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  • richardmitnick 8:49 am on April 9, 2016 Permalink | Reply
    Tags: , , DNA,   

    From AAAS: “Virus fighter may have played a key role in human evolution” 

    AAAS

    AAAS

    Apr. 7, 2016
    Elizabeth Pennisi

    1
    Some of the body’s antiviral proteins can mess with our DNA—for better or worse. iStock

    A virus-fighting protein in humans and other primates triggers an explosion in genetic mutations that may have sped up the evolution of our species, according to a new study.

    “In some sense, this is scary,” says Kelley Harris, a geneticist at Stanford University in Palo Alto, California, who was not involved with the work. Random mutations are often harmful. But there could be a silver lining: These changes also “provide raw material for evolution to happen” and that may enable individuals besieged by viruses to come up with better antiviral defenses, she says. “The paper doesn’t prove that it’s beneficial for humans to mutate their own DNA when they are infected by viruses, but it’s an interesting possibility.”

    Since the beginning of time, viruses have been inserting their genetic material into the genomes of their hosts, tricking the cell’s machinery into making more virus. Today, our genomes are riddled with these interlopers, called retroviruses and transposable elements, but many now just sit there, unable to generate additional copies of themselves. That’s because our bodies have a group of proteins that have mutated this DNA. These so-called APOBEC proteins seek out certain combinations of the letters that make up DNA (called bases), and, in DNA of viral origin, chemically convert the base cytosine into the base uracil—a change in the genetic alphabet from C to U that can disrupt a gene.

    In 2012, researchers discovered that certain APOBEC proteins do the same in some cancer cells. “You can see they are very active and affect the DNA in the tumor tremendously, causing lots of mutations that may further the cells’ uncontrolled growth, says Alon Keinan, a computational biologist at Cornell University. Because those cancer cells are part of the lungs, kidney, liver, or other organs, the mutations only affect those tissues. But if an APOBEC protein was active in germline cells—those destined to become eggs and sperm—then these mutations could possibly affect future generations, and ultimately alter the course of evolution.

    To see whether this has been the case with one APOBEC protein, APOBEC3G, Erez Levanon, a computational biologist at Bar-Ilan University in Ramat Gan, Israel, contacted Keinan, whose team specializes in comparing genomes to discern patterns of evolution. The group matched the genomes of a modern human, a Denisovan, a Neandertal, and a chimp up against genomes of a mouse, a rhesus macaque, and an orangutan to look for places in the human and chimp genomes with an unusual concentration of changes from a cytosine to another base. They focused only on changes along stretches of DNA with the APOBEC3G protein’s favorite sequence targets. For example, one such favorite is a series of three Cs in a row; the APOBEC3G protein frequently swaps out the third C for a different base.

    All together, the researchers found about 37,000 mutations* occurring in 10,000 clusters in the chimp and human genomes that they think were caused by these proteins, they report today in Genome Research. These mutations were Cs in the orangutan, macaque, and mouse, but a different base in each of the other four species. Many of the clusters were located in key places in the genome, such as regions important for regulating gene activity or protein-coding parts of genes. For example, more than 33% of the base changes they found in coding regions also alter its protein product. Many other changes likely happened in the germlines during the evolution of these species, detrimental ones likely disappeared, whereas those that provided some survival benefit persisted. “It shows that this primate-specific antiviral mechanism also led to the shaping of our and our relatives’ genomes,” Keinan says.

    “It’s surprising to see this impact on all these primate genomes,” says Jeffrey Kidd, a geneticist at the University of Michigan, Ann Arbor, who was not involved with the work. “It makes us realize that nothing comes for free,” and the trade-off of having a mechanism to thwart viral DNA is disruptions in our own DNA, he says. “It raises the question of how that balance is worked out.”

    There are related proteins that may likewise cause mutations; “this might just be the tip of the iceberg,” Keinan says. He and his colleagues are now calculating what percentage of the genetic changes that made us human were caused by APOBEC proteins. Typically, a newborn is expected to have 70 new mutations in its genome, but just one of these proteins “can introduce potentially thousands [of them]” in close proximity and in one generation, Keinan says.

    And that’s a lot of new material for evolution to work on.

    Science Paper:
    Clustered mutations in hominid genome evolution are consistent with APOBEC3G enzymatic activity

    Science team:
    Yishay Pinto1,3; Orshay Gabay 1,3; Leonardo Arbiza 2; Aaron J. Sams 2; Alon Keinan 2,4; and Erez Y. Levanon 1,4

    Author Affiliations

    1 Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel;
    2 Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, New York 14853, USA

    3,4 not reported

    Corresponding authors: erez.levanon@biu.ac.il, alon.keinan@cornell.edu

    See the full article here .

    The American Association for the Advancement of Science is an international non-profit organization dedicated to advancing science for the benefit of all people.

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  • richardmitnick 5:16 pm on March 23, 2016 Permalink | Reply
    Tags: , DNA, ,   

    From LBL: “Unlocking the Secrets of Gene Expression” 

    Berkeley Logo

    Berkeley Lab

    March 23, 2016
    Julie Chao (510) 486-6491
    JHChao@lbl.gov

    Your DNA governs more than just what color your eyes are and whether you can curl your tongue. Your genes contain instructions for making all your proteins, which your cells constantly need to keep you alive. But some key aspects of how that process works at the molecular level have been a bit of a mystery—until now.

    Using cryo-electron microscopy (cryo-EM), Lawrence Berkeley National Laboratory (Berkeley Lab) scientist Eva Nogales and her team have made a significant breakthrough in our understanding of how our molecular machinery finds the right DNA to copy, showing with unprecedented detail the role of a powerhouse transcription factor known as TFIID.

    1
    Berkeley Lab scientists Eva Nogales and Robert Louder at the electron microscope. (Credit: Roy Kaltschmidt/Berkeley Lab)

    This finding is important as it paves the way for scientists to understand and treat a host of malignancies. “Understanding this regulatory process in the cell is the only way to manipulate it or fix it when it goes bad,” said Nogales. “Gene expression is at the heart of many essential biological processes, from embryonic development to cancer. One day we’ll be able to manipulate these fundamental mechanisms, either to correct for expression of genes that should or should not be present or to take care of malignant states where the process has gone out of control.”

    Their study has been published online in the journal Nature in an article titled, Structure of promoter-bound TFIID and insight into human PIC assembly. The lead author is Robert Louder, a biophysics graduate student in Nogales’ lab, and other authors are Yuan He, José Ramón López-Blanco, Jie Fang, and Pablo Chacón.

    Nogales, a biophysicist who also has appointments at Howard Hughes Medical Institute and UC Berkeley, has been studying gene expression for 18 years. While she and her team have made several significant findings in recent years, she calls this the biggest breakthrough so far. “This is something that will go in biochemistry textbooks,” she said. “We now have the structure of the whole protein organization that is formed at the beginning of every gene. This is something no one has come close to doing because it is really very difficult to study by traditional methodologies.”

