow and cyan), Cornell researchers led by professor and director of applied and engineering physics Lois Pollack found that the release of histone proteins is guided by unwrapping DNA. Joshua Tokuda/Provided

    Nucleosomes are tightly packed bunches of DNA and protein which, when linked together as chromatin, form each of the 46 chromosomes found in human cells.

    The organization of DNA in nucleosomes is important not just for DNA packaging; it also forms the basis for the regulation of gene expression. By controlling the access to DNA, nucleosomes help facilitate all kinds of gene activity, from RNA transcription to DNA replication and repair.

    A research group led by Lois Pollack, professor of applied and engineering physics, used a combination of X-ray and fluorescence-based approaches to study how the shapes and compositions of nucleosomes change after being destabilized.

    The group’s paper, Asymmetric unwrapping of nucleosomal DNA propagates asymmetric opening and dissociation of the histone core, is published online in Proceedings of the National Academy of Sciences. Co-lead authors are postdoctoral researcher Yujie Chen and doctoral student Joshua Tokuda.

    Using FRET, small-angle X-ray scattering and other methods, the group was able to get a clear picture of the DNA activity during unwrapping of the histone core. It was found that different DNA shapes were produced during the unwrapping process, most notably a “teardrop” shape that seemed to promote protein activity.

    The histone core goes from eight protein molecules to six when the DNA unwraps into the teardrop shape. “It’s as if having the DNA in this shape is a signal to the protein: ‘Hey, now’s the time. You want to change it up? Go ahead,’” Pollack said.

    This finding suggests that the molecular transition is guided by this specific type of unwrapping. It’s a step toward better understanding of DNA access during transcription, replication and repair.

    “The reason why these structures are so important, in addition to packaging, is that it also gives cells the opportunity to control which genes are on and off,” Tokuda said.

    Tokuda adds that misregulation of chromatin remodeling is also implicated in many human diseases, from neuro-development and degenerative disorders to immunodeficiency syndromes and cancer.

    “We hope that by developing these tools to investigate the fundamental mechanism of remodeler proteins,” he said, “we may be able to provide insight that will aid in the development of new therapeutic strategies for these diseases.”

    This work was supported by grants from the National Institutes of Health.

    See the full article here .

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    Once called “the first American university” by educational historian Frederick Rudolph, Cornell University represents a distinctive mix of eminent scholarship and democratic ideals. Adding practical subjects to the classics and admitting qualified students regardless of nationality, race, social circumstance, gender, or religion was quite a departure when Cornell was founded in 1865.

    Today’s Cornell reflects this heritage of egalitarian excellence. It is home to the nation’s first colleges devoted to hotel administration, industrial and labor relations, and veterinary medicine. Both a private university and the land-grant institution of New York State, Cornell University is the most educationally diverse member of the Ivy League.

    On the Ithaca campus alone nearly 20,000 students representing every state and 120 countries choose from among 4,000 courses in 11 undergraduate, graduate, and professional schools. Many undergraduates participate in a wide range of interdisciplinary programs, play meaningful roles in original research, and study in Cornell programs in Washington, New York City, and the world over.

     
  • richardmitnick 9:34 pm on December 23, 2016 Permalink | Reply
    Tags: Aquaporin, , DNA studies,   

    From Caltech: “Visualizing Gene Expression with MRI” 

    Caltech Logo
    Caltech

    12/23/2016

    Lori Dajose
    (626) 395-1217
    ldajose@caltech.edu

    1
    An illustration of aquaporin’s effect on cells. Credit: M. Shapiro Laboratory/Caltech

    Genes tell cells what to do—for example, when to repair DNA mistakes or when to die—and can be turned on or off like a light switch. Knowing which genes are switched on, or expressed, is important for the treatment and monitoring of disease. Now, for the first time, Caltech scientists have developed a simple way to visualize gene expression in cells deep inside the body using a common imaging technology.

    Researchers in the laboratory of Mikhail Shapiro, assistant professor of chemical engineering and Heritage Medical Research Institute Investigator, have invented a new method to link magnetic resonance imaging (MRI) signals to gene expression in cells—including tumor cells—in living tissues. The technique, which eventually could be used in humans, would allow gene expression to be monitored non-invasively, requiring no surgical procedures such as biopsies.

    The work appears in the December 23 online edition of the journal Nature Communications.

    In MRI, hydrogen atoms in the body—atoms that are mostly contained in water molecules and fat—are excited using a magnetic field. The excited atoms, in turn, emit signals that can be used to create images of the brain, muscle, and other tissues, which can be distinguished based on the local physical and chemical environment of the water molecules. While this technique is widely used, it usually provides only anatomical snapshots of tissues or physiological functions such as blood flow rather than observations of the activity of specific cells.

    “We thought that if we could link signals from water molecules to the expression of genes of interest, we could change the way the cell looks under MRI,” says Arnab Mukherjee, a postdoctoral scholar in chemical engineering at Caltech and co-lead author on the paper.

    The group turned to a protein that naturally occurs in humans, called aquaporin. Aquaporin sits within the membrane that envelops cells and acts as a gatekeeper for water molecules, allowing them to move in and out of the cell. Shapiro’s team realized that increasing the number of aquaporins on a given cell made it stand out in MRI images acquired using a common clinical technique called diffusion-weighted imaging, which is sensitive to the movement of water molecules. They then linked aquaporin to genes of interest, making it what scientists call a reporter gene. This means that when a gene of interest is turned on, the cell will overexpress aquaporin, making the cell look darker under diffusion-weighted MRI.

    The researchers showed that this technique was successful in monitoring gene expression in a brain tumor in mice. After implanting the tumor, they gave the mice a drug to trigger the tumor cells to express the aquaporin reporter gene, which made the tumor look darker in MRI images.

    “Overexpression of aquaporin has no negative impact on cells because it is exclusive to water and simply allows the molecules to go back and forth across the cell membrane,” Shapiro says. Under normal physiological conditions the number of water molecules entering and exiting an aquaporin-expressing cell is the same, so that the total amount of water in each cell does not change. “Aquaporin is a very convenient way to genetically change the way that cells look under MRI.”

