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  • richardmitnick 8:44 am on August 3, 2017 Permalink | Reply
    Tags: , CRISPR-Cas9, , ,   

    From Salk: “Early gene-editing success holds promise for preventing inherited diseases” 

    Salk Institute bloc

    Salk Institute for Biological Studies

    August 2, 2017

    Scientists have, for the first time, corrected a disease-causing mutation in early stage human embryos with gene editing. The technique, which uses the CRISPR-Cas9 system, corrected the mutation for a heart condition at the earliest stage of embryonic development so that the defect would not be passed on to future generations.

    The work, which is described in Nature on August 2, 2017, is a collaboration between the Salk Institute, Oregon Health and Science University (OHSU) and Korea’s Institute for Basic Science and could pave the way for improved in vitro fertilization (IVF) outcomes as well as eventual cures for some of the thousands of diseases caused by mutations in single genes.

    “Thanks to advances in stem cell technologies and gene editing, we are finally starting to address disease-causing mutations that impact potentially millions of people,” says Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory and a corresponding author of the paper. “Gene editing is still in its infancy so even though this preliminary effort was found to be safe and effective, it is crucial that we continue to proceed with the utmost caution, paying the highest attention to ethical considerations.”

    Though gene-editing tools have the power to potentially cure a number of diseases, scientists have proceeded cautiously, in part to avoid introducing unintended mutations into the germ line (cells that become eggs or sperm). Izpisua Belmonte is uniquely qualified to speak to the ethics of genome editing in part because, as a member of the committee on human gene editing of the National Academies of Sciences, Engineering and Medicine, he helped author the 2016 roadmap “Human Genome Editing: Science, Ethics, and Governance.” The research in the current study is fully compliant with recommendations made in that document, and adheres closely to guidelines established by OHSU’s Institutional Review Board and additional ad-hoc committees set up for scientific and ethical review.

    1
    Newly fertilized eggs before gene editing (left) and embryos after gene editing and a few rounds of cell division (right). Credit: OHSU/Mitalipov lab.

    Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden death in otherwise healthy young athletes, and affects approximately 1 in 500 people overall. It is caused by a dominant mutation in the MYBPC3 gene, but often goes undetected until it is too late. Since people with a mutant copy of the MYBPC3 gene have a 50 percent chance of passing it on to their own children, being able to correct the mutation in embryos would prevent the disease not only in affected children, but also in their descendants.

    The researchers generated induced pluripotent stem cells from a skin biopsy donated by a male with HCM and developed a gene-editing strategy based on CRISPR-Cas9 that would specifically target the mutated copy of the MYBPC3 gene for repair. The targeted mutated MYBPC3 gene was cut by the Cas9 enzyme, allowing the donor’s cells’ own DNA-repair mechanisms to fix the mutation during the next round of cell division by using either a synthetic DNA sequence or the non-mutated copy of MYBPC3 gene as a template.

    Using IVF techniques, the researchers injected the best-performing gene-editing components into healthy donor eggs newly fertilized with the donor’s sperm. Then they analyzed all the cells in the early embryos at single-cell resolution to see how effectively the mutation was repaired.

    The scientists were surprised by just how safe and efficient the method was. Not only did a high percentage of embryonic cells get repaired, but also gene correction didn’t induce any detectable off-target mutations and genome instability—major concerns for gene editing. In addition, the researchers developed a robust strategy to ensure the repair occurred consistently in all the cells of the embryo. (Spotty repairs can lead to some cells continuing to carry the mutation.)

    “Even though the success rate in patient cells cultured in a dish was low, we saw that the gene correction seems to be very robust in embryos of which one copy of the MYBPC3 gene is mutated,” says Jun Wu, a Salk staff scientist and one of the paper’s first authors. This was in part because, after CRISPR-Cas9 mediated enzymatic cutting of the mutated gene copy, the embryo initiated its own repairs. Instead of using the provided synthetic DNA template, the team found, surprisingly, that the embryo preferentially used the available healthy copy of the gene to repair the mutated part. “Our technology successfully repairs the disease-causing gene mutation by taking advantage of a DNA repair response unique to early embryos” says Wu.