    2
    Cryo-EM model of the human transcription pre-initiation complex. (Credit: Robert Louder/Berkeley Lab )

    How genetic information flows in living organisms is referred to as the “central dogma of molecular biology.” Cells are constantly turning genes on and off in response to what’s happening in their environment, and to do that, the cell uses its DNA, the big library of genetic blueprints, finds the correct section, and makes a copy in the form of messenger RNA; the mRNA is then used to produce the needed protein.

    The problem with this “library” is that it has no page numbers or table of contents. However, markers are present in the form of specific DNA sequences (called core promoter motifs) to indicate where a gene starts and ends. So how does the polymerase, the enzyme that carries out the transcription, know where to start? “DNA is a huge, huge molecule. Out of this soup, you have to find where this gene starts, so the polymerase knows where to start copying,” Nogales said. “This transcription factor, TFIID, is the protein complex that does exactly that, by recognizing and binding to DNA core promoter regions.”

    What Nogales and her team have been able to do is to visualize, with unprecedented detail, TFIID bound to DNA as it recognizes the start, or promoter, region of a gene. They have also found how it serves as a sort of landing pad for all the molecular machinery that needs to assemble at this position—this is called the transcription pre-initiation complex (PIC). This PIC ultimately positions the polymerase so it can start transcribing.

    3
    TFIID (blue) as it contacts the DNA and recruits the polymerase (grey) for gene transcription. The start of the gene is shown with a flash of light. (Credit: Eva Nogales/Berkeley Lab)

    “TFIID has to do not only the binding of the DNA, recruitment, and serving as landing pad, it has to somehow do all that differently for different genes at any given point in the life of the organism,” Nogales said.

    Added Louder: “We have generated the first ever structural model of the full human TFIID-based PIC. Our model yields novel insights into human PIC assembly, including the role of TFIID in recruiting other components of the PIC to the promoter DNA and how the long observed conformational flexibility of TFIID plays a role in the regulation of transcription initiation.”

    Proteins have traditionally been studied using X-ray crystallography, but that technique has not been possible for this kind of research. “TFIID has not been accessible to protein crystallography because there’s not enough material to crystallize it, it has very flexible elements, and it is of a huge size,” Nogales said. “All of those things we can overcome through cryo-EM.”

    Cryo-EM, in which samples are imaged at cryogenic temperatures without need for dyes or fixatives, has been used since the 1980s in structural biology. With extensive computational analysis of the images researchers are able to obtain three-dimensional structures. However, cryo-EM has undergone a revolution in the last few years with the advent of new detectors—developed, in fact, at Berkeley Lab—that improve resolution and reduce the amount of data needed by up to a hundred-fold.

    “Many biological systems we had thought were impossible to study at high resolution have become accessible,” she said. “Now the resolution allows us to get atomic details. This is an area in which Berkeley Lab has been one of the leaders.”

    While this study has revealed important new insights into gene expression, Nogales notes that much work remains to be done. Next she plans to investigate how TFIID is able to recognize different sequences for different gene types and also how it is regulated by cofactors and activators.

    “We are just at the beginning,” she said. “This complex, TFIID, is very, very critical. Now we have broken barriers in the sense that we can start generating atomic models and get into details of how DNA is being bound.”

    This research was supported by the National Institutes of Health’s National Institute of General Medical Sciences and by the Spanish Ministry of Economy and Competitiveness. Computational work was carried out at the National Energy Research Scientific Computing Center (NERSC), a DOE Office of Science User Facility hosted at Berkeley Lab. Nogales is a Senior Faculty Scientist in Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging Division; additional information on her lab can be found here.

    See the full article here .

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

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  • richardmitnick 6:50 am on February 6, 2016 Permalink | Reply
    Tags: , , DNA, SPIE   

    From SPIE: “Tracking DNA damage with electrochemical sensing” 

    SPIE

    SPIE

    2.6.16
    Jason D. Slinker
    The University of Texas at Dallas
    Richardson, TX

    DNA, the fundamental biomolecule of life, is constantly subject to damage that threatens the vitality of cells and the integrity of the genome. Without enzymatic intervention, this damage can produce mutations that lead to cancerous tumors. Furthermore, many current and developing treatments of cancer and disease rely on the generation of DNA damage products, which—from a chemical standpoint—are very subtle. For example, 8-oxoguanine, the most prevalent oxidative DNA damage product, involves the addition of a single oxygen bond to a guanine base. Remarkably, enzymes in cells recognize and remove this damage and other products of degradation. Biological assays that follow repair of this subtle DNA damage assist cancer studies by advancing fundamental understanding of DNA-protein interactions, connecting damage to diagnosis, and informing options for treatment.

    We have demonstrated devices that follow DNA damage repair in real time, with a convenient, low-cost package (see Figure 1).1 In this device, DNA is bound to the circular electrodes of multielectrode chips, and a redox probe at the top of the DNA reports charge transfer through it. DNA is the natural recognition element not only for the binding of repair proteins but also for their repair activity, and it can be synthesized with or without damage/lesion sites to establish controls. Furthermore, DNA can also serve as an electrical transducing element when modified with a redox-active probe and self-assembled on a working electrode, as first demonstrated by the Barton group.2 We have combined these features of DNA, using them to form devices capable of selectively detecting oxidative DNA damage repair (see Figure 1) and changes in DNA stability.1 The devices give a direct measure of molecular-level repair, providing a window into intracellular DNA repair by DNA-binding proteins.

    DNA Device
    Figure 1. Top: Schematic of detection of oxidative damage removal. Bottom: Image of the device used to study DNA-damaging drugs. (Photo by Randy Anderson). FPG: Formamidopyrimidine DNA glycosylase. e-: Electron.

    Specifically, we have used our approach to show sensitive and selective electrochemical sensing of 8-oxoguanine and uracil repair glycosylase activity. We produced sensors on electrospun fibers as low-cost devices with improved dynamic range. Our experiments compared electroactive, probe-modified DNA monolayers containing a base defect with the rational control of defect-free monolayers. We found damage-specific limits of detection on the order of femtomoles of proteins, corresponding to mere nanograms of the enzymes. The DNA chips enabled the real-time observation of protein activity, and we observed base excision activity on the order of seconds. We also demonstrated damage-specific detection in a mixture of enzymes and in response to environmental oxidative damage. We showed how nanofibers may behave similarly to conventional gold-on-silicon devices, revealing the potential of these low-cost devices for sensing applications. This device approach enables sensitive, selective, and rapid assay of repair protein activity, allowing biological interrogation of DNA damage repair.