    Though the work was done in mice, it has the potential for clinical translation, according to Shapiro. Aquaporin is a naturally occurring gene and will not cause an immune reaction. Previously developed reporter genes for MRI have been much more limited in their capabilities, requiring the use of specific metals that are not always available in some tissues.

    “An effective reporter gene for MRI is a ‘holy grail’ in biomedical imaging because it would allow cellular function to be observed non-invasively,” says Shapiro. “Aquaporins are a new way to think about this problem. It is remarkable that simply allowing water molecules to more easily get into and out of cells in a tissue gives us the ability to remotely see those cells in the middle of the body.”

    The paper is titled Non-invasive imaging using reporter genes altering cellular water permeability. In addition to Shapiro and Mukherjee, other coauthors include Caltech graduate students Di Wu (MS ’16 and co-lead author) and Hunter Davis. The work was funded by the Dana Foundation, a Burroughs Wellcome Career Award at the Scientific Interface, the Heritage Medical Research Institute, and the National Institutes of Health.

    See the full article here .

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    Caltech campus
    The California Institute of Technology (commonly referred to as Caltech) is a private research university located in Pasadena, California, United States. Caltech has six academic divisions with strong emphases on science and engineering. Its 124-acre (50 ha) primary campus is located approximately 11 mi (18 km) northeast of downtown Los Angeles. “The mission of the California Institute of Technology is to expand human knowledge and benefit society through research integrated with education. We investigate the most challenging, fundamental problems in science and technology in a singularly collegial, interdisciplinary atmosphere, while educating outstanding students to become creative members of society.”

     
  • richardmitnick 5:30 am on September 10, 2016 Permalink | Reply
    Tags: , DNA studies, , ,   

    From ICL: “Activity of Huntington’s disease gene curbed for six months in mice” 

    Imperial College London
    Imperial College London

    09 September 2016
    Hayley Dunning

    1
    Healthy brain (L) and Huntington’s brain (R). No image credit.

    A single injection of a new treatment has reduced the activity of the gene responsible for Huntington’s disease for several months in a trial in mice.

    Huntington’s disease is a genetic disorder that affects around 1 in every 10,000 people and damages nerve cells in the brain. This causes neurological symptoms affecting movement, cognition and behaviour.

    Huntington’s usually only begins to show symptoms in adulthood. There is currently no cure and no way to slow the progression of the disease. Symptoms typically progress over 10-25 years until the person eventually dies.

    Now, the EU-funded FINGERS4CURE project team led by researchers at Imperial College London have engineered a therapeutic protein called a ‘zinc finger’.

    Huntington’s disease is caused by a mutant form of a single gene called Huntingtin. The zinc finger protein works by targeting the mutant copies of the Huntingtin gene, repressing its ability to express and create harmful proteins.

    In the new study involving mice, published in the journal Molecular Neurodegeneration, the injection of zinc finger repressed the mutant copies of the gene for at least six months.

    In a previous study in mice, the team had curbed the mutant gene’s activity for just a couple of weeks. By tweaking the ingredients of the zinc finger in the new study they were able to extend its effects to several months, repressing the disease gene over that period without seeing any harmful side effects. This involved making the zinc finger as invisible to the immune system as possible.

    A lot of promise

    Project lead Dr Mark Isalan from the Department of Life Sciences at Imperial said: “We are extremely excited by our latest results, which show a lot of promise for treating Huntington’s disease.

    “However, while these encouraging results in mice mean that the zinc finger looks like a good candidate to take forward to human trials, we still need to do a lot of work first to answer important questions around the safety of the intervention, whether repeat treatments are effective, whether there might be longer-term side effects, and whether we can extend and increase the benefits beyond six months.

    “In this study we weren’t looking at how repressing the gene activity affected the symptoms of the disease and this is obviously a critical question as well. However, we have reason to be confident from our previous studies that repressing the gene does in fact significantly reduce symptoms.

    “If all goes well and we have further positive results, we would aim to start clinical trials within five years to see whether the treatment could be safe and effective in humans. We are urgently looking for industry partners and funding to achieve this.”

    Cut off at the source

    The mutant Huntingtin gene is thought to cause toxic levels of protein to aggregate in the brain. Preventing the activity of this gene could theoretically halt the disease, but this has been difficult to achieve.

    The gene is present in many different cell types in the brain, making it difficult to target, and every patient also has a non-mutant copy of the gene, which scientists need to avoid targeting with any intervention in order to prevent unwanted side effects.

    The zinc finger protein sticks to the DNA of the mutant Huntingtin gene and turns off the gene’s expression. “We don’t know exactly how the mutant Huntingtin gene causes the disease, so the idea is that targeting the gene expression cuts off the problem at its source – preventing it from ever having the potential to act,” said Dr Isalan.

    By targeting the fundamental DNA of the gene, the zinc finger therapy also has the advantage over other potential Huntington’s therapies of needing less frequent treatments.

    Lengthening effect

    In the study, the researchers gave a single injection of zinc finger to 12 mice with Huntingdon’s disease. They examined the brains of the mice at different intervals after the initial injection and found that on average, 77 per cent of the ‘bad’ gene expression was repressed in mouse brains three weeks after injection of the zinc finger, 61 per cent repressed at six weeks, and 48 per cent repressed at 12 weeks.

    By 24 weeks after the initial injections, there was still 23 per cent repression, which is thought to still be useful therapeutically. The team are now working on ways to lengthen the repression period even further.

    The study was funded by a European Research Council Proof-of-Concept Award (ERC-2014-PoC 641232 FINGERS4CURE) and involved researchers from Imperial College London, Centre for Genomi Regulation (CRG) in Spain, and Universitat Pompeu Fabra in Spain.

    See the full article here .

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    Imperial College London

    Imperial College London is a science-based university with an international reputation for excellence in teaching and research. Consistently rated amongst the world’s best universities, Imperial is committed to developing the next generation of researchers, scientists and academics through collaboration across disciplines. Located in the heart of London, Imperial is a multidisciplinary space for education, research, translation and commercialisation, harnessing science and innovation to tackle global challenges.

