Stanford bioengineers develop tool for reprogramming genetic code

Stanford bioengineers have developed a new tool that allows them to preferentially activate or deactivate genes in living cells. VITSTUDIO/SHUTTERSTOCK

Biology relies upon the precise activation of specific genes to work properly. If that sequence gets out of whack, or one gene turns on only partially, the outcome can often lead to a disease.

Now, bioengineers at Stanford and other universities have developed a sort of programmable genetic code that allows them to preferentially activate or deactivate genes in living cells. The work is published in the current issue of Cell, and could help usher in a new generation of gene therapies.

The technique is an adaptation of CRISPR, itself a relatively new genetic tool that makes use of a natural defense mechanism that bacteria evolved over millions of years to slice up infectious virus DNA.

Standard CRISPR consists of two components: a short RNA that matches a particular spot in the genome, and a protein called Cas9 that snips the DNA in that location. For the purposes of gene editing, scientists can control where the protein snips the genome, insert a new gene into the cut and patch it back together.

Inserting new genetic code, however, is just one way to influence how the genome is expressed. Another involves telling the cell how much or how little to activate a particular gene, thus controlling how much protein a cell produces from that gene and altering its behavior.

It's this action that Lei Stanley Qi, an assistant professor of bioengineering and of chemical and systems biology at Stanford, and his colleagues aim to manipulate.

Influencing the genome
In the new work, the researchers describe how they have designed the CRISPR molecule to include a second piece of information on the RNA, instructing the