    Izpisua Belmonte and Wu emphasize that, although promising, these are very preliminary results and more research will need to be done to ensure no unintended effects occur.

    “Our results demonstrate the great potential of embryonic gene editing, but we must continue to realistically assess the risks as well as the benefits,” adds Izpisua Belmonte.

    Future work will continue to assess the safety and effectiveness of the procedure and efficacy of the technique with other mutations.

    Other authors included: Keiichiro Suzuki of the Salk Institute; Hong Ma, Nuria Marti-Gutierrez, Yeonmi Lee, Amy Koski, Dongmei Ji, Tomonari Hayama, Riffat Ahmed, Hayley Darby, Crystal Van Dyken, Ying Li, Eunju Kang, David Battaglia, Sacha A. Krieg, David M. Lee, Diana H. Wu, Don P. Wolf, Stephen B. Heitner, Paula Amato, Sanjiv Kaul and Shoukhrat Mitalipov of Oregon Health and Science University; Sang-Wook Park, A-Reum Park, Sang-Tae Kim and Jin-Soo Kim of Korea’s Institute for Basic Science; Daesik Kim of Seoul National University; and Jianhui Gong, Ying Gu and Xun Xu of BGI, China.

    The work was funded by: Oregon Health and Science University, the Institute for Basic Science, the G. Harold and Leila Y. Mathers Charitable Foundation, the Moxie Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, and Shenzhen Municipal Government of China.

    See the full article here .

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    Salk Institute Campus

    Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

     
  • richardmitnick 10:42 am on July 31, 2017 Permalink | Reply
    Tags: , , CRISPR-Cas9, , , It wouldn’t have been obvious that PTPN2 is a good drug target for the immunotherapy of cancer, , PD-1 checkpoint inhibitors have transformed the treatment of many cancers   

    From HMS: “Attack Mode “ 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    7.31.17
    KAT MCALPINE

    1
    Genetic screening for cancer immunotherapy targets. Cancer cells (colored shapes), each with a different CRISPR-Cas9-mediated gene knocked out. T cells (red) destroy the cancer cells that have had essential immune evasion genes knocked out. Image: Haining Lab, Dana-Farber/Boston Children’s.

    A novel screening method developed by a team at Harvard Medical School and Dana-Farber/Boston Children’s Cancer and Blood Disorders Center—using CRISPR-Cas9 genome editing technology to test the function of thousands of tumor genes in mice—has revealed new drug targets that could potentially enhance the effectiveness of PD-1 checkpoint inhibitors, a promising new class of cancer immunotherapy.

    In findings published online today by Nature http://www.nature.com/nature/journal/v547/n7664/full/nature23270.html the Dana-Farber/Boston Children’s team, led by pediatric oncologist W. Nick Haining, reports that deletion of the PTPN2 gene in tumor cells made them more susceptible to PD-1 checkpoint inhibitors. PD-1 blockade is a drug that “releases the brakes” on immune cells, enabling them to locate and destroy cancer cells.

    “PD-1 checkpoint inhibitors have transformed the treatment of many cancers,” said Haining, HMS associate professor of pediatrics at Dana-Farber/Boston Children’s, associate member of the Broad Institute of MIT and Harvard, and senior author on the new paper. “Yet despite the clinical success of this new class of cancer immunotherapy, the majority of patients don’t reap a clinical benefit from PD-1 blockade.”

    That, Haining said, has triggered a rush of additional trials to investigate whether other drugs, when used in combination with PD-1 inhibitors, can increase the number of patients whose cancer responds to the treatment.