     
  • richardmitnick 8:57 am on September 9, 2016 Permalink | Reply
    Tags: , , DNA studies, , Genetic Engineering to Clash With Evolution,   

    From Quanta: “Genetic Engineering to Clash With Evolution” 

    Quanta Magazine
    Quanta Magazine

    September 8, 2016
    Brooke Borel

    In a crowded auditorium at New York’s Cold Spring Harbor Laboratory in August, Philipp Messer, a population geneticist at Cornell University, took the stage to discuss a powerful and controversial new application for genetic engineering: gene drives.

    Gene drives can force a trait through a population, defying the usual rules of inheritance. A specific trait ordinarily has a 50-50 chance of being passed along to the next generation. A gene drive could push that rate to nearly 100 percent. The genetic dominance would then continue in all future generations. You want all the fruit flies in your lab to have light eyes? Engineer a drive for eye color, and soon enough, the fruit flies’ offspring will have light eyes, as will their offspring, and so on for all future generations. Gene drives may work in any species that reproduces sexually, and they have the potential to revolutionize disease control, agriculture, conservation and more. Scientists might be able to stop mosquitoes from spreading malaria, for example, or eradicate an invasive species.

    The technology represents the first time in history that humans have the ability to engineer the genes of a wild population. As such, it raises intense ethical and practical concerns, not only from critics but from the very scientists who are working with it.

    Messer’s presentation highlighted a potential snag for plans to engineer wild ecosystems: Nature usually finds a way around our meddling. Pathogens evolve antibiotic resistance; insects and weeds evolve to thwart pesticides. Mosquitoes and invasive species reprogrammed with gene drives can be expected to adapt as well, especially if the gene drive is harmful to the organism — it’ll try to survive by breaking the drive.

    “In the long run, even with a gene drive, evolution wins in the end,” said Kevin Esvelt, an evolutionary engineer at the Massachusetts Institute of Technology. “On an evolutionary timescale, nothing we do matters. Except, of course, extinction. Evolution doesn’t come back from that one.”

    Gene drives are a young technology, and none have been released into the wild. A handful of laboratory studies show that gene drives work in practice — in fruit flies, mosquitoes and yeast. Most of these experiments have found that the organisms begin to develop evolutionary resistance that should hinder the gene drives. But these proof-of-concept studies follow small populations of organisms. Large populations with more genetic diversity — like the millions of swarms of insects in the wild — pose the most opportunities for resistance to emerge.

    It’s impossible — and unethical — to test a gene drive in a vast wild population to sort out the kinks. Once a gene drive has been released, there may be no way to take it back. (Some researchers have suggested the possibility of releasing a second gene drive to shut down a rogue one. But that approach is hypothetical, and even if it worked, the ecological damage done in the meantime would remain unchanged.)

    The next best option is to build models to approximate how wild populations might respond to the introduction of a gene drive. Messer and other researchers are doing just that. “For us, it was clear that there was this discrepancy — a lot of geneticists have done a great job at trying to build these systems, but they were not concerned that much with what is happening on a population level,” Messer said. Instead, he wants to learn “what will happen on the population level, if you set these things free and they can evolve for many generations — that’s where resistance comes into play.”

    At the meeting at Cold Spring Harbor Laboratory, Messer discussed a computer model his team developed, which they described in a paper posted in June on the scientific preprint site biorxiv.org. The work is one of three theoretical papers on gene drive resistance submitted to biorxiv.org in the last five months — the others are from a researcher at the University of Texas, Austin, and a joint team from Harvard University and MIT. (The authors are all working to publish their research through traditional peer-reviewed journals.) According to Messer, his model suggests “resistance will evolve almost inevitably in standard gene drive systems.”

    It’s still unclear where all this interplay between resistance and gene drives will end up. It could be that resistance will render the gene drive impotent. On the one hand, this may mean that releasing the drive was a pointless exercise; on the other hand, some researchers argue, resistance could be an important natural safety feature. Evolution is unpredictable by its very nature, but a handful of biologists are using mathematical models and careful lab experiments to try to understand how this powerful genetic tool will behave when it’s set loose in the wild.

    1
    Lucy Reading-Ikkanda for Quanta Magazine

    Resistance Isn’t Futile

    Gene drives aren’t exclusively a human technology. They occasionally appear in nature. Researchers first thought of harnessing the natural versions of gene drives decades ago, proposing to re-create them with “crude means, like radiation” or chemicals, said Anna Buchman, a postdoctoral researcher in molecular biology at the University of California, Riverside. These genetic oddities, she adds, “could be manipulated to spread genes through a population or suppress a population.”

    In 2003, Austin Burt, an evolutionary geneticist at Imperial College London, proposed a more finely tuned approach called a homing endonuclease gene drive, which would zero in on a specific section of DNA and alter it.

    Burt mentioned the potential problem of resistance — and suggested some solutions — both in his seminal paper and in subsequent work. But for years, it was difficult to engineer a drive in the lab, because the available technology was cumbersome.

    With the advent of genetic engineering, Burt’s idea became reality. In 2012, scientists unveiled CRISPR, a gene-editing tool that has been described as a molecular word processor. It has given scientists the power to alter genetic information in every organism they have tried it on. CRISPR locates a specific bit of genetic code and then breaks both strands of the DNA at that site, allowing genes to be deleted, added or replaced.

    CRISPR provides a relatively easy way to release a gene drive. First, researchers insert a CRISPR-powered gene drive into an organism. When the organism mates, its CRISPR-equipped chromosome cleaves the matching chromosome coming from the other parent. The offspring’s genetic machinery then attempts to sew up this cut. When it does, it copies over the relevant section of DNA from the first parent — the section that contains the CRISPR gene drive. In this way, the gene drive duplicates itself so that it ends up on both chromosomes, and this will occur with nearly every one of the original organism’s offspring.

    Just three years after CRISPR’s unveiling, scientists at the University of California, San Diego, used CRISPR to insert inheritable gene drives into the DNA of fruit flies, thus building the system Burt had proposed. Now scientists can order the essential biological tools on the internet and build a working gene drive in mere weeks. “Anyone with some genetics knowledge and a few hundred dollars can do it,” Messer said. “That makes it even more important that we really study this technology.”