    Given the ability of these devices to follow induced oxidative damage, we are further using them to follow DNA-damaging anticancer drug activity. We are working with the group of David Boothman of the University of Texas Southwestern Medical Center to sense DNA repair activity in conjunction with a novel drug therapy that selectively produces oxidative damage of DNA in cancer cells, bringing about selective cancer cell death. We represent key features of a living system to reproduce DNA damaging and repair activity pathways on the chip. Recent results have shown that we can follow specific drug-induced DNA damage excision and subsequent DNA repair with our devices. Furthermore, the multiple electrodes of the chip allowed us to perform controls of each associated enzyme and to obtain high statistical confidence of results. Given this success, we have launched studies of other DNA damaging drugs to explore the generality of this technique.

    In summary, we have designed and fabricated low-cost devices that are capable of electrochemical sensing of 8-oxoguanine and uracil repair glycosylase activity. Ultimately, in addition to their utility in bioassays of DNA-protein interactions, our devices have potential in a number of applications for public health, and our future work will focus on realizing these. The prevalence of high damage repair sites can be an indication of cancers and disease states, and these devices could provide statistically significant diagnosis. Additionally, as a number of cancer treatments involve DNA-damaging agents, our devices can be used to improve treatment outcomes. These devices could be used to sample the activity of multiple drugs with a small volume patient sample, enabling a tailored treatment based on DNA-damaging effectiveness. Similarly, they may also be used to follow the course of cancer treatment through characteristic measures of enzymatic activity of cancer cells versus healthy cells.

    See the full article here.

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  • richardmitnick 2:07 am on January 23, 2016 Permalink | Reply
    Tags: , DNA,   

    From Vanderbilt: “Faulty building blocks in DNA” 

    Vanderbilt U Bloc

    Vanderbilt University

    Jan. 22, 2016
    Bill Snyder

    Temp 1
    (iStock)

    Enzymes called DNA polymerases assemble DNA from 2´-deoxyribonucleoside triphosphate building blocks in the cell. Normally they can distinguish DNA building blocks from the ribonucleotides that make up RNA, but sometimes they misinsert ribonucleotides into DNA, generating “DNA lesions.”

    Yan Su, Ph.D., Martin Egli, Ph.D., and F. Peter Guengerich, Ph.D., have provided an important glimpse into how this happens. They studied human DNA polymerase eta (hpol-eta), which is directly related to a human genetic disorder, xeroderma pigmentosum, associated with an increased risk of skin and other cancers.

    In a paper published online this month by the Journal of Biological Chemistry, they show that hpol-eta can incorporate ribonucleotides into DNA with relatively high selectivity but low efficiency. They also crystallized the enzyme and obtained what appears to be the first crystal structure of an incoming ribonucleotide opposite a DNA lesion within a DNA polymerase. Based on these findings, “it is highly possible that hpol-eta inserts a considerable amount of ribonucleotides into DNA,” they conclude.

    This work was supported by National Institutes of Health.

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  • richardmitnick 2:56 pm on January 6, 2016 Permalink | Reply
    Tags: , , CRISPR gene editing, DNA   

    From AAAS: “Researchers rein in slice-happy gene editor, CRISPR” 

    AAAS

    AAAS

    6 January 2016
    Kelly Servick

    Temp 1
    Adapted from H. Nishimasu et al., Cell, 156, 5 (2014); Wikimedia/Creative Commons

    Changes to the DNA-cutting enzyme Cas9 make CRISPR more precise.

    Keith Joung remembers the first time he took CRISPR for a spin. In late 2012, the pathologist at Massachusetts General Hospital in Boston assembled the components of the new gene-editing technology and fiddled with the DNA of a zebrafish embryo. “It was so easy to do,” he says. “It was just stunning.”

    CRISPR—the highly efficient set of molecular scissors recently selected as Science’s Breakthrough of the Year—might be easy to use, but it’s not perfect. Joung and his colleagues soon found that these scissors could get too slice-happy, cutting DNA in unexpected and unwanted locations. In early experiments, the group observed that these off-target effects could occur at some DNA sites with nearly the same frequency as the desired edits. That’s a problem if CRISPR is to form the basis of human therapies, for example, repairing the defective genes that cause muscular dystrophy or hereditary liver disease. Researchers’ primary concern is that cutting into an unwanted gene could cause uncontrolled growth and cancer.

    Now, Joung and colleagues have found a way to make CRISPR more precise. In a new study, they modified its cutting enzyme to reduce off-target effects below detectable levels.

    “I think that this is a potential breakthrough,” says Jin-Soo Kim, a molecular biologist at Seoul National University who was not involved with the work. But the quest to perfect CRISPR doesn’t have a clear end. “No drugs are free of off-target effects,” he notes. With CRISPR-based therapies still far from human testing, no one knows just how precise is precise enough.

    CRISPR relies on a DNA-cutting enzyme called Cas9 attached to a short strand of RNA that guides it to specific point in the genome. When the RNA finds a complementary—or nearly complementary—sequence, Cas9 makes its slice. There are already several approaches to prevent unintended slicing. Shortening the length of the guide RNA makes it more sensitive to mismatched sequences, but it can also create entirely new off-target effects. Some labs have experimented with a version of Cas9 that cuts through a single DNA strand instead of two. That means two Cas9 enzymes bearing two different guide RNAs have to recognize their target sequences to cut both strands—a more demanding matching process. But doubling the number of RNA guides adds bulk, which could make it harder to deliver a CRISPR-based treatment into cells.

    In the new work, published online today in Nature, Joung and colleagues took a different approach. They modified the Cas9 enzyme itself to change the way it interacts with DNA. They first altered some of the “residues” on the enzyme’s surface that presumably help the guide RNA pair with its matching DNA strand. One set of modifications created a new variant of Cas9, called Cas9-HF1, that appears to be much more discriminating in its cuts. The researchers made seven different edits guided by seven different RNA strands, each known to produce off-target effects with Cas9. But Cas9-HF1 showed no detectable off-target effects in six of these cases—and just one errant slice in the seventh, they report. Joung adds that the apparent slice could actually be the result of a sequencing error.

    The results come on the heels of a similar feat, led by CRISPR pioneer Feng Zhang of Harvard University and the Broad Institute in Cambridge, Massachusetts, published last month in Science. That team modified Cas9 to change how it interacts with a different part of a cell’s DNA. It, too, dramatically improved CRISPR’s specificity. But it’s hard to compare those results directly with the new paper because they used slightly different methods to measure off-target effects.

    Joung claims his group’s measurements are roughly 10-fold more sensitive than the one used in the Science paper. Both studies rely on methods that attach molecular tags to all points in the genome where a double-stranded break has occurred, before sequencing the short, flagged segments to count the cuts in various genes. Joung’s team claims to detect edits that occur in at least 0.1% of the genome. Zhang says the method used in his paper has been validated down 0.3%, and it may be even more sensitive.

    Does detecting just a couple of faulty cuts in a thousand matter? Absolutely, Joung says. “A lot of therapeutic strategies envision manipulating millions, tens of millions, even hundreds of millions of cells, potentially. So one in 1000 sounds pretty good, but that number can become quite large.” He argues that the field needs tests that root out these potentially harmful effects at frequencies of 0.01% or even lower.