    “The challenge so far has been finding the most effective immunotherapy targets and prioritizing those that work best when combined with PD-1 inhibitors,” Haining said. “So, we set out to develop a better system for identifying new drug targets that might aid the body’s own immune system in its attack against cancer.

    “Our work suggests that there’s a wide array of biological pathways that could be targeted to make immunotherapy more successful,” Haining continued. “Many of these are surprising pathways that we couldn’t have predicted. For instance, without this screening approach, it wouldn’t have been obvious that PTPN2 is a good drug target for the immunotherapy of cancer.”

    Sifting through thousands of potential targets

    To cast a wide net, the paper’s first author Robert Manguso, a graduate student in Haining’s lab, designed a genetic screening system to identify genes used by cancer cells to evade immune attack. He used CRISPR-Cas9, a genome editing technology that works like a pair of molecular scissors to cleave DNA at precise locations in the genetic code, to systematically knock out 2,368 genes expressed by melanoma skin cancer cells. Manguso was then able to identify which genes, when deleted, made the cancer cells more susceptible to PD-1 blockade.

    Manguso started by engineering the melanoma skin cancer cells so that they all contained Cas9, the cutting enzyme that is part of the CRISPR editing system. Then, using a virus as a delivery vehicle, he programmed each cell with a different single-guide-RNA (sgRNA) sequence of genetic code. In combination with the Cas9 enzyme, the sgRNA codes—about 20 amino acids in length—enabled 2,368 different genes to be eliminated.

    By injecting the tumor cells into mice and treating them with PD-1 checkpoint inhibitors, Manguso was then able to tally up which modified tumor cells survived. Those that perished had been sensitized to PD-1 blockade as a result of their missing gene.

    Using this approach, Manguso and Haining first confirmed the role of two genes already known to be immune evaders—PD-L1 and CD47, drug inhibitors that are already in clinical trials. They then discovered a variety of new immune evaders that, if inhibited therapeutically, could enhance PD-1 cancer immunotherapy. One such newly found gene of particular interest is PTPN2.

    “PTPN2 usually puts the brakes on the immune signaling pathways that would otherwise smother cancer cells,” Haining said. “Deleting PTPN2 ramps up those immune signaling pathways, making tumor cells grow slower and die more easily under immune attack.”

    Gaining more ground

    With the new screening approach in hand, Haining’s team is quickly scaling up their efforts to search for additional novel drug targets that could boost immunotherapy.

    Haining says the team is expanding their approach to move from screening thousands of genes at a time to eventually being able to screen the whole genome and to move beyond melanoma to colon, lung, renal carcinoma and more. He’s assembled a large team of scientists spanning Dana-Farber/Boston Children’s and the Broad to tackle the technical challenges that accompany screening efforts on such a large scale.

    In the meantime, while more new potential drug targets are likely around the corner, Haining’s team is taking action based on their findings about PTPN2.

    “We’re thinking hard about what a PTPN2 inhibitor would look like,” said Haining. “It’s easy to imagine making a small molecule drug that turns off PTPN2.”

    This work was supported by the Broad Institute of Harvard and MIT (BroadIgnite and Broadnext10 awards) and the National Institute of General Medical Sciences (T32GM007753).

    See the full article here .

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    Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

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    Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

    Harvard University has 12 degree-granting Schools in addition to the Radcliffe Institute for Advanced Study. The University has grown from nine students with a single master to an enrollment of more than 20,000 degree candidates including undergraduate, graduate, and professional students. There are more than 360,000 living alumni in the U.S. and over 190 other countries.

     
  • richardmitnick 1:21 pm on July 8, 2017 Permalink | Reply
    Tags: , , , CRISPR-Cas9, , , , , , UCSD Comet supercomputer   

    From Science Node: “Cracking the CRISPR clock” 

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    Science Node

    05 Jul, 2017
    Jan Zverina

    SDSC Dell Comet supercomputer

    Capturing the motion of gyrating proteins at time intervals up to one thousand times greater than previous efforts, a team led by University of California, San Diego (UCSD) researchers has identified the myriad structural changes that activate and drive CRISPR-Cas9, the innovative gene-splicing technology that’s transforming the field of genetic engineering.