    Although there are many different ways gene drives could work in practice, two approaches have garnered the most attention: replacement and suppression. A replacement gene drive alters a specific trait. For example, an anti-malaria gene drive might change a mosquito’s genome so that the insect no longer had the ability to pick up the malaria parasite. In this situation, the new genes would quickly spread through a wild population so that none of the mosquitoes could carry the parasite, effectively stopping the spread of the disease.

    A suppression gene drive would wipe out an entire population. For example, a gene drive that forced all offspring to be male would make reproduction impossible.

    But wild populations may resist gene drives in unpredictable ways. “We know from past experiences that mosquitoes, especially the malaria mosquitoes, have such peculiar biology and behavior,” said Flaminia Catteruccia, a molecular entomologist at the Harvard T.H. Chan School of Public Health. “Those mosquitoes are much more resilient than we make them. And engineering them will prove more difficult than we think.” In fact, such unpredictability could likely be found in any species.

    2
    A sample of malaria-infected blood contains two Plasmodium falciparum parasites. CDC/PHIL

    The three new biorxiv.org papers use different models to try to understand this unpredictability, at least at its simplest level.

    The Cornell group used a basic mathematical model to map how evolutionary resistance will emerge in a replacement gene drive. It focuses on how DNA heals itself after CRISPR breaks it (the gene drive pushes a CRISPR construct into each new organism, so it can cut, copy and paste itself again). The DNA repairs itself automatically after a break. Exactly how it does so is determined by chance. One option is called nonhomologous end joining, in which the two ends that were broken get stitched back together in a random way. The result is similar to what you would get if you took a sentence, deleted a phrase, and then replaced it with an arbitrary set of words from the dictionary — you might still have a sentence, but it probably wouldn’t make sense. The second option is homology-directed repair, which uses a genetic template to heal the broken DNA. This is like deleting a phrase from a sentence, but then copying a known phrase as a replacement — one that you know will fit the context.

    Nonhomologous end joining is a recipe for resistance. Because the CRISPR system is designed to locate a specific stretch of DNA, it won’t recognize a section that has the equivalent of a nonsensical word in the middle. The gene drive won’t get into the DNA, and it won’t get passed on to the next generation. With homology-directed repair, the template could include the gene drive, ensuring that it would carry on.

    The Cornell model tested both scenarios. “What we found was it really is dependent on two things: the nonhomologous end-joining rate and the population size,” said Robert Unckless, an evolutionary geneticist at the University of Kansas who co-authored the paper as a postdoctoral researcher at Cornell. “If you can’t get nonhomologous end joining under control, resistance is inevitable. But resistance could take a while to spread, which means you might be able to achieve whatever goal you want to achieve.” For example, if the goal is to create a bubble of disease-proof mosquitoes around a city, the gene drive might do its job before resistance sets in.

    The team from Harvard and MIT also looked at nonhomologous end joining, but they took it a step further by suggesting a way around it: by designing a gene drive that targets multiple sites in the same gene. “If any of them cut at their sites, then it’ll be fine — the gene drive will copy,” said Charleston Noble, a doctoral student at Harvard and the first author of the paper. “You have a lot of chances for it to work.”

    The gene drive could also target an essential gene, Noble said — one that the organism can’t afford to lose. The organism may want to kick out the gene drive, but not at the cost of altering a gene that’s essential to life.

    The third biorxiv.org paper, from the UT Austin team, took a different approach. It looked at how resistance could emerge at the population level through behavior, rather than within the target sequence of DNA. The target population could simply stop breeding with the engineered individuals, for example, thus stopping the gene drive.

    “The math works out that if a population is inbred, at least to some degree, the gene drive isn’t going to work out as well as in a random population,” said James Bull, the author of the paper and an evolutionary biologist at Austin. “It’s not just sequence evolution. There could be all kinds of things going on here, by which populations block [gene drives],” Bull added. “I suspect this is the tip of the iceberg.”

    Resistance is constrained only by the limits of evolutionary creativity. It could emerge from any spot along the target organism’s genome. And it extends to the surrounding environment as well. For example, if a mosquito is engineered to withstand malaria, the parasite itself may grow resistant and mutate into a newly infectious form, Noble said.

    Not a Bug, but a Feature?

    If the point of a gene drive is to push a desired trait through a population, then resistance would seem to be a bad thing. If a drive stops working before an entire population of mosquitoes is malaria-proof, for example, then the disease will still spread. But at the Cold Spring Harbor Laboratory meeting, Messer suggested the opposite: “Let’s embrace resistance. It could provide a valuable safety control mechanism.” It’s possible that the drive could move just far enough to stop a disease in a particular region, but then stop before it spread to all of the mosquitoes worldwide, carrying with it an unknowable probability of unforeseen environmental ruin.

    Not everyone is convinced that this optimistic view is warranted. “It’s a false security,” said Ethan Bier, a geneticist at the University of California, San Diego. He said that while such a strategy is important to study, he worries that researchers will be fooled into thinking that forms of resistance offer “more of a buffer and safety net than they do.”

    And while mathematical models are helpful, researchers stress that models can’t replace actual experimentation. Ecological systems are just too complicated. “We have no experience engineering systems that are going to evolve outside of our control. We have never done that before,” Esvelt said. “So that’s why a lot of these modeling studies are important — they can give us a handle on what might happen. But I’m also hesitant to rely on modeling and trying to predict in advance when systems are so complicated.”

    Messer hopes to put his theoretical work into a real-world setting, at least in the lab. He is currently directing a gene drive experiment at Cornell that tracks multiple cages of around 5,000 fruit flies each — more animals than past studies have used to research gene drive resistance. The gene drive is designed to distribute a fluorescent protein through the population. The proteins will glow red under a special light, a visual cue showing how far the drive gets before resistance weeds it out.

    Others are also working on resistance experiments: Esvelt and Catteruccia, for example, are working with George Church, a geneticist at Harvard Medical School, to develop a gene drive in mosquitoes that they say will be immune to resistance. They plan to insert multiple drives in the same gene — the strategy suggested by the Harvard/MIT paper.

