News Article | August 26, 2016
The genome-editing system known as CRISPR allows scientists to delete or replace any target gene in a living cell. MIT researchers have now added an extra layer of control over when and where this gene editing occurs, by making the system responsive to light. With the new system, gene editing takes place only when researchers shine ultraviolet light on the target cells. This kind of control could help scientists study in greater detail the timing of cellular and genetic events that influence embryonic development or disease progression. Eventually, it could also offer a more targeted way to turn off cancer-causing genes in tumor cells. “The advantage of adding switches of any kind is to give precise control over activation in space or time,” said Sangeeta Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Electrical Engineering and Computer Science at MIT and a member of MIT’s Koch Institute for Integrative Cancer Research and its Institute for Medical Engineering and Science. Bhatia is the senior author of a paper describing the new technique in the journal Angewandte Chemie. The paper’s lead author is Piyush Jain, a postdoc in MIT’s Institute for Medical Engineering and Science. Before coming to MIT, Jain developed a way to use light to control a process called RNA interference, in which small strands of RNA are delivered to cells to temporarily block specific genes. “While he was here, CRISPR burst onto the scene and he got very excited about the prospect of using light to activate CRISPR in the same way,” Bhatia said. CRISPR relies on a gene-editing complex composed of a DNA-cutting enzyme called Cas9 and a short RNA strand that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut. When Cas9 and the guide RNA are delivered into cells, a specific cut is made in the genome; the cells’ DNA repair processes glue the cut back together but permanently delete a small portion of the gene, making it inoperable. In previous efforts to create light-sensitive CRISPR systems, researchers have altered the Cas9 enzyme so that it only begins cutting when exposed to certain wavelengths of light. The MIT team decided to take a different approach and make the binding of the RNA guide strand light-sensitive. For possible future applications in humans, it could be easier to deliver these modified RNA guide strands than to program the target cells to produce light-sensitive Cas9, Bhatia said. “You really don’t have to do anything different with the cargo you were planning to deliver except to add the light-activated protector,” she said. “It’s an attempt to make the system much more modular.” To make the RNA guide strands light-sensitive, the MIT team created “protectors” consisting of DNA sequences with light-cleavable bonds along their backbones. These DNA strands can be tailored to bind to different RNA guide sequences, forming a complex that prevents the guide strand from attaching to its target in the genome. When the researchers expose the target cells to light with a wavelength of 365 nanometers (in the ultraviolet range), the protector DNA breaks into several smaller segments and falls off the RNA, allowing the RNA to bind to its target gene and recruit Cas9 to cut it. In this study, the researchers demonstrated that they could use light to control editing of the gene for green fluorescent protein (GFP) and two genes for proteins normally found on cell surfaces and overexpressed in some cancers. “If this is really a generalizable scheme, then you should be able to design protector sequences against different target sequences,” Bhatia said. “We designed protectors against different genes and showed that they all could be light-activated in this way. And in a multiplexed experiment, when a mixed population of protectors was used, the only targets that were cleaved after light exposure were those being photo-protected.” This precise control over the timing of gene editing could help researchers study the timing of cellular events involved in disease progression, in hopes of determining the best time to intervene by turning off a gene. “CRISPR-Cas9 is a powerful technology that scientists can use to study how genes affect cell behavior,” said James Dahlman, an assistant professor of biomedical engineering at Georgia Tech, who was not involved in the research. “This important advance will enable precise control over those genetic changes. As a result, this work gives the scientific community a very useful tool to advance many gene editing studies.” Bhatia’s lab is also pursuing medical applications for this technique. One possibility is using it to turn off cancerous genes involved in skin cancer, which is a good target for this approach because the skin can be easily exposed to ultraviolet light. The team is also working on a “universal protector” that could be used with any RNA guide strand, eliminating the need to design a new one for each RNA sequence, and allowing it to inhibit CRISPR-Cas9 cleavage of many targets at once. The research was funded by the Ludwig Center for Molecular Oncology, the Marie-D. and Pierre Casimir-Lambert Fund, a Koch Institute Support Grant from the National Cancer Institute, and the Marble Center for Cancer Nanomedicine.