    Others are less focused on increasingly sensitive tests. Because CRISPR will never fully be rid of off-target effects, the key question for a given therapy is not strictly how many unwanted cuts it makes, but whether it disrupts any essential genes, says Jiing-Kuan Yee, a molecular biologist at the research center City of Hope in Duarte, California. Each therapeutic application will require its own carefully selected Cas9 molecule—and modifications like those in the two recent papers might be combined.

    “Pretty soon, I think everybody’s going to start using these modified Cas9s,” he says. “The [off-target] problem will still be there, but it’s going to be much, much reduced.”

    See the full article here .

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  • richardmitnick 5:14 pm on December 28, 2015 Permalink | Reply
    Tags: , DNA, ,   

    From NOVA: “The Man Who Rewrote the Tree of Life” 2014 but Interesting and Important 

    PBS NOVA

    NOVA

    30 Apr 2014
    Carrie Arnold

    Carl Woese may be the greatest scientist you’ve never heard of. “Woese is to biology what [Albert] Einstein is to physics,” says Norman Pace, a microbiologist at the University of Colorado, Boulder. A physicist-turned-microbiologist, Woese specialized in the fundamental molecules of life—nucleic acids—but his ambitions were hardly microscopic. He wanted to create a family tree of all life on Earth.

    Woese certainly wasn’t the first person with this ambition. The desire to classify every living thing is ageless. The Ancient Greeks and Romans worked to develop a system of classifying life. The Jewish people, in writing the Book of Genesis, set Adam to the task of naming all the animals in the Garden of Eden. And in the mid-1700s, Swedish botanist Carl von Linné published Systema Naturae, introducing the world to a system of Latin binomials—Genus species—that scientists use to this day.

    Temp 1
    Carl Woese in his later years. Photo credits: Jason Lindsey/University of Illinois, Tim Bocek/Flickr (CC BY-NC-SA)

    What Woese was proposing wasn’t to replace Linnaean classification, but to refine it. During the late 1960s, when Woese first started thinking about this problem as a young professor at the University of Illinois, biologists were relying a lot on guesswork to determine how organisms were related to each other, especially microbes. At the time, researchers used the shapes of microbes—their morphologies—and how they turned food into energy—their metabolisms—to sort them into bins. Woese was underwhelmed. To him, the morphology-metabolism approach was like trying to create a genealogical history using only photographs and drawings. Are people with dimples on their right cheeks and long ring fingers all members of the same family? Maybe, but probably not.

    “If you wanted to build a tree of life prior to what Woese did, there was no way to put something together that was based upon actual data,” says Jonathan Eisen, an evolutionary microbiologist at the University of California Davis.

    Just as outward appearances aren’t the best way to determine family relations, Woese believed that morphology and metabolism were inadequate classifiers for life on Earth. Instead, he figured that DNA could sketch a much more accurate picture. Today, that approach may seem like common sense. But in the late 60s and early 70s, this was no easy task. Gene sequencing was a time-consuming, tedious task. Entire PhDs were granted for sequencing just one gene. To create his tree of life, Woese would need to sequence the same gene in hundreds, if not thousands, of different species.

    So Woese toiled in his lab, sometimes with his postdoc George Fox but often alone, hunched over a light box with a magnifying glass, sequencing genes nucleotide by nucleotide. It took more than a decade. “When Woese first announced his results, I thought he was exaggerating at first,” Fox recalls. “Carl liked to think big, and I thought this was just another of his crazy ideas. But then I looked at the data and the enormity of what we had discovered hit me.”

    Woese and Fox published their results in 1977 in a well-respected journal, the Proceedings of the National Academy of Science. They had essentially rewritten the tree of life. But Woese still had a problem: few scientists believed him. He would spend the rest of his life working to convince the biological community that his work was correct.

    Animal, Vegetable, Mineral

    Following the publication of Linnaeus’s treatise in the 18th century, taxonomy progressed incrementally. The Swedish botanist had originally sorted things into three “kingdoms” of the natural world: animal, vegetable, and mineral. He placed organisms in their appropriate cubbyholes by looking at similarities in appearance. Plants with the same number of pollen-producing stamens were all lumped together, animals with the same number of teeth per jaw were grouped, and so on. With no knowledge of evolution and natural selection, he didn’t have a better way to comprehend the genealogy of life on Earth.

    The publication of [Charles]Darwin’s On the Origin of Species in 1859, combined with advances in microscopy, forced scientists to revise Linnaeus’s original three kingdoms to include the tiniest critters, including newly visible ones like amoebae and E. coli. Scientists wrestled with how to integrate microbial wildlife into the tree of life for the next 100 years. By the mid-20th century, however, biologists and taxonomists had mostly settled on a tree with five major branches: protists, fungi, plants, animals, and bacteria. It’s the classification system that many people learned in high school biology class.

    Woese and other biologists weren’t convinced, though. Originally a physics major at Amherst College in Massachusetts and having received a PhD in biophysics from Yale in 1953, Woese believed that there had to be a more objective, data-driven way to classify life. Woese was particularly interested in how microbes fit into the classification of life, which had escaped a rigorous genealogy up until that point.

    He arrived at the University of Illinois Urbana-Champaign as a microbiologist in the mid-1960s, shortly after James Watson and Francis Crick won the Nobel prize for their characterization of DNA’s double-helix form. It was the heyday of DNA. Woese was enthralled. He believed that DNA could unlock the hidden relationships between different organisms. In 1969, Woese wrote a letter to Crick, stating that:

    ” …this can be done by using the cell’s ‘internal fossil record’—i.e., the primary structures of various genes. Therefore, what I want to do is to determine primary structures for a number of genes in a very diverse group of organisms, on the hope that by deducing rather ancient ancestor sequences for these genes, one will eventually be in the position of being able to see features of the cell’s evolution….”

    This type of thinking was “radically new,” says Norman Pace, a microbiologist at the University of Colorado, Boulder. “No one else was thinking in this direction at the time, to look for sequence-based evidence of life’s diversity.”

    Evolution’s Timekeeper

    Although the field of genetics was still quite young, biologists had already figured out some of the basics of how evolution worked at the molecular level. When a cell copies its DNA before dividing in two, the copies aren’t perfectly identical. Mistakes inevitably creep in. Over time, this can lead to significant changes in the sequence of nucleotides and the proteins they code for. By finding genes with sites that mutate at a known rate—say 4 mutations per site per million years—scientists could use them as an evolutionary clock that would give biologists an idea of how much time had passed since two species last shared a common ancestor.

    To create his evolutionary tree of life, then, Woese would need to choose a gene that was present in every known organism, one that was copied from generation to generation with a high degree of precision and mutated very slowly, so he would be able to track it over billions of years of evolution.