    Such experiments will likely guide the next generation of computer models, to help tailor them more precisely to a large wild population.

    “I think it’s been interesting because there is this sort of going back and forth between theory and empirical work,” Unckless said. “We’re still in the early days of it, but hopefully it’ll be worthwhile for both sides, and we’ll make some informed and ethically correct decisions about what to do.”

    See the full article here .

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    Formerly known as Simons Science News, Quanta Magazine is an editorially independent online publication launched by the Simons Foundation to enhance public understanding of science. Why Quanta? Albert Einstein called photons “quanta of light.” Our goal is to “illuminate science.” At Quanta Magazine, scientific accuracy is every bit as important as telling a good story. All of our articles are meticulously researched, reported, edited, copy-edited and fact-checked.

     
  • richardmitnick 10:14 am on September 7, 2016 Permalink | Reply
    Tags: , , , DNA studies,   

    From Brown: “Brown to lead $9.7M grant to advance theory of aging” 

    Brown University
    Brown University

    September 6, 2016
    David Orenstein
    david_orenstein@brown.edu
    401-863-1862

    A new multi-university research effort will seek to determine whether rogue elements of DNA promote or even cause aging and whether interventions against them could help people live longer and more healthfully.

    Over the last few years, scientists — including a team at Brown University — have produced mounting evidence that mobility within genome of potentially harmful DNA snippets, called retrotransposable elements, may cause health problems associated with aging.

    With a new $9.67 million, five-year grant from the National Institutes of Health, researchers at Brown University, New York University and the University of Rochester will collaborate to further strengthen the evidence and to advance toward the goal of applying the findings medically.

    “There are a lot of provocative data and a lot of very cool ideas, but the issue now is how to nail this down,” said John Sedivy, the Hermon C. Bumpus Professor of Biology at Brown University and principal investigator of the grant. “Let’s take the bull by the horns and see what’s really going on. Is this a legitimate mechanism of aging and can we control it for therapeutic purposes?”

    Previous studies have shown that as cells age, or become senescent, they lose their ability to prevent retrotransposable elements from spreading into new places in the genome. In three new projects supported by two core facilities, the grant will spur the study of how retrotransposable elements function in cells and how their activity might cause specific diseases, and test possible ways of suppressing that activity. The researchers will not only work with human cells but also with mice and fruit flies, or Drosophila, where they can ask more direct questions, obtain faster answers and therefore better inform eventual interventions for people.

    Sedivy will lead one of the effort’s three projects, “Regulation of Retrotransposable Element Activity in Cellular Senescence and Aging,” and the administrative core. Dr. Stephen Helfand, a fellow Brown professor of biology, will lead the second project, “Regulation of Retrotransposable Element Activity in Drosophila.”

    Professor Jef Boeke of NYU will lead the Retrotransposon Engineering and Genomics Core. Rochester Associate Professor Andrei Seluanov will lead the Mouse Intervention and Aging Core, while Rochester Professor Vera Gorbunova will lead the third project, “Repression of Retrotransposable Elements by the Longevity Gene SIRT6.”

    The results could shed important light on the health consequences of retrotransposable element activity, Sedivy said. Experiments demonstrating the best interventions could then become translated into future human clinical trials.

    In fact, Sedivy notes, some drugs already exist and are widely used against one well known retrotransposable element, HIV. Brown researchers have already shown that some of these drugs also suppress some endogenous retrotransposable elements, although they were never intended for that purpose.

    “Hence, if endogenous retrotransposable elements do promote aging in some contexts, we already have pretty good drugs that could be tested right away,” Sedivy said. “What you want to come out with is a therapeutic that is directed against retrotransposable elements and then use that therapeutic to target a number of diseases. But at this point, we don’t really know which human diseases are linked with these retrotransposition events.”

    The new grant, Sedivy said, will help the field get there.

    See the full article here .

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    Welcome to Brown

    Brown U Robinson Hall
    Located in historic Providence, Rhode Island and founded in 1764, Brown University is the seventh-oldest college in the United States. Brown is an independent, coeducational Ivy League institution comprising undergraduate and graduate programs, plus the Alpert Medical School, School of Public Health, School of Engineering, and the School of Professional Studies.

    With its talented and motivated student body and accomplished faculty, Brown is a leading research university that maintains a particular commitment to exceptional undergraduate instruction.

    Brown’s vibrant, diverse community consists of 6,000 undergraduates, 2,000 graduate students, 400 medical school students, more than 5,000 summer, visiting and online students, and nearly 700 faculty members. Brown students come from all 50 states and more than 100 countries.

    Undergraduates pursue bachelor’s degrees in more than 70 concentrations, ranging from Egyptology to cognitive neuroscience. Anything’s possible at Brown—the university’s commitment to undergraduate freedom means students must take responsibility as architects of their courses of study.

     
  • richardmitnick 4:32 pm on July 29, 2016 Permalink | Reply
    Tags: , Bacterial Pathogenicity, DNA studies,   

    From LBL: “Study Finds Molecular Switch That Triggers Bacterial Pathogenicity” 

    Berkeley Logo

    Berkeley Lab

    July 29, 2016
    Sarah Yang
    scyang@lbl.gov
    (510) 486-4575

    1
    The top two rows show illustrations of crystals and solution structures of bacterial HU proteins with DNA represented by X-ray crystallography and small angle X-ray scattering, respectively. DNA strands are yellow and HU proteins are shades of blue. Soft X-ray tomography was used to visualize bacterial chromatin (in yellow) in wild type and invasive E. coli cells, shown in the bottom row. (Credit: Michal Hammel/Berkeley Lab)

    Scientists have revealed for the first time the molecular steps that turn on bacteria’s pathogenic genes. Using an array of high-powered X-ray imaging techniques, the researchers at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) showed that histone-like proteins that bind to DNA are related to the physical twisting of the genetic strand, and that the supercoiling of the chromosome can trigger the expression of genes that make a microbe invasive.

    The study, published Friday, July 29, in the journal Science Advances, could open up new avenues in the development of drugs to prevent or treat bacterial infection, the study authors said.