Researchers have identified a gene that increases the risk of schizophrenia, and they say they have a plausible theory as to how this gene may cause the devastating mental illness. After conducting studies in both humans and mice, the researchers said this new schizophrenia risk gene, called C4, appears to be involved in eliminating the connections between neurons — a process called "synaptic pruning," which, in humans, happens naturally in the teen years. It's possible that excessive or inappropriate "pruning" of neural connections could lead to the development of schizophrenia, the researchers speculated. This would explain why schizophrenia symptoms often first appear during the teen years, the researchers said. Further research is needed to validate the findings, but if the theory holds true, the study would mark one of the first times that researchers have found a biological explanation for the link between certain genes and schizophrenia. It's possible that one day, a new treatment for schizophrenia could be developed based on these findings that would target an underlying cause of the disease, instead of just the symptoms, as current treatments do, the researchers said. "We're far from having a treatment based on this, but it's exciting to think that one day, we might be able to turn down the pruning process in some individuals and decrease their risk" of developing the condition, Beth Stevens, a neuroscientist who worked on the new study, and an assistant professor of neurology at Boston Children's Hospital, said in a statement. The study, which also involved researchers at the Broad Institute's Stanley Center for Psychiatric Research at Harvard Medical School, is published today (Jan. 27) in the journal Nature. [Top 10 Mysteries of the Mind] From previous studies, the researchers knew that one of the strongest genetic predictors of people's risk of schizophrenia was found within a region of DNA located on chromosome 6. In the new study, the researchers focused on one of the genes in this region, called complement component 4, or C4, which is known to be involved in the immune system. Using postmortem human brain samples, the researchers found that variations in the number of copies of the C4 gene that people had, and the length of their gene, could predict how active the gene was in the brain. The researchers then turned to a genome database, and pulled information about the C4 gene in 28,800 people with schizophrenia, and 36,000 people without the disease, from 22 countries. From the genome data, they estimated people's C4 gene activity. They found that the higher the levels of C4 activity were, the greater a person's risk of developing schizophrenia was. The researchers also did experiments in mice, and found that the more C4 activity there was, the more synapses were pruned during brain development. Previous studies found that people with schizophrenia have fewer synapses in certain brain areas than people without the condition. But the new findings "are the first clear evidence for a molecular and cellular mechanism of synaptic loss in schizophrenia," said Jonathan Sebat, chief of the Beyster Center for Molecular Genomics of Neuropsychiatric Diseases at the University of California, San Diego, who was not involved in the study. Still, Sebat said that the studies in mice are preliminary. These experiments looked for signs of synaptic pruning in the mice but weren't able to directly observe the process occurring. More detailed studies of brain maturation are now needed to validate the findings, Sebat said. In addition, it remains to be seen whether synaptic pruning could be a target for antipsychotic drugs, but "it's promising," Sebat said. There are drugs in development to activate the part of the immune system in which C4 is involved, Sebat noted. Copyright 2016 LiveScience, a Purch company. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
Sometimes chemists set themselves up for a surprise. Following sets of experiments in which something doesn’t happen and doesn’t seem likely to happen, they soon believe it never will. Until it does. Chemists have traditionally thought of cyclopentadienyl ligands as being “innocent,” which means they offer electronic support to a metal catalyst but generally don’t do anything chemically. The two groups were studying reactions involving Cp*Rh(bipyridine), often used in hydrogenation reactions and in hydrogen-forming reactions, when they found that the expected metal hydride intermediate was followed by formation of an unexpected intermediate in which the hydrogen had migrated to one of the carbon atoms in the Cp* ring. “These two reports showing that the seemingly innocent Cp* ligand can reversibly form a C–H bond by proton transfer from rhodium hydride are remarkable,” comments chemistry professor David