    “This would let him make a direct measure of evolutionary history,” Pace says. “By tracking these gene sequences over time, he could calculate the evolutionary distance between two organisms and make a map of how life on Earth may have evolved.”

    Some of the most ancient genes are those coding for molecules known as ribosomal RNAs. In ribosomes, parts of the cell that float around the soupy cytoplasm, proteins and ribosomal RNA, or rRNA, work together to crank out proteins. Each ribosome is composed of large and small subunits, which are similar in both simple, single-celled prokaryotes and more complex eukaryotes. Woese had several different rRNA molecules to choose from in the various subunits, which are classified based on their length. At around 120 nucleotides long, 5S rRNA wasn’t big enough to use to compare lots of different organisms. On the other end of the spectrum, 23S rRNA was more than 2300 nucleotides long, making it far too difficult for Woese to sequence using the technologies of the time. The Goldilocks molecule—long enough to allow for meaningful comparisons but not too long and difficult to sequence—was 16S rRNA in prokaryotes and its slightly longer eukaryotic equivalent, 18S rRNA. Woese decided to use these to create his quantitative tree of life.

    His choice was especially fortuitous, Eisen says, because of several factors inherent in 16S rRNA that Woese couldn’t have been aware of at the time, including its ability to measure evolutionary time on several different time scales. Certain parts of the 16S rRNA molecule mutate at different speeds. Changes to 16S rRNA are, on the whole, still extremely slow (humans share about 50% of their 16S rRNA sequence with the bacterium E. coli), but one portion mutates much more slowly than the other. It’s as if the 16S rRNA clock has both an hour hand and a minute hand. The very slowly evolving “hour hand” lets biologists study the long-term changes to the molecule, whereas the more quickly evolving “minute hand” provides a more recent history. “This gives this gene an advantage because it lets use ask questions about deep evolutionary history and more recent history at the same time,” Eisen says.

    Letter by letter

    Selecting the gene was just Woese’s first challenge. Now he had to sequence it in a variety of different organisms. In the late 60s and early 70s, when Woese began his work, DNA sequencing was far from automated. Everything, down to the last nucleotide, had to be done by hand. Woese used a method to catalog short pieces of RNA developed in 1965 by British scientist Frederick Sanger, which used enzymes to chop RNA into small pieces. These small pieces were sequenced, and then scientists had to reassemble the overlapping pieces to determine the overall sequence of the entire molecule—a process that was tedious, expensive, and time-consuming, but that was seen as a minor annoyance to a workhorse like Woese, Fox says. “All he cared about was getting the answer.”

    Woese started with prokaryotes, the single-celled organisms that were his primary area of interest. He and his lab started by growing bacteria in a solution of radioactive phosphate, which the cells incorporated into backbones of their RNA molecules. This made the 16S rRNA radioactive. Then, Woese and Fox extracted the RNA from the cells and chopped it into smaller pieces using enzymes that acted like scissors. The enzymatic scissors would only cut at certain sequences. If a sequence was present in one organism but missing in a second, the scissors would pass over the second one’s sequence. Its fragment would be longer.

    Since RNA’s sugar-phosphate backbone is negatively charged, the researchers could use a process known as electrophoresis to separate the different length pieces. As electricity coursed through gels containing samples, it pulled the smaller, lighter bits farther through the gels than the longer, heavier chunks. The result was distinct bands of different lengths of RNA. Woese and Fox then exposed each gel to photographic paper over several days. The radioactive bands in the gel transferred marks to the paper. This created a Piet Mondrian-esque masterpiece of black bands on a white background. Each different organism left its own mark. “To Carl, each spot was a puzzle that he would solve,” Fox says.

    After developing each image, Woese and Fox returned to the gel and neatly cut out each individual blotch that contained fragments of a certain length. They then chopped up these fragments with another set of enzymes until they were about five to 15 nucleotides long, a length that made sequencing easier. For some of the longer fragments, it took several iterations of the process before they were successfully sequenced. The sequences were then recorded on a set of 80-column IBM punch cards. The cards were then run through a large computer to compare band patterns and RNA sequences among different organisms to determine evolutionary relationships. At the beginning, it took Woese and Fox months to obtain a single 16S rRNA fingerprint.

    “This process was a huge breakthrough,” says Peter Moore, an RNA chemist at Yale University who worked with Woese on other research relating to RNA’s structure. “It gave biologists a tool for sorting through microorganisms and giving them a conceptual way to understand the relationship between them. At the time, the field was just a total disaster area. Nobody knew what the hell was going on.”

    RNA is so fundamental to life that some scientists think it’s the spark that started it all. To learn more about RNA, visit NOVA’s RNA Lab.

    By the spring of 1976, Woese and Fox had created fingerprints of a variety of bacterial species when they turned to an oddball group of prokaryotes: methanogens. These microbes produce methane when they break down food for energy. Because even tiny amounts of oxygen are toxic to these prokaryotes, Woese and Fox had to grow them under special conditions.

    After months of trial and error, the two scientists were finally able to obtain an RNA fingerprint of one type of methanogen. When they finally analyzed its fingerprint, however, it looked nothing like any of the other bacteria Woese and Fox had previously analyzed. All of the previous bacterial gels contained two large splotches at the bottom. They were entirely absent from these new gels. Woese knew instantly what this meant.

    To fellow microbiologist Ralph Wolfe, who worked in the lab next door, Woese announced, “I don’t even think these are bacteria, Wolfe.”

    He dropped the full bombshell on Fox. “The methanogens didn’t have any of the spots he was expecting to see. When he realized this wasn’t a mistake, he just went nuts. He ran into my lab and told me we had discovered a new form of life,” Fox recalls.

    The New Kingdom

    The methanogens Woese and Fox had analyzed looked superficially like other bacteria, yet their RNA told a different story, sharing more in common with nucleus-containing eukaryotes than with other bacteria. After more analysis of his RNA data, Woese concluded that what he was tentatively calling Archaea (from Latin, meaning primitive) wasn’t a minor twig on the tree of life, but a new main branch. It wasn’t just Bacteria and Eukarya any more .

    To prove to their critics that these prokaryotes really were a separate domain on the tree of life, Woese and Fox knew the branch needed more than just methanogens. Fox knew enough about methanogen biology to know that their unique RNA fingerprint wasn’t the only thing that made them strange. For one thing, their cell walls lacked a mesh-like outer layer made of peptidoglycan. Nearly every other bacterium Fox could think of contained peptidoglycan in its cell wall—until he recalled a strange fact he had learned as a graduate student—another group of prokaryotes, the salt-loving halophiles, also lacked peptidoglycan.

    2
    Grand Prismatic Spring in Yellowstone National Park is home to many species of thermophilic archaea.

    Fox turned to the research literature to search for other references to prokaryotes that lack peptidoglycan. He found two additional examples: Thermoplasma and Sulfolobus. Other than the missing peptidoglycan, these organisms and the methanogens seemed nothing alike. Methanogens were found everywhere from wetlands to the digestive tracts, halophiles flourished in salt, Thermoplasma liked things really hot, and Sulfolobus are often found in volcanoes and hot, acidic springs.

