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Archive - Dec 3, 2015

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Broad & MIT Scientists Engineer Slightly Altered Cas9 Enzyme to Dramatically Reduce Off-Target Effects of CRISPR/Cas9 Genome Editing; Modified Enzyme Made Available to Labs Worldwide

Researchers at the Broad Institute of MIT and Harvard and at the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR/Cas9 genome editing system that significantly cut down on "off-target" editing errors. The refined technique addresses one of the major technical issues in the use of genome editing. The CRISPR/Cas9 system works by making a precisely targeted modification in a cell's DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA strand whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, Cas9 can bind to and cut at additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a gene out entirely, which might lead to the development of cancer or other problems. In a paper published online on December 1, 2015 in Science, Feng Zhang, Ph.D., and his colleagues report that changing just 3 of the approximately 1,400 amino acids that make up the Cas9 enzyme from S. pyogenes dramatically reduced "off-target editing" to undetectable levels in the specific cases examined. The Science article is titled “Rationally Engineered Cas9 Nucleases with Improved Specificity.” Dr. Zhang and his colleagues used knowledge about the structure of the Cas9 protein to decrease off-target cutting. DNA, which is negatively charged, binds to a groove in the Cas9 protein that is positively charged. Knowing the structure, the scientists were able to predict that replacing some of the positively charged amino acids with neutral ones would decrease the binding of "off target" sequences much more than the binding of "on target" sequences.

Targeted, Exosome-Mediated Delivery of Opioid Receptor siRNA for Treatment of Morphine Addiction; Results Suggest “Brand New Strategy to Treat Drug Relapse and Diseases of the Central Nervous System”

Scientists at the Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology at Nanjing University in China have shown that exosomes modified to express the neuron-specific rabies viral glycoprotein (RVG) peptide on their membrane surface can be used to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. In an open-access article published online today (December 3, 2015) in Scientific Reports, the researchers described results demonstrating that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in these exosomes. The modified exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain, the scientists said. Functionally, the siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. The new article is titled “Targeted Exosome-Mediated Delivery of Opioid Receptor Mu siRNA for the Treatment of Morphine Relapse.” As background, the researchers said that MOR is a major target of opioid drugs and appears to play critical roles in mediating the major effects of these drugs, including analgesia, tolerance, abuse, dependence, and respiratory depression. It has been reported, the authors said, that the rewarding effect of morphine, mediated by MOR, is abolished in MOR-deficient mice and that a MOR antagonist diminished the consequences of an initial opioid drug relapse. Therefore, the researchers selected the MOR as a target for the treatment of drug addiction.

First Report of Hibernating Primate Existing Outside Madagascar; Pygmy Loris in Southeast Asia Newly Shown to Hibernate; Possibly Major Clue to Better Understanding of Evolution of Hibernation

Up until now, three species of lemurs on Madagascar were the only primates known to hibernate. Researchers at Vetmeduni Vienna in Austria, have now reported, for the first time, that another primate species, the pygmy slow loris that lives in Vietnam, Cambodia, Laos, and China, the pygmy slow loris, also uses hibernation to save energy. The results were published online today (December 3, 2015) in an open-access article in Scientific Reports. The article is titled “Hibernation in the Pygmy Slow Loris (Nycticebus Pygmaeus): Multiday Torpor In Primates Is Not Restricted to Madagascar.” Hibernation is a state of energy conservation during which body temperature and metabolism are drastically reduced. If this state lasts longer than 24 hours, it is called hibernation. Shorter periods are called daily torpor. There are many mammals that hibernate. However, among primates hibernation is a rare capability, as it had been previously found in only three species of lemurs. Lemurs live exclusively on the island of Madagascar off the east coast of Africa, where they hibernate during the dry season, mainly to conserve water. Now a team at the Research Institute of Wildlife Ecology at the Vetmeduni Vienna, collaborating with colleagues from the Vietnamese Endangered Primate Rescue Center, has discovered another primate that hibernates: the pygmy slow loris (Nycticebus pygmaeus). These animals belong to the group of so-called “wet nosed primates.” They reach a body length of 7.7 inches to 9.1 inches and a body mass (adult males) of approximately 15 ounces. They live in Southeast Asia and are nocturnal, tree-living (arboreal) animals. The researchers investigated the body temperatures of five pygmy lorises in fall, winter, and spring in a Vietnam primate reservation.