Milstein of the Weizmann Institute of Science, who was not involved in the research. “Considering the ubiquity of cyclopentadienyl metal complexes in homogeneous catalysis, this pathway should be seriously considered in the design and understanding of reactions in which proton/hydride transfer may be involved.” Alexander J. M. Miller of the University of North Carolina, Chapel Hill, who led one of the teams, says chemists had previously worked out mechanisms involving hydride intermediates that made sense and thought the story ended there. But they did not exercise due diligence and poke around enough to see that a protonated Cp* intermediate, denoted Cp*H, could be involved as well. “What’s more surprising,” Miller points out, “the Cp*H complex is not a dead end. This diene complex is still an active catalyst.” Miller’s group came across the Cp*H intermediate while investigating hydride transfer reactions with the cellular enzyme cofactor nicotinamide adenine dinucleotide (NAD+) to form the reduced product NADH (Chem. Commun. 2016, DOI: 10.1039/c6cc00575f). Meanwhile, a team led by Harry B. Gray and Jay R. Winkler at Caltech and James D. Blakemore at the University of Kansas discovered the Cp*H intermediate while investigating the coupling of protons to form H when treating Cp*Rh(bipyridine) with acid (Proc. Natl. Acad. Sci. USA 2016, DOI: 10.1073/pnas.1606018113). “These discoveries illustrate the versatility of mechanisms by which protons and hydrides can be delivered to and from metals,” comments Morris Bullock, director of the Center for Molecular Electrocatalysis at Pacific Northwest National Laboratory. “While these examples are for rhodium, the prevalence of cyclopentadienyl ligands in organometallic catalysts raises the possibility that similar reactivity could be widespread and involve other metals, and may be intentionally exploited in the design of new catalysts.”
Cardiac glycosides, which are bioactive natural products found in certain plants and insects, aid in cardiac treatment because they cause the heart to contract and increase cardiac output. They are used in prescription medications such as Digitoxin and Strophanthin. Now researchers at Yale have also discovered that cardiac glycosides block the repair of DNA in tumor cells. Because tumor cells are rapidly dividing, their DNA is more susceptible to damage, and inhibition of DNA repair is a promising strategy to selectively kill these cells. Several other researchers have noted that cardiac glycosides possess anticancer properties, but the basis for these effects was not well known. The Yale scientists showed that cardiac glycosides inhibit two key pathways that are involved in the repair of DNA. "We performed a high-content drug screen with the Yale Center for Molecular Discovery, which identified some interesting cardiac drugs that affect DNA repair," said Ranjit Bindra, assistant professor of therapeutic radiology and of pathology at the Yale School of Medicine. "This has many therapeutic implications for new cancer drugs." Bindra and Yale professor of chemistry Seth Herzon are the principal investigators of the study, which appears in the Journal of the American Chemical Society. Herzon and Bindra also are members of the Yale Cancer Center. "Our approach focused on damaging the cancer cells' DNA using radiation, and then measuring the rate of repair in the presence of different compounds. All in all, we evaluated 2,400 compounds," Herzon said. "Surprisingly, we think that the cardiac glycosides inhibit the retention of a key DNA repair protein known as 53BP1 at the site of DNA double-strand breaks. This is a very interesting activity that was unexpected." Herzon and Bindra said the same approach can be applied to screen hundreds of thousands of compounds. "We are partnering with industry to gain access to their large compound collections. Not only will this help us find new anticancer agents, it can help us elucidate more of the fundamental biology underlying DNA repair," Herzon said. The next step in their research will be to improve the cancer-fighting properties of cardiac glycosides, while modulating their other biological effects. Explore further: Rare byproduct of marine bacteria kills cancer cells by snipping their DNA