    Despite their apparent differences, they all metabolized food in the same, unusual way—unlike anything seen in other bacteria—and the fats in the cell membrane were alike, too. When Woese and Fox sequenced the 16S rRNA of these organisms, they found that these prokaryotes were most similar to the methanogens.

    “Once we had the fingerprints, it all fell together,” Fox says.

    Woese believed his findings were going to revolutionize biology, so he organized a press conference when the paper was published in PNAS in 1977. It landed Woese on the front page of the New York Times, and created animosity among many biologists. “The write-ups were ludicrous and the reporters got it all wrong,” Wolfe says. “No biologists wanted anything to do with him.”

    It wasn’t just distaste for what looked like a publicity stunt that was working against Woese. He had spent most of the last decade holed up in his third floor lab, poring over RNA fingerprints. His reclusive nature had given him the reputation of a crank. It also didn’t help that he had single-handedly demoted many biologists’ favorite species. Thanks to Woese, Wolfe says, “Microbes occupy nearly all of the tree. Then you have one branch at the very end where all the animals and plants were. And the biologists just couldn’t believe that all the plants and all the animals were really just one tiny twig on one branch.”

    Although some specialists were quick to adopt Woese’s new scheme, the rest of biology remained openly hostile to the idea. It wasn’t until the mid-1980s that other microbiologists began to warm to the idea, and it took well over another decade for other areas of biology to follow suit. Woese had grown increasingly bitter that so many other scientists were so quick to reject his claims. He knew his research and ideas were solid. But he was left to respond to what seemed like an endless stream of criticism. Shying from these attacks, Woese retreated to his office for the next two decades.

    “He was a brash, iconoclastic outsider, and his message did not go down well,” says Moore, the Yale RNA chemist.

    Woese’s cause wasn’t helped by his inability to engage critics in dialogue and discussion. Both reticent and abrupt, he preferred his lab over conferences and presentations. In place of public appearances to address his detractors, he sent salvos of op-eds and letters to the editor. Still, nothing seemed to help. The task of publicly supporting this new tree of life fell to Woese’s close colleagues, especially Norman Pace.

    But as technology improved, scientists began to obtain the sequences of an increasing number of 16S rRNAs from different organisms. More and more of their analyses supported Woese’s hypothesis. As sequencing data poured in from around the world, it became clear to nearly everyone in biology that Woese’s initial tree was, in fact, been correct.

    Now, when scientists try to discover unknown microbial species, the first gene they sequence is 16S rRNA. “It’s become one of the fundamentals of biology,” Wolfe says. “After more than 20 years, Woese was finally vindicated.”

    Woese died on December 30, 2012, at the age of 84 of complications from pancreatic cancer. At the time of his death, he had won some of biology’s most prestigious awards and had become one of the field’s most respected scientists. Thanks to Woese’s legacy, we now know that most of the world’s biodiversity is hidden from view, among the tiny microbes that live unseen in and around us, and in them, the story of how life first evolved on this planet.

    See the full article here .

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  • richardmitnick 6:27 am on October 27, 2015 Permalink | Reply
    Tags: , DNA,   

    From MIT: “Mapping the 3-D structure of DNA” 


    MIT News

    October 26, 2015
    Julia Sklar

    1
    Abe Weintraub Photo: M. Scott Brauer

    PhD student Abe Weintraub helps identify when DNA folding is helpful, and when it might cause cancer.

    For graduate student Abe Weintraub, the magic and intrigue of DNA is all in the packaging.

    Imagine trying to fit 24 miles of string into a tennis ball, the PhD student in biology says: That is, in essence, what it’s like inside every cell nucleus in the human body, each of which contains about 2 meters’ worth of DNA strands. But, as Weintraub is finding, this packaging sometimes goes awry, which may be the basis for disease.

    Although the genetic code that resides in DNA has traditionally been thought of as linear, Weintraub is contributing to a body of knowledge about its 3-D organization. Two genes that may exist far apart when a strand is stretched out straight could actually be right next to each other when the strand is folded inside a cell nucleus — and the same applies to regulatory elements, which tell genes to turn on or off.

    Looking at DNA as a 3-D phenomenon may yield insights about how certain genes get turned on or off, and thus how cells differentiate — in other words, DNA’s 3-D structure might actually be what’s behind one cell becoming a skin cell, while another becomes a lung cell.

    Weintraub has now been part of the lab of Richard Young, a professor of biology, for one and a half years; his research began in figuring out how DNA gets folded up the way it does, and has more recently shifted to the consequences of improper folding.

    DNA gets packed tightly in organized loops, rather than being haphazardly crammed into cell nuclei. Weintraub helped find what causes this ordered looping.

    “When we zoomed out, we could see that what creates the particular 3-D structure of DNA are basically large loops, constraining small loops. Science can be pretty meta,” Weintraub jokes.

    This looping system looks fairly consistent across cells, and is how the same genes and regulatory elements end up adjacent to each other in every skin cell, for example. To study the 3-D structure of DNA, Weintraub works with mouse embryonic stem cells. He is now working on assembling maps that show normal patterns and gene organization across different types of healthy cells.

    Bad packaging

    While there is a general consistency to DNA’s 3-D structure, Weintraub also noticed variations as he worked to create these maps. He began to wonder whether DNA’s packaging affects its functionality, beyond just allowing it to fit inside a nucleus. So he shifted his research slightly, and now focuses on how slight changes in the way that DNA strands are folded can cause serious problems, like cancer.

    In particular, he’s interested in T-cell acute lymphoblastic leukemia.

    “That’s a disease that primarily affects children,” Weintraub says. “So it keeps me motivated in my research.”

    Outside of the lab, his research also resonates with physicians at Massachusetts General Hospital and Boston Children’s Hospital, grounding this aspect of his work in therapeutic discovery — although Weintraub remains staunchly connected to the importance of basic science, too.

    “It’s kind of the best of both research worlds,” he says of his research with DNA, as his project mapping healthy cells’ 3-D DNA structure has broader applications.

    Historical context

    For Weintraub, finding his place in the historical context of this field has been interesting. Through reading peer-reviewed journal articles from the 1970s and 1980s, he noticed a number of researchers postulating that DNA had a distinct 3-D structure that affected its functionality, but were unable to prove this hypothesis. With today’s precise technology, he gets to be part of a team that’s finally confirming this notion.

    “I like the idea that what I’m doing is identifying principles behind something as central to our biology as DNA,” Weintraub says. “I like that there’s still room for that kind of discovery here.”

    While Weintraub grew up and attended college in California, he says he may end up settling in Boston because of the area’s vast resources for biotech research. But with several years left until he completes his PhD, it’s too early to tell whether he’ll choose to go into academia or industry.