    The researchers looked at how the long strands of DNA are wound tight, a necessity if they are to fit into compact spaces. For eukaryotes, the strands wrap around histone proteins to fit inside a nucleus. For single-celled prokaryotes, which include bacteria, HU proteins serve as the histones, and the chromosomes bunch up in the nucleoid, which lacks a membrane.

    When the normal twists and turns of DNA compaction turn into supercoiling, trouble can begin.

    “It has been known that DNA supercoiling leads to pathogenicity in bacteria, but exactly how the bacterial chromosome is condensed, organized, and ultimately segregated has been a puzzle for over half a century,” said study lead author Michal Hammel, research scientist in Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging Division. “What we did for the first time was to visualize in E. coli how this packing is done, and we also discovered that the way HU proteins pack the chromosomes can trigger gene expression. That is new.”

    Capturing HU in action

    Elucidating these molecular mechanisms entailed imaging HU proteins at different resolutions and stages using two beamlines at Berkeley Lab’s Advanced Light Source [ALS], a DOE Office of Science User Facility.

    LBL ALS interior
    ALS

    The Structurally Integrated Biology for Life Sciences (SIBYLS) beamline, directed by senior scientist John Tainer, combines X-ray crystallography and small angle X-ray scattering (SAXS) capabilities. The crystallography provided atomic-level details of how the HU proteins interacted with the bacterial DNA, while SAXS was able to show how the HU proteins assembled and affected the longer strands of DNA in a solution.

    3
    Berkeley Lab scientists Michal Hammel and Carolyn Larabell in front of the SIBYLS Beamline at the Advanced Light Source. (Credit: Paul Mueller/Berkeley Lab)

    To get a clear sense of how that twisting and packing manifests at the cellular level, Hammel teamed up with Berkeley Lab faculty scientist Carolyn Larabell, director of the National Center for X-ray Tomography (NCXT), which is also based at the Advanced Light Source.

    “We needed the interaction of these different techniques to get the overall picture of how the HU interactions with DNA were affecting the bacteria,” added Larabell, who is also a professor of anatomy at UC San Francisco. “With X-ray tomography, we’re able to see the natural contrast in organic material in as close to a living state as possible, and we can provide quantitative comparisons of how compacted the chromosomes were in pathogenic and normal strains of E. coli.”

    Larabell calculated that the genetic material in the pathogenicE. coli is so tightly packed that it consumes less than one-half the volume compared with its non-mutant counterpart.

    A target to control pathogenesis

    Before this paper, it had been believed that the enzyme topoisomerase was the primary driver of DNA coiling in bacteria. This new study shows that, independent of topoisomerase, changing the assembly of HU proteins was enough to induce changes in DNA coiling at different stages of bacterial growth.

    “What is notable about HU proteins as a trigger for gene expression is that it’s quick,” said Hammel. “This makes sense as a survival mechanism for bacteria, which need to adapt quickly to different environments.”

    The study results also beg the question: If pathogenicity can be switched on, could it also be switched off?

    “We certainly expect to answer that question in future studies,” said Hammel. “These HU interactions could be an attractive target for drugs that control pathogenesis, not only of bacteria, but of other microbes with comparable genetic structures.”

    Other study co-authors include researchers from the National Cancer Institute’s Center for Cancer Research and the University of Texas.

    The National Institutes of Health and the DOE Office of Science supported this research.

    See the full article here .

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    A U.S. Department of Energy National Laboratory Operated by the University of California

    University of California Seal

    DOE Seal

     
  • richardmitnick 10:02 am on June 28, 2016 Permalink | Reply
    Tags: , DNA studies,   

    From Rice U: “Core proteins exert control over DNA function” 

    Rice U bloc

    Rice University

    June 21, 2016
    Mike Williams

    Rice University-based models simulate how nucleosomes facilitate gene exposure

    1
    Rice University scientists simulated a nucleosome coiled in DNA to discover the interactions that control its unwinding. The DNA double helix binds tightly to proteins (in red, blue, orange and green) that make up the histone core, which exerts control over the exposure (center and right) of genes for binding. Courtesy of the Wolynes Lab

    The spools at the center of nucleosomes, the fundamental unit of DNA organization, are histone protein core complexes. Nucleosomes are buried deep within a cell’s nucleus. About 147 DNA base pairs (from the more than 3 billion in the human genome) wrap around each histone core 1.7 times. The double helix moves on to spiral around the next core, and the next, with linker sections of 20 to 90 base pairs in between.

    The structure helps squeeze a 6-foot-long strand of DNA in each cell into as compact a form as possible while facilitating the controlled exposure of genes along the strand for protein expression.

    The spools consist of two pairs of heterodimers, macromolecules that join to form the core. The core is stable until genes along the DNA are called upon by transcription factors or RNA polymerases; the researchers’ goal was to simulate what happens as the DNA unwinds from the core, making itself available to bind to outside proteins or make contact with other genes along the strand.

    The researchers used their energy landscape models to simulate the nucleosome disassembly mechanism based on the energetic properties of its constituent DNA and proteins. The landscape maps the energies of all the possible forms a protein can take as it folds and functions. Conceptual insights from energy landscape theory have been implemented in an open-source biomolecular modeling framework called AWSEM Molecular Dynamics, which was jointly developed by the Papoian and Wolynes groups.

    Wolynes said most studies elsewhere treated the histone core as if it were rigid and irreversibly disassociated when DNA unwrapped. But more recent experimental studies that involved gently pulling strands of DNA or used fluorescent resonance energy transfer, which measures energy moving between two molecules, showed the protein core is flexible and does not completely disassemble during unwrapping.

    In their simulations, the researchers found the core changed its shape as the DNA unwound. Without DNA, they found the histone core was completely unstable in physiological conditions.

    Their simulations showed that histone tails – the terminal regions of the core proteins – play a crucial role in nucleosome stability. The tails are highly charged and bind tightly with DNA, keeping its genomic content from being exposed until necessary. Their models predicted a faster unwrapping for tail-less nucleosomes, as seen in experiments.

    The nucleosome study is part of a larger effort both by Papoian at Maryland and by Wolynes with his colleagues at CTBP to understand the mechanics of DNA, from how it functions to how it reproduces during mitosis. Wolynes said the new study and another new one by his lab on DNA during mitosis represent the opposite ends of the size scale.