Scientists designed, created, and tested a chromium (Cr) complex, finding that a novel phosphorus-containing ring structure helps chromium turn dinitrogen and acid into ammonia. This work is part of efforts to develop molecular complexes to control electrons and protons for use in turning renewable energy into storable fuels. Credit: Jonathan Darmon, PNNL Underappreciated compared to its heavier metal counterparts, chromium failed for more than 30 years to turn nitrogen gas into ammonia, a reaction that involves breaking one tough bond and making six new ones. But scientists at the Center for Molecular Electrocatalysis thought chromium was up to the job; it just needed a little support. At the center, one of DOE's Energy Frontier Research Centers (EFRCs), the scientists created a 12-atom ring structure called a ligand that partially surrounds the metal and offers a stable environment for the metal to drive the reaction. By creating this ligand structure, the team demonstrated the importance of the environment supporting chromium. Often a key to controlling metal reactivity, the structure encircling the chromium causes the normally unreactive dinitrogen to become more reactive when it binds to the metal. "This research required the synergy of experimental and computational efforts in an EFRC," said the study's lead Dr. Michael Mock at DOE's Pacific Northwest National Laboratory. "Studying this challenging reaction has benefited from the multiple years of funding that an EFRC enables." The EFRCs are funded by the Office of Basic Energy Sciences at DOE's Office of Science. Producing ammonia for fertilizer consumes vast quantities of energy, an issue that this work may one day help solve. However, this study is focused on another important challenge: storing intermittent wind and solar energy. Solar panels and wind turbines produce electrons that flow along power lines to energize appliances around our homes. But, solar power levels drop when the clouds roll in. What if those electrons could be stored inside a chemical bond, as an energy-dense storage option? This study, which is complemented by two previous reports focused on understanding dinitrogen reactivity with chromium, may someday lead to the development of a system with this common metal as a hard-working catalyst. "This research shows how important it is to move six electrons and six protons in the right order," said Dr. Roger Rousseau, who led the computational studies. "It is rather like herding cats-and very difficult cats at that." There is a long tradition of turning dinitrogen (N2) into ammonia (NH3) using complex molecular catalysts, materials that reduce the roadblocks to make the reaction occur and aren't consumed in the process. Of the metals studied in the column known as group 6 transition metals, chromium supported by phosphorus ligands didn't work. In fact, papers from 1970 to the present day reported failures using chromium even in an environment that was thought to goad it into working. However, Mock and his team focused on the stabilizing effect from the phosphorus atoms of a 12-membered ligand that partially surrounded the chromium metal. Every fourth atom in the ring is a phosphorus atom that forms a bond with the chromium atom. The chemical bonds formed with three phosphorus atoms of the large ring together with two additional phosphorus donor atoms of a second ligand make the chromium atom very electron rich, which then can bind the dinitrogen. Once bound, the dinitrogen triple bond is weakened by coordination to the metal. The team showed that the correct surroundings enhance chromium's ability to bind and activate dinitrogen. In fact, the dinitrogen molecule in this case is more activated than in similar complexes with the heavier metals, molybdenum and tungsten, which have similar properties to chromium. However, breaking the dinitrogen triple bond is still a delicate task. The team found that managing the number of phosphorus atoms and the electron-donating ability of these atoms was crucial. The team ran the reactions with acid at -50°C so that certain intermediate products containing nitrogen-hydrogen bonds didn't fall apart. In these reactions, hydrogen ions from the acid surrounding the complex formed only a small amount of ammonia. They showed that adding acid caused the protons to favor binding with the metal, an unwanted connection. Additional optimization of the chromium complex and the conditions is required to control the formation of the desired nitrogen-hydrogen bonds. The reaction still has secrets to reveal. The team is digging into two of them. First, how do the 12-membered rings that support the chromium form? In the experiments, the rings self-assemble around the chromium. What factors dictate that formation? Also, how can the protons be controlled to prevent them from binding to the electron-rich chromium and form additional bonds with nitrogen? Answering these questions could lead to learning how to control the reaction's environs and lead to a catalyst that is fast, efficient, and long lasting, to convert nitrogen to ammonia. Explore further: Converting Nitrogen to a More Useful Form More information: Michael T. Mock et al. Protonation Studies of a Mono-Dinitrogen Complex of Chromium Supported by a 12-Membered Phosphorus Macrocycle Containing Pendant Amines, Inorganic Chemistry (2015). DOI: 10.1021/acs.inorgchem.5b00351