    “I don’t want to limit myself just yet,” he says. “There’s still a lot to learn.”

    See the full article here .

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  • richardmitnick 3:24 am on October 16, 2015 Permalink | Reply
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    From EPFL: “The future of farming depends on local breeds” 

    EPFL bloc

    Ecole Polytechnique Federale Lausanne

    16.10.15
    Jan Overney

    1
    The dwindling genetic diversity of farm animals is increasingly becoming a threat to livestock production. Although new DNA technologies can help us address this problem, changing habits to preserve local lineages may prove to be challenging. No image credit.

    It is hard to overestimate the importance of global livestock production to society and the economy. It constitutes the main source of income for 1.3 billion farmers, providing vital food for 800 million subsistence farmers, and making up 40% of global agricultural GDP. But overbreeding and dwindling genetic diversity could limit the ability of livestock populations to adapt the environmental changes, such as global warming and related new diseases. Currently on the sidelines, lesser-known livestock breeds and the DNA they carry could become key to securing the future of livestock farming.

    For four years leading up to 2014, a European research project chaired by EPFL took stock of the past, present, and future of farm animal genetic resources and outlined the questions of highest priority for research, infrastructure and policy development for the coming decade. A selection of the project’s scientific output has now been published by the open access journal Frontiers in Genetics and is available online in the form of 31 research papers.

    A shrinking genetic reservoir

    Over the past 100 years, many local breeds have gone extinct, as more productive industrial breeds have taken over. Even within these breeds, the genetic diversity between individuals is shrinking. So why does this matter? “A reduction of genetic diversity goes hand with hand with a reduction of the species’ capacity to adapt to new diseases, warmer temperatures, or new food sources,” says Stéphane Joost, the project’s chair.

    “Studying 1,200 sheep from 32 old, native breeds from around the world, we previously identified a specific gene involved in regulating their metabolism, whose presence correlated strongly with the amount of incident solar radiation – a genetic trait that made them better adapted to their environment than cosmopolitan breeds that are more productive in the short term,” says Joost. If breeds carrying such specific adaptations disappear, so too will the coping strategies they acquired throughout evolution.

    The better choice?

    Joost’s advice to farmers: “Farmers should keep their local, well adapted breeds,” he urges. They may be less productive than their industrially bred cousins, but in developing countries with extreme climates sticking to them is often the wiser choice – a lesson that many farmers learn the hard way. After investing their savings to crossbreed a species of cow local to West Africa with an industrial breed, farmers in Burkina Faso first reaped the fruits of their investment, until they realized that all of the mixed breed’s offspring were poorly adapted to their climate and eventually died. “Only local breeds are adapted to resist to such harsh environments and withstand diseases such as trypanosomiasis, spread by the tsetse fly,” says Joost.

    An archive of adaptation

    Understanding the genetic history of today’s breeds could help us find ways of adapting in the future, says Joost. “What ancestral animals conferred the species with a specific trait? And what can we do today to recover that same trait?” he asks. Knowing, for example, exactly which native species were crossbred to produce today’s breeds could help pinpoint certain well-adapted genes present in the native species that may have been lost. In the same way, well-adapted local breeds that were abandoned to the point of extinction could be recreated by cross-breeding the ancestral species they emerged from.

    To ensure that the research carried out in this project finds its way into the agricultural community, the 31 studies will be compiled into an e-book, which will also be made available in print and distributed to stakeholders in developing countries by the FAO. But changing habits will be an uphill battle, as it involves sacrificing short-term profits for long-term sustainability – a problem that Joost and the co-organizers of the research project are well aware of. “Throughout this project, we emphasized the need to work with social scientists to effectively influence the habits of the breeders associations and other stakeholders. This is one front on which we still have much to do,” he concludes.

    See the full article here .

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    EPFL is Europe’s most cosmopolitan technical university. It receives students, professors and staff from over 120 nationalities. With both a Swiss and international calling, it is therefore guided by a constant wish to open up; its missions of teaching, research and partnership impact various circles: universities and engineering schools, developing and emerging countries, secondary schools and gymnasiums, industry and economy, political circles and the general public.

     
  • richardmitnick 2:33 pm on September 25, 2015 Permalink | Reply
    Tags: , , DNA, ,   

    From MIT: “New system for human genome editing has potential to increase power and precision of DNA engineering” 


    MIT News

    September 25, 2015
    Broad Institute

    1
    CRISPR systems are found in many different bacterial species, and have evolved to protect host cells against infection by viruses. Image courtesy of Broad Institute/Science Photo Images

    CRISPR-Cpf1 offers simpler approach to editing DNA; technology could disrupt scientific and commercial landscape.

    A team including the scientist who first harnessed the CRISPR-Cas9 system for mammalian genome editing has now identified a different CRISPR system with the potential for even simpler and more precise genome engineering.

    In a study published today in Cell, Feng Zhang and his colleagues at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT, with co-authors Eugene Koonin at the National Institutes of Health, Aviv Regev of the Broad Institute and the MIT Department of Biology, and John van der Oost at Wageningen University, describe the unexpected biological features of this new system and demonstrate that it can be engineered to edit the genomes of human cells.

    “This has dramatic potential to advance genetic engineering,” says Eric Lander, director of the Broad Institute. “The paper not only reveals the function of a previously uncharacterized CRISPR system, but also shows that Cpf1 can be harnessed for human genome editing and has remarkable and powerful features. The Cpf1 system represents a new generation of genome editing technology.”

    CRISPR sequences were first described in 1987, and their natural biological function was initially described in 2010 and 2011. The application of the CRISPR-Cas9 system for mammalian genome editing was first reported in 2013, by Zhang and separately by George Church at Harvard University.

    In the new study, Zhang and his collaborators searched through hundreds of CRISPR systems in different types of bacteria, searching for enzymes with useful properties that could be engineered for use in human cells. Two promising candidates were the Cpf1 enzymes from bacterial species Acidaminococcus and Lachnospiraceae, which Zhang and his colleagues then showed can target genomic loci in human cells.

    “We were thrilled to discover completely different CRISPR enzymes that can be harnessed for advancing research and human health,” says Zhang, the W.M. Keck Assistant Professor in Biomedical Engineering in MIT’s Department of Brain and Cognitive Sciences.

    The newly described Cpf1 system differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics, as well as for business and intellectual property:

    First: In its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.

    Second, and perhaps most significantly: Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving “blunt ends” that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.

    Third: Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be recut, allowing multiple opportunities for correct editing to occur.

    Fourth: The Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting some genomes, such as in the malaria parasite as well as in humans.

    “The unexpected properties of Cpf1 and more precise editing open the door to all sorts of applications, including in cancer research,” says Levi Garraway, an institute member of the Broad Institute, and the inaugural director of the Joint Center for Cancer Precision Medicine at the Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and the Broad Institute. Garraway was not involved in the research.