    By shedding light on the biophysical details governing the mechanics of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) activity, the study provides a fundamental framework for designing a more efficient and accurate genome-splicing technology that doesn’t yield ‘off-target’ DNA breaks currently frustrating the potential of the CRISPR-Cas9- system, particularly for clinical uses.


    Shake and bake. Gaussian accelerated molecular dynamics simulations and state-of-the-art supercomputing resources reveal the conformational change of the HNH domain (green) from its inactive to active state. Courtesy Giulia Palermo, McCammon Lab, UC San Diego.

    “Although the CRISPR-Cas9 system is rapidly revolutionizing life sciences toward a facile genome editing technology, structural and mechanistic details underlying its function have remained unknown,” says Giulia Palermo, a postdoctoral scholar with the UC San Diego Department of Pharmacology and lead author of the study [PNAS].

    See the full article here
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    Science Node is an international weekly online publication that covers distributed computing and the research it enables.

    “We report on all aspects of distributed computing technology, such as grids and clouds. We also regularly feature articles on distributed computing-enabled research in a large variety of disciplines, including physics, biology, sociology, earth sciences, archaeology, medicine, disaster management, crime, and art. (Note that we do not cover stories that are purely about commercial technology.)

    In its current incarnation, Science Node is also an online destination where you can host a profile and blog, and find and disseminate announcements and information about events, deadlines, and jobs. In the near future it will also be a place where you can network with colleagues.

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  • richardmitnick 11:56 am on June 20, 2017 Permalink | Reply
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    From COSMOS: “Precision gene editing may deliver biofuel promise” 

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    COSMOS

    20 June 2017
    Elizabeth Finkel

    1
    Biofuels from algae come with great promise. Santiago Urquijo/Getty Images

    For decades now, we’ve been promised cheap biofuels from algae. But there’s no free lunch. Growing these mini oil factories in vast ponds requires fertiliser and mechanical aeration; and then the oil has to be extracted. It all costs energy and money so the yields need to be high to make it worthwhile.

    One promising industrial species is Nannocholoropsis gaditana, which can produce a lipid – the oil and fat energy store – content up to about 60% of the algae’s ash-free dry weight. But Eric Moellering and colleagues at the company Synthetic Genomics Inc in California, wanted to do better.

    Starving the algae of nitrogen, paradoxically, boosts oil production. The problem is that the plants also curtail their growth so there’s no net gain. Ever since the 1970s, scientists have been trying to genetically engineer their way out of this quandary. But they’ve had to wait for the right tool: the bacterially derived CRISPR–Cas9 enzyme that has transformed ham-fisted genetic engineering into precision gene-editing.

    Moellering’s group identified a gene, called ZnCys, that was deactivated when the algae was starved of nitrogen. When the researchers completely disabled that gene, they saw oil production double, even without starving the algae.

    But the algae still grew poorly, so the scientists made use of the finesse of CRISPR–Cas9 to finely edit the DNA code of the ZnCys gene instead of disabling it competely. As they report in Nature Biotechnology this led to doubling oil yield without dampening algae growth.

    The final yield was up to five grams per square metre of algae per day.

    Amazing what a good editor can do!

    See the full article here .

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  • richardmitnick 10:13 am on March 9, 2017 Permalink | Reply
    Tags: , CRISPR-Cas9, , Embryos can be repaired, in vitro fertilization, Triple helix,   

    From Yale: “Gene editing opens the door to a “revolution” in treating and preventing disease” 

    Yale University bloc

    Yale University

    March 8, 2017
    John Dent Curtis

    Today, in vitro fertilization provides a way for couples to avoid passing potentially disease-causing genes to their offspring. A couple will undergo genetic screening. Tests will determine whether their unborn children are at risk. If embryos created through IVF show signs of such a genetic mutation, they can be discarded.