    “We can understand things at each end of the scale, but there’s a no-man’s land in between,” he said. “We’ll have to see whether the phenomena in the present-day no-man’s land can be understood. I don’t believe in magic; I believe they eventually will.”

    Wolynes is the D.R. Bullard-Welch Foundation Professor of Science, a professor of chemistry, of biochemistry and cell biology, of physics and astronomy and of materials science and nanoengineering at Rice and a senior investigator of the National Science Foundation (NSF)-funded CTBP. Papoian is the Monroe Martin Professor and chemical physics director at the University of Maryland. Zhang will join the Massachusetts Institute of Technology as an assistant professor in July.

    The research was supported by the NSF, the CTBP and the National Institute of General Medical Sciences.

    The researchers used the NSF-supported DAVinCI supercomputer administered by Rice’s Ken Kennedy Institute for Information Technology.

    2
    IBM iDataPlex DAVinCI supercomputer

    See the full article here .

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    Rice U campus

    In his 1912 inaugural address, Rice University president Edgar Odell Lovett set forth an ambitious vision for a great research university in Houston, Texas; one dedicated to excellence across the range of human endeavor. With this bold beginning in mind, and with Rice’s centennial approaching, it is time to ask again what we aspire to in a dynamic and shrinking world in which education and the production of knowledge will play an even greater role. What shall our vision be for Rice as we prepare for its second century, and how ought we to advance over the next decade?

    This was the fundamental question posed in the Call to Conversation, a document released to the Rice community in summer 2005. The Call to Conversation asked us to reexamine many aspects of our enterprise, from our fundamental mission and aspirations to the manner in which we define and achieve excellence. It identified the pressures of a constantly changing and increasingly competitive landscape; it asked us to assess honestly Rice’s comparative strengths and weaknesses; and it called on us to define strategic priorities for the future, an effort that will be a focus of the next phase of this process.

     
  • richardmitnick 7:20 am on June 22, 2016 Permalink | Reply
    Tags: DNA studies, , , Second layer of information hidden in our DNA   

    From Science Alert: “Physicists confirm there’s a second layer of information hidden in our DNA 

    ScienceAlert

    Science Alert

    9 JUN 2016
    FIONA MACDONALD

    1
    Liya Graphics/Shutterstock.com

    Theoretical physicists have confirmed that it’s not just the information coded into our DNA that shapes who we are – it’s also the way DNA folds itself that controls which genes are expressed inside our bodies.

    That’s something biologists have known for years, and they’ve even been able to figure out some of the proteins responsible for folding up DNA. But now a group of physicists have been able to demonstrate for the first time through simulations how this hidden information controls our evolution.

    Let’s back up for a second here, because although it’s not necessarily news to many scientists, this second level of DNA information might not be something you’re familiar with.

    As you probably learnt in high school, Watson and Crick discovered in 1953 the double helix structure of DNA. Since then we’ve learnt that the DNA code that determines who we are is made up of a sequence of the letters G, A, C, and T.

    The order of these letters determines which proteins are made in our cells. So, if you have brown eyes, it’s because your DNA contains a particular series of letters that encodes for a protein that makes the dark pigment inside your iris.

    But that’s not the whole story, because all the cells in your body start out with the exact same DNA code, but every organ has a very different function – your stomach cells don’t need to produce the brown eye protein, but they do need to produce digestive enzymes. So how does that work?

    Since the ’80s, scientists have found that the way DNA is folded up inside our cells actually controls this process. Environmental factors can play a big role in this process too, with things like stress known to turn certain genes on and off through something known as epigenetics.

    But the mechanics of the DNA folding is an incredibly important control mechanism. That’s because every single cell in our body contains around 2 metres of DNA, so to fit inside us, it has to be tightly wrapped up into a bundle called a nucleosome – like a thread around a spool.

    And the way the DNA is wrapped up controls which genes are ‘read’ by the rest of the cell – genes that are all wrapped on the inside won’t be expressed as proteins, but those on the outside will. This explains why different cells have the same DNA but different functions.

    In recent years, biologists have even started to isolate the mechanical cues that determine the way DNA is folded, by ‘grabbing onto’ certain parts of the genetic code or changing the shape of the ‘spool’ the DNA is wrapped around.

    So far, so good, but what do theoretical physicists have to do with all this?

    A team from Leiden University in the Netherlands has now been able to step back and look at the process on a whole-genome scale, and back up through computer simulations that these mechanical cues are actually coded into our DNA.

    The physicists, led by Helmut Schiessel, did this by simulating the genomes of both baker’s yeast and fission yeast, and then randomly assigning them a second level of DNA information, complete with mechanical cues.

    They were able to show that these cues affected how the DNA was folded and which proteins are expressed – further evidence that the mechanics of DNA are written into our DNA, and they’re just as important in our evolution as the code itself.

    This means the researchers have shown that there’s more than one way that DNA mutations can affect us: by changing the letters in our DNA, or simply by changing the mechanical cues that arrange the way a strand is folded.

    “The mechanics of the DNA structure can change, resulting in different packaging and levels of DNA accessibility,” they explain, “and therefore differing frequency of production of that protein.”

    Again, this is simply backing up what many biologists already knew, but what’s really exciting from a purely speculative point of view is the fact that the computer simulations open up the possibility for scientists to model and maybe one day even manipulate the mechanical cues that shape our genetic code.

    There’s no evidence that we can do that just yet, but what we do know is that the more scientists understand about how our DNA is controlled and folded, the closer we get to being able to improve upon it.

    The research has been published in PLOS ONE.

    See the full article here .

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  • richardmitnick 8:09 am on June 14, 2016 Permalink | Reply
    Tags: , , DNA studies, ,   

    From NC State: “What Is CRISPR? And How Can it Be Used to Turn Genes ‘Off’?” 

    NC State bloc

    North Carolina State University

    1

    June 13, 2016
    Matt Shipman

    CRISPR systems have been a hot research topic since they were shown to have utility as genetic engineering tools in 2012. And they’re often explained in a way that most folks can understand. But those explanations often overlook key details – like the fact that scientists are still in the process of discovering the fundamental rules of how these systems work.