    An open approach to empower research

    Zhang, along with the Broad Institute and MIT, plan to share the Cpf1 system widely. As with earlier Cas9 tools, these groups will make this technology freely available for academic research via the Zhang lab’s page on the plasmid-sharing website Addgene, through which the Zhang lab has already shared Cas9 reagents more than 23,000 times with researchers worldwide to accelerate research. The Zhang lab also offers free online tools and resources for researchers through its website.

    The Broad Institute and MIT plan to offer nonexclusive licenses to enable commercial tool and service providers to add this enzyme to their CRISPR pipeline and services, further ensuring availability of this new enzyme to empower research. These groups plan to offer licenses that best support rapid and safe development for appropriate and important therapeutic uses.

    “We are committed to making the CRISPR-Cpf1 technology widely accessible,” Zhang says. “Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome editing advances.”

    See the full article here .

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  • richardmitnick 5:40 pm on September 10, 2015 Permalink | Reply
    Tags: , DNA,   

    From The Atlantic: “How Data-Wranglers Are Building the Great Library of Genetic Variation” 

    Atlantic Magazine

    The Atlantic Magazine

    Sep 9, 2015
    Ed Yong

    1

    A huge project unexpectedly led to a way of finding disease genes without needing to know about diseases.

    Let’s say you have a patient with a severe inherited muscle disorder, the kind that Daniel MacArthur from the Broad Institute of Harvard and MIT specializes in. They’re probably a child, with debilitating symptoms and perhaps no diagnosis. To discover the gene(s) that underlie the kid’s condition, you sequence their genome, or perhaps just their exome: the 1 percent of their DNA that codes for proteins. The results come back, and you see tens of thousands of variants—sites where, say, the usual A has been replaced by a T, or the typical C is instead a G.

    You’d then want to know if those variants have ever been associated with diseases, and how common they are in the general population. (The latter is especially important because most variants are so common that they can’t possibly be plausible culprits behind rare genetic diseases.) “To make sense of a single patient’s genome, you need to put it in the context of many people’s genomes,” says MacArthur. In an ideal world, you would compare all of a patient’s variants against “every individual who has ever been sequenced in the history of sequencing.”

    This is not that world, at least not yet. When Macarthur launched his lab in 2012, he started by sequencing the exomes of some 300 patients with rare muscle diseases. But he quickly realized that he had nothing decent to compare them against. It has never been easier, cheaper, or quicker to sequence a person’s genome, but interpreting those sequences is tricky, absent a comprehensive reference library of human genetic variation. No such library existed, or at least nothing big or diverse enough. So, MacArthur started making one.

    It was hard work, not because the data didn’t exist, but because it was scattered. To date, scientists have probably sequenced at least 5,000 full genomes and some 500,000 exomes, but most are completely inaccessible to other researchers. There might be intellectual-property restrictions, or issues around consent. There’s the logistical hassle of shipping huge volumes of data on hard drives. And some scientists are just plain competitive.

    Fortunately, MacArthur’s colleagues at the Broad Institute and beyond had deciphered so many exomes that he could gather thousands of sequences by personally popping into offices. Buoyed by that success, he started contacting people who were studying the genomes of people with cancer, heart disease, diabetes, schizophrenia, and more. “There’s a big swath of human genetics where people have learned that you either fail by yourself or succeed together, so they’re committed to sharing data,” MacArthur says.

    By 2014, he had amassed more than 90,000 exomes from around a dozen sources, collectively called the Exome Aggregation Consortium. Then, he had to munge them together.

    That was the worst bit. Researchers use very different technologies to sequence and annotate genomes, so combining disparate data sets is like mushing together the dishes from separate restaurants and hoping that the results will be palatable. Often, they won’t be.

    Monkol Lek, a postdoc in MacArthur’s lab who himself has a genetic muscle disease, solved this problem by essentially starting from scratch. He took the raw data from some 60,706 patients and analyzed their exomes, one position at a time. The raw sequences took up a petabyte of memory, and the final compressed file filled a three-terabyte hard disk.

    The prize from all this data-wrangling was one of the most thorough portraits of human genetic variation ever produced. MacArthur went through the main results in the opening talk of this week’s Genome Science 2015 conference, in Birmingham, U.K. His team had identified around 10 million genetic variants scattered throughout the exome, most of which had never been described before. And most turned up just once in the data, meaning that they lurk within just one in every 60,000 people. “Human variation is dominated by these extremely rare variants,” says MacArthur. That’s where the secrets of many rare genetic disorders reside.

    But unexpectedly, the most interesting variants turned out to be the ones that weren’t there.

    The graduate student Kaitlin Samocha developed a mathematical model to predict how many variants you’d expect to find in a given gene, in a population of 60,000 people. The model was remarkably accurate at estimating neutral variants, which don’t change the protein that’s encoded by the gene, and so have minimal impact. But the model often wildly overestimated the number of “loss-of-function variants,” which severely disrupt the gene in question. Repeatedly, the ExAc data revealed far fewer of these variants than Samocha’s model predicted.

    Why? Because many of these loss-of-function variants are so destructive that their carriers develop debilitating disorders, or die before they’re even born. So, the difference between prediction and reality reflects the brutal hand of natural selection. The variants are simply not around to be sequenced because they have long been expunged from the gene pool.

    For example, the team expected to find 161 loss-of-function variants in a gene called DYNC1H1. By contrast, the ExAc data revealed only four—and indeed, DYNC1H1 is associated with several severe inherited neurodevelopmental disorders.

    The model also predicted 125 loss-of-function variants in the UBR5 gene—and the data revealed just one. That’s far more interesting because UBR5 has never before been linked to a human disease.

    A full quarter of human genes are like this: They have a lower-than-expected number of loss-of-function variants. And while some of them are known “diseases genes,” the rest have never been pinpointed as such. So, if you find one of these variants in a patient with a severe genetic disorder, the chances are good that you’ve found a genuine culprit.

    That blew my mind. Here is a way of identifying potential disease-related genes, without needing to know anything about the diseases in question. Or, as MacArthur said in his talk, “We should soon be able to say, with high precision: If you have a mutation at this site, it will kill you. And we’ll be able to say that without ever seeing a person with that mutation.”

    These results speak to one of the greatest challenges of modern genomics: weaving together existing sets of data in useful ways. They also vindicate the big, expensive studies that have searched for variants behind common diseases like type 2 diabetes, heart disease, and schizophrenia. These endeavors have indeed found several variants, but with such small effects that they explain just a tiny fraction of the risk of each condition. But “all this data can be re-purposed for analyzing rare diseases,” says MacArthur. “Without those large-scale studies, we’d have no chance of doing something like ExAc.”

    “His talk really shows that you can’t anticipate what these data sets will show you until you put them together,” says Nick Loman from the University of Birmingham. “Our ability to interrogate biology if you can put hundreds of thousands, or millions, of genomes together is massive.”

    See the full article here .

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