    Flash forward a few years, and, instead of being discarded, those embryos can be repaired with new gene editing technologies. And those repairs will affect not only those children, but all their descendants.

    “This is definitely new territory,” said Pasquale Patrizio, M.D., director of the Yale Fertility Center and Fertility Preservation Program. “We are at the verge of a huge revolution in the way disease is treated.”

    We are at the verge of a huge revolution in the way disease is treated.”
    Pasquale Patrizio, M.D., director of the Yale Fertility Center and Fertility Preservation Program

    In a move that seems likely to help clear the path for the use of gene editing in the clinical setting, on February 14 the Committee on Human Gene Editing, formed by the National Academy of Medicine and the National Academy of Sciences, recommended that research into human gene editing should go forward under strict ethical and safety guidelines. Among their concerns were ensuring that the technology be used to treat only serious diseases for which there is no other remedy, that there be broad oversight, and that there be equal access to the treatment. These guidelines provide a framework for discussion of technology that has been described as an “ethical minefield” and for which there is no government support in the United States.

    A main impetus for the committee’s work appears to be the discovery and widespread use of CRISPR-Cas9, a defense that bacteria use against viral infection. Scientists including former Yale faculty member Jennifer Doudna, Ph.D., now at the University of California, Berkeley, and Emmanuelle Charpentier, Ph.D., of the Max Planck Institute for Infection Biology in Berlin, discerned that the CRISPR enzyme could be harnessed to make precision cuts and repairs to genes. Faster, easier, and cheaper than previous gene editing technologies, CRISPR was declared the breakthrough of the year in 2015 by Science magazine, and has become a basic and ubiquitous laboratory research tool. The committee’s guidelines, said scientists, physicians, and ethicists at Yale, could pave the way for thoughtful and safe use of this and other human gene editing technologies. In addition to CRISPR, the committee described three commonly used gene editing techniques; zinc finger nucleases, meganucleases, and transcription activator-like effector nucleases.

    Patrizio, professor of obstetrics, gynecology, and reproductive sciences, said the guidelines are on the mark, especially because they call for editing only in circumstances where the diseases or disabilities are serious and where there are not alternative treatments. He and others cited such diseases as cystic fibrosis, sickle cell anemia, and thalassemia as targets for gene editing. Because they are caused by mutations in a single gene, repairing that one gene could prevent disease.

    Peter Glazer, M.D. ’87, Ph.D. ’87, HS ’91, FW ’91, chair and the Robert E. Hunter Professor of Therapeutic Radiology and professor of genetics, said, “The field will benefit from guidelines that are thoughtfully developed. This was a step in the right direction.”

    The panel recommended that gene editing techniques should be limited to deal with genes proven to cause or predispose to specific diseases. It should be used to convert mutated genes to versions that are already prevalent in the population. The panel also called for stringent oversight of the process and for a prohibition against use of the technology for “enhancements,” rather than to treat disease. “As physicians, we understand what serious diseases are. Many of them are very well known and well characterized on a genetic level,” Glazer said. “The slippery slope is where people start thinking about modifications in situations where people don’t have a serious disorder or disease.”

    Mark Mercurio, M.D., professor of pediatrics (neonatology), and director of the Program for Biomedical Ethics, echoed that concern. While he concurs with the panel’s recommendations, he urged a clear definition of disease prevention and treatment. “At some point we are not treating, but enhancing.” This in turn, he said, conjures up the nation’s own medical ethical history, which includes eugenics policies in the early 20th century that were later adopted in Nazi Germany. “This has the potential to help a great many people, and is a great advance. But we need to be cognizant of the history of eugenics in the United States and elsewhere, and need to be very thoughtful in how we use this technology going forward,” he said.

