A team led by Massachusetts General Hospital (MGH) researchers has developed a faster way to engineer synthetic enzymes that target specific DNA sequences for inactivation, repair or alteration. The report from the MGH Molecular Pathology Unit, being published online in Nature Methods, describes a highly effective but less labor-intensive way to generate powerful tools called zinc-finger nucleases (ZFNs).
"With our approach, called context-dependent assembly, any scientist can use either standard molecular biology techniques or commercial DNA synthesis to design ZFNs for their target gene of interest," says J. Keith Joung, MD, PhD, associate chief for Research in MGH Pathology, the study's senior author. "ZFNs are broadly applicable, powerful tools for manipulating the genomes of cells from various organisms – including humans – and may provide a way to efficiently correct gene mutations responsible for human disease, avoiding problems resulting from the imprecise nature of current gene therapy approaches using viral vectors."
Most human transcription factors that control whether a genetic signal is translated into a protein bind to specific DNA sequences using peptides called zinc fingers. Zinc-finger nucleases are synthetic "designer" proteins combining a zinc-finger domain, engineered to bind a particular DNA sequence, with an enzyme that breaks both DNA strands at the targeted site. While ZFNs have great potential, creating the customized proteins has been challenging.
In the simplest approach, called modular assembly, individual peptides are linked together like beads on a string to create a multi-finger protein theoretically able to recognize long DNA segments. Joung and others have shown that, in practice, modular assembly has a very low success rate for creating multi-finger proteins. This high failure rate is most likely due to "context-dependent" effects that individual zinc fingers can have on the DNA-binding activities of their neighboring fingers. Assembling peptides that don't work well together would be like trying to put together jigsaw puzzle pieces that don't fit.
In 2008, Joung and colleagues at the University of Minnesota and other institutions, members of the Zinc Finger Consortium, reported developing a method called OPEN (Oligomerized Pool ENgineering), which takes these context-dependent effects into account. But although OPEN works well, it can be labor intensive and extremely time consuming – requiring up to a year for a lab to establish the technology and two months of work to generate desired ZFNs. To address these limitations, the MGH research team has assembled an extensive archive of zinc fingers known to work well when positioned together – in essence puzzle pieces that already have been put together. Using this context-dependent method, the investigators were able to assemble dozens of ZFNs in as little as four days.
"With this archive in hand, any researcher can easily generate their own ZFNs in less than a week, and no special expertise is needed," Joung explains. "In addition to being much faster, context-dependent assembly can generate large numbers of ZFNs simultaneously, which is hard to do with OPEN because it is more labor intensive." As was the case with OPEN, the Joung lab and the Zinc Finger Consortium (http://www.zincfingers.org) will make the software and reagents required to practice context-dependent assembly available to all academic laboratories.
"One of the holy grails of genetics is the ability to make targeted changes to individual genes," says Laurie Tompkins, PhD, who over sees genetics grants at the National Institute of General Medical Sciences, one of the National Institutes of Health and a major supporter of this study. "Dr. Joung and his colleagues have developed an extraordinarily simple, efficient strategy for using zinc finger technology to swap out altered versions of genes for normal ones – or vice versa – providing basic scientists and clinicians alike with a broadly applicable research tool."
Adds Joung, an associate professor of Pathology at Harvard Medical School, "At this point, I believe that context-dependent assembly will have the biggest impact on researchers using ZFNs to genetically manipulate model organisms, possibly even models developed from pluripotent stem cells. Other big impacts should be enabling researchers to create knockout mutations in a large series of genes involved in a common pathway or related to a specific disease and to use ZFNs to create comprehensive collections of mutants for every gene in an organism." Joung is also a member of the MGH Center for Computational and Integrative Biology and Center for Cancer Research
The challenges posed to scientists interested in using ZFNs in their investigations were described in an article in the Fall 2010 issue of the MGH-sponsored magazine Proto, which can be accessed at http://protomag.com/assets/zinc-fingers-entry-fee.
Jeffry Sander, PhD, of the MGH Molecular Pathology Unit is lead author of the Nature Methods report. Additional co-authors are Elizabeth Dahlborg, Mathew Goodwin, Jessica Blackburn, Stacey Thibodeau-Beganny, Morgan Maeder, Cyd Khayter and David Langenau, MGH Molecular Pathology; Lindsay Cade, Randall Peterson and Jing-Ruey Yeh, MGH Cardiovascular Research Center; Feng Zhang, Shaun Curtin, Yiping Qi, Christopher Pierick, Robert Stupar and Daniel Voytas, University of Minnesota; Daniel Cifuentes, Ellen Hoffman and Antonio Giraldez, Yale University; and Deepak Reyon and Drena Dobbs, Iowa State University. Support for the study includes grants from the National Institutes of Health and the National Science Foundation.
Massachusetts General Hospital, established in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $600 million and major research centers in AIDS, cardiovascular research, cancer, computational and integrative biology, cutaneous biology, human genetics, medical imaging, neurodegenerative disorders, regenerative medicine, systems biology, transplantation biology and photomedicine.
Researchers identify potentially druggable mutant p53 proteins that promote cancer growth
09.12.2016 | Cold Spring Harbor Laboratory
Plant-based substance boosts eyelash growth
09.12.2016 | Fraunhofer-Institut für Angewandte Polymerforschung IAP
Physicists of the University of Würzburg have made an astonishing discovery in a specific type of topological insulators. The effect is due to the structure of the materials used. The researchers have now published their work in the journal Science.
Topological insulators are currently the hot topic in physics according to the newspaper Neue Zürcher Zeitung. Only a few weeks ago, their importance was...
In recent years, lasers with ultrashort pulses (USP) down to the femtosecond range have become established on an industrial scale. They could advance some applications with the much-lauded “cold ablation” – if that meant they would then achieve more throughput. A new generation of process engineering that will address this issue in particular will be discussed at the “4th UKP Workshop – Ultrafast Laser Technology” in April 2017.
Even back in the 1990s, scientists were comparing materials processing with nanosecond, picosecond and femtosesecond pulses. The result was surprising:...
Have you ever wondered how you see the world? Vision is about photons of light, which are packets of energy, interacting with the atoms or molecules in what...
A multi-institutional research collaboration has created a novel approach for fabricating three-dimensional micro-optics through the shape-defined formation of porous silicon (PSi), with broad impacts in integrated optoelectronics, imaging, and photovoltaics.
Working with colleagues at Stanford and The Dow Chemical Company, researchers at the University of Illinois at Urbana-Champaign fabricated 3-D birefringent...
In experiments with magnetic atoms conducted at extremely low temperatures, scientists have demonstrated a unique phase of matter: The atoms form a new type of quantum liquid or quantum droplet state. These so called quantum droplets may preserve their form in absence of external confinement because of quantum effects. The joint team of experimental physicists from Innsbruck and theoretical physicists from Hannover report on their findings in the journal Physical Review X.
“Our Quantum droplets are in the gas phase but they still drop like a rock,” explains experimental physicist Francesca Ferlaino when talking about the...
16.11.2016 | Event News
01.11.2016 | Event News
14.10.2016 | Event News
09.12.2016 | Life Sciences
09.12.2016 | Ecology, The Environment and Conservation
09.12.2016 | Health and Medicine