    For example, here’s a simplified explanation: CRISPR-Cas systems protect bacteria from invaders such as viruses. They do this by creating small strands of RNA that match DNA sequences specific to a given invader. When those CRISPR RNAs find a match, they unleash Cas proteins that chop up the invader’s DNA, preventing it from replicating.

    But of course it’s more complex than that. For example, there are six different types of CRISPR systems (that we know of). One of the most widely-studied CRISPR systems is CRISPR-Cas9, which is a Type II CRISPR system.

    But the most common CRISPR systems in nature are Type I. And new research from NC State is shedding light on some of the fundamental rules that govern Type I CRISPR systems – such as how long that CRISPR RNA can be, and how changing the length of the CRISPR RNA affects the behavior of the system.

    To learn more, we talked to Chase Beisel and Michelle Luo, who recently published a paper on the work in Nucleic Acids Research, in collaboration with two groups at Montana State University. Beisel is an assistant professor of chemical and biomolecular engineering at NC State; Luo is a Ph.D. student in Beisel’s lab.

    The Abstract: Why are Type I CRISPR systems of particular interest?
    Michelle Luo1
    Michelle Luo

    Michelle Luo: As you mentioned, Type I systems are the most common type of CRISPR-Cas systems. They account for over half of known systems. This is of particular interest as we look into co-opting an organism’s own system for other purposes. While CRISPR-Cas9 is undeniably a revolutionary genetic tool, it relies on importing this foreign Cas9 protein into an organism. This is a non-trivial task. However, if you use an organism’s own CRISPR-Cas proteins, as shown in our earlier work, you can avoid the challenges of expressing a non-natural protein. Because Type I systems are so prevalent, they offer a promising route to explore how a natural CRISPR-Cas system can be exploited for other means.

    The Abstract: In your recent work, you were evaluating how and whether you could modify the RNA in Type I CRISPR systems. Specifically, you were looking at whether you could modify the length of RNA in Type I CRISPR systems. Why would you want to change the length of the RNA?

    Luo: Two years ago, a number of papers were published detailing the crystal structures of Type I protein complexes that bind and help degrade target DNA. These publications hinted at the CRISPR RNA serving as a scaffold to assemble the different proteins in the complex. In other words, the RNA serves as a framework for these proteins to grab onto. Thus, we hypothesized that if we changed the length of the CRISPR RNA, we could change the size and composition of the Type I protein complex, and possibly the complex’s behavior.

    The Abstract: How, or why, might expanding the protein complexes used in DNA recognition be useful?
    Chase Beisel2
    Chase Beisel

    Chase Beisel: Going into the project, we didn’t know if the longer RNAs would allow the complex to even assemble, let alone function properly. We were surprised to find that the longer RNAs still formed a stable complex that could bind and direct the cutting of DNA. Because this complex is larger and recognizes a longer target sequence, we originally envisioned that the complex could be used for more specific DNA editing or for controlling gene expression.

    The Abstract: When I think of CRISPR, I think of a system that either leaves DNA alone or cuts it up. What do you mean when you say that changing the length of the RNA is more effective at gene repression?

    Luo: Your summary is on point. Normally, CRISPR-Cas systems survey the DNA landscape, and if they detects a target, they will cut up the DNA with tiny molecular scissors. If the target is not identified, the DNA will be left alone. Our earlier work demonstrated that we can prevent the cutting of the DNA by removing the scissors from the equation. We do this by deleting the cas3 gene from the genomic Type I locus. Now, instead of cutting the DNA, the CRISPR-Cas system simply binds the DNA. If we direct these modified systems to a gene, it will block the expression of that gene. Our most recent work shows that changing the length of the RNA can affect how strongly that silencing occurs. For certain regions, the longer the CRISPR RNA, the stronger the repression.

    The Abstract: Does that make the CRISPR system more specific? I.e., does it allow the system to be more targeted in terms of the DNA it “attacks”?

    Beisel: We wondered the same thing. We did in fact explore how longer RNAs impact specificity as part of the publication, although the results were mixed. On one hand, more of the RNA was involved in base pairing, where more base pairing would necessarily mean greater specificity. On the other hand, we found the longer RNAs were accommodating to mismatches with the target sequence, suggesting weaker specificity. In the end, more experiments will be needed to explore the question of specificity and how it impacts any downstream uses of Type I systems.

    The Abstract: How might that gene repression function be used? Are there any potential applications?

    Luo: Absolutely! This is particularly promising for metabolic engineering. If you want to make a microbial factory to produce a valuable product of interest, such as a biofuel, you have to alter the metabolism of an organism. This requires overexpressing genes that lead to production and turning off genes that compete with production. Our system allows researchers to turn off genes in a way that is potent, site-specific, reversible, and multiplexed. Our latest discovery suggests that you can fine-tune the extent of CRISPR-based gene repression simply by altering the length of the CRISPR RNA. That’s what our recent paper in Nucleic Acids Research is about.

    The Abstract: What are the future directions for this research?

    Beisel: Aside from the applications Michelle mentioned, we’re interested in why nature only uses RNA of a fixed length, given that longer RNAs make perfectly functional complexes. We’re also interested in whether this phenomenon applies across the many different flavors of Type I systems, from those that use far fewer proteins in the complex to those found in organisms living at extreme temperatures.

    See the full article here .

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    NC State campus

    NC State was founded with a purpose: to create economic, societal and intellectual prosperity for the people of North Carolina and the country. We began as a land-grant institution teaching the agricultural and mechanical arts. Today, we’re a pre-eminent research enterprise that excels in science, technology, engineering, math, design, the humanities and social sciences, textiles and veterinary medicine.

    NC State students, faculty and staff take problems in hand and work with industry, government and nonprofit partners to solve them. Our 34,000-plus high-performing students apply what they learn in the real world by conducting research, working in internships and co-ops, and performing acts of world-changing service. That experiential education ensures they leave here ready to lead the workforce, confident in the knowledge that NC State consistently rates as one of the best values in higher education.

     
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