    The new technology, he said, can lead to uncharted ethical waters. “Pediatric ethics are more difficult,” Mercurio said. “It is one thing to decide for yourself–is this a risk I’m willing to take—and another thing to decide for a child. It is another thing still further, which we have never had to consider, to decide for future generations.”

    Myron Genel, M.D., emeritus professor of pediatrics and senior research scientist, served on Connecticut’s stem cell commission and four years on the Health and Human Services Secretary’s Advisory Committee on Human Research Protections. He believes that Connecticut’s guidelines on stem cell research provide a framework for addressing the issues associated with human gene editing. “There is a whole regulatory process that has been evolved governing the therapeutic use of stem cells,” he said. “There are mechanisms that have been put in place for effective local oversight and national oversight for stem cell research.”

    Although CRISPR has been the subject of a bitter patent dispute between Doudna and Charpentier and The Broad Institute in Cambridge, Mass., a recent decision by the U.S. Patent Trial and Appeal Board in favor of Broad is unlikely to affect research at Yale and other institutions. Although Broad, an institute of Harvard and the Massachusetts Institute of Technology, can now claim the patent, universities do not typically enforce patent rights against other universities over research uses.

    At Yale, scientists and physicians noted that gene editing is years away from human trials, and that risks remain. The issue now, said Glazer, is “How do we do it safely? It is never going to be risk-free. Many medical therapies have side effects and we balance the risks and benefits.” Despite its effectiveness, CRISPR is also known for what’s called “off-target risk,” imprecise cutting and splicing of genes that could lead to unforeseen side effects that persist in future generations. “CRISPR is extremely potent in editing the gene it is targeting,” Glazer said. “But it is still somewhat promiscuous and will cut other places. It could damage a gene you don’t want damaged.”

    Glazer has been working with a gene editing technology called triple helix that hijacks DNA’s own repair mechanisms to fix gene mutations. Triple helix, as its name suggests, adds a third strand to the double helix of DNA. That third layer, a peptide nucleic acid, binds to DNA and provokes a natural repair process that copies a strand of DNA into a target gene. Unlike CRISPR and other editing techniques, it does not use nucleases that cut DNA. “This just recruits a process that is natural. Then you give the cell this piece of DNA, this template that has a new sequence,” Glazer said, adding that triple helix is more precise than CRISPR and leads to fewer off-target effects, but is a more complex technology that requires advanced synthetic chemistry.

    Along with several scientists across Yale, Glazer is studying triple helix as a potential treatment for cystic fibrosis, HIV/AIDS, spherocytosis, and thalassemia.

    Adele Ricciardi, a student in her sixth year of the M.D./Ph.D. program, is working with Glazer and other faculty on use of triple helix to make DNA repairs in utero. She also supports the panel’s decision, but believes that more public discussion is needed to allay fears of misuse of the technology. In a recent presentation to her lab mates, she noted that surveys show widespread public concern about such biomedical advances. One study found that most of those surveyed felt it should be illegal to change the genes of unborn babies, even to prevent disease.

    “There is, I believe, a misconception of what we are using gene editing for,” Ricciardi said. “We are using it to edit disease-causing mutations, not to improve the intelligence of our species or get favorable characteristics in babies. We can improve quality of life in kids with severe genetic disorders.”

    See the full article here .

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    Yale University comprises three major academic components: Yale College (the undergraduate program), the Graduate School of Arts and Sciences, and the professional schools. In addition, Yale encompasses a wide array of centers and programs, libraries, museums, and administrative support offices. Approximately 11,250 students attend Yale.

     
  • richardmitnick 3:16 pm on January 20, 2016 Permalink | Reply
    Tags: , CRISPR-Cas9, ,   

    From Berkeley: “Advance improves cutting and pasting with CRISPR-Cas9 gene editing” 

    UC Berkeley

    UC Berkeley

    January 20, 2016
    Robert Sanders

    Temp 1
    A view of the Cas9 protein (red and blue) bound to a double strand of DNA (purple and grey). After both strands are cut, one DNA strand (purple dots) is free and able to bind with a piece of DNA to be inserted at the break.This behavior can be utilized to significantly boost the efficiency of gene editing. Image by Christopher Richardson, UC Berkeley, based on structure solved by Martin Jinek’s lab.

    UC Berkeley researchers have made a major improvement in CRISPR-Cas9 technology that achieves an unprecedented success rate of 60 percent when replacing a short stretch of DNA with another.

    The improved technique is especially useful when trying to repair genetic mutations that cause hereditary diseases, such as sickle cell disease or severe combined immune deficiency. The technique allows researchers to patch an abnormal section of DNA with the normal sequence and potentially correct the defect and is already working in cell culture to improve ongoing efforts to repair defective genes.

    “The exciting thing about CRISPR-Cas9 is the promise of fixing genes in place in our genome, but the efficiency for that can be very low,” said Jacob Corn, scientific director of the Innovative Genomics Initiative at UC Berkeley, a group that focuses on next-generation genome editing and gene regulation for lab and clinical application. “If you think of gene editing as a word processor, we know how to cut, but we need a more efficient way to paste and glue a new piece of DNA where we make the cut.”

    “In cases where you want to change very small regions of DNA, up to 30 base pairs, this technique would be extremely effective,” said first author Christopher Richardson, an IGI postdoc.

    Problems in short sections of DNA, including single base-pair mutations, are typical of many genetic diseases. Base pairs are the individual building blocks of DNA, strung end-to-end in a strand that coils around a complementary strand to make the well-known helical, double-stranded DNA molecule.

    Richardson, Corn and their IGI colleagues describe the new technique in the Jan. 21 issue of the journal Nature Biotechnology.

    Grabbing onto a loose strand

    Richardson invented the new approach after finding that the Cas9 protein, which does the actual DNA cutting, remains attached to the chromosome for up to six hours, long after it has sliced through the double-stranded DNA. Richardson looked closely at the Cas9 protein bound to the two strands of DNA and discovered that while the protein hangs onto three of the cut ends, one of the ends remains free.


    Jennifer Doudna explains how CRISPR-Cas9 edits genes. Video by Roxanne Makasdjian and Stephen McNally, UC Berkeley.
    Watch/download mp4 video here .

    When Cas9 cuts DNA, repair systems in the cell can grab a piece of complementary DNA, called a template, to repair the cut. Researchers can add templates containing changes that alter existing sequences in the genome — for example, correcting a disease-causing mutation.

    Richardson reasoned that bringing the substitute template directly to the site of the cut would improve the patching efficiency, and constructed a piece of DNA that matches the free DNA end and carries the genetic sequence to be inserted at the other end. The technique worked extremely well, allowing successful repair of a mutation with up to 60 percent efficiency.

    “Our data indicate that Cas9 breaks could be different at a molecular level from breaks generated by other targeted nucleases, such as TALENS and zinc-finger nucleases, which suggests that strategies like the ones we are using can give you more efficient repair of Cas9 breaks,” Richardson said.

    The researchers also showed that variants of the Cas9 protein that bind DNA but do not cut also can successfully paste a new DNA sequence at the binding site, possibly by forming a “bubble” structure on the target DNA that also acts to attract the repair template. Gene editing using Cas9 without genome cutting could be safer than typical gene editing by removing the danger of off-target cutting in the genome, Corn said.

    Co-authors with Richardson and Corn are IGI researchers Jordan Ray, Mark DeWitt and Gemma Curie. The work was funded by the Li Ka Shing Foundation.

    See the full article here .

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  • richardmitnick 2:31 pm on January 13, 2016 Permalink | Reply
    Tags: , , CRISPR-Cas9, ,   

    From MIT Tech Review: “CRISPR Dispute to Be Decided by Patent Office” 

    MIT Technology Review
    M.I.T Technology Review

    January 12, 2016
    Jacob S. Sherkow

    A “patent interference” proceeding will determine who controls foundational patents on gene editing.

    As this magazine and others have detailed, CRISPR-Cas9—the powerful gene-editing technology being hailed as molecular biology’s “holy grail”—is the subject of a contentious dispute between the widely celebrated Jennifer Doudna at the University of California, Berkeley, and wunderkind Feng Zhang at the Broad Institute and MIT.

    The central question: who invented it first?

    Yesterday, that dispute became official in the eyes of the U.S. Patent and Trademark Office when an administrative patent judge officially declared an “interference” between Doudna’s pending patent application and a dozen of Zhang’s already issued patents. The interference proceeding sets up a legal showdown that may strip Zhang of his patents and see the two scientists deposed under oath.

    Even among patent attorneys—generally, friends of the arcane and hypertechnical—interference proceedings are famous for their complexity. The U.S. patent office now grants patents on a “first to file” basis. But before 2013 this was not the case. Historically, U.S. patent law instead recognized that patent rights should go to whoever could prove they were “first to invent” an idea. Because there is a lag between when patent applications are filed and when they are issued—roughly, three years—this gave rise to the possibility that a later inventor could be awarded a patent before the patent office had time to process an earlier inventor’s application. In that circumstance, the later inventor’s patent “interferes” with the earlier inventor’s ability to rightfully obtain theirs.

    This is precisely what occurred between Doudna and Zhang, whose patents are covered by the older rule. Doudna, with colleagues in Europe, filed a provisional patent application on her early iteration of the CRISPR editing technology on May 25, 2012; Zhang did the same on December 12, 2012. But Zhang’s attorneys requested that the patent office expedite its review of his application under a procedure—funnily named a Petition to Make Special—that allows inventors a quick up-or-down vote on simplified patent applications. As a result, Zhang was awarded his first patent on April 15, 2014, while Doudna’s patent application remained in limbo. Shortly thereafter, Zhang was awarded over a dozen patents on various forms of the technology.

    Perhaps fearing that they were losing the great biotech patent race of the century, Doudna’s attorneys amended her application in order to directly conflict with Zhang’s patents. Specifically, Doudna’s attorneys claimed that her patent application covered gene-editing in mammalian cells—including humans—even though her original filing didn’t detail that aspect of the technology. Yesterday, to the delight of watchers of patent dockets everywhere, an administrative patent judge with a PhD in molecular biology, Judge Deborah Katz, officially declared the interference.

    Despite these seemingly dry technicalities, the CRISPR patent dispute has been spiced with intrigue. During the examination of Doudna’s patent application, several unidentified third parties filed papers with the patent office seeking to block it, arguing that she was not the first to invent her CRISPR technique, while the Broad Institute unleashed its own volley of legal papers, lab notebooks, even copies of private e-mails between scientists. If Doudna’s original application did not command the focus of patent office supervisors when it was filed, it sure does now.

    What comes next? A panel of three patent judges will get to decide who gets the patent rights to CRISPR-Cas9 editing in animal cells. Their decision is likely to center on a few core issues. One is whether Doudna’s original patent application really covered working with human cells. Another is the earliest date either scientist can prove they performed their breakthrough work.

    Yesterday’s declaration of an interference proceeding already provides a few hints. First, it lists Doudna as the “senior party” and Zhang as the “junior party”—an initial determination that the administrative patent judge agrees that Doudna was the first inventor. This means that the burden of proof rests on Zhang, much like how, in a criminal trial, the government—not a criminal defendant—must prove its case beyond a reasonable doubt. Second, the declaration puts at issue all of the patent claims; none are left out. This suggests that the interference proceeding—assuming it retains its current scope—will be an all-or-nothing affair: Zhang will either get to keep all of his patents or lose all of them. This may mean that there is little room, legally, for the patent office to keep both sides happy. But as the dispute has shown us thus far, there is always room for surprises.

    See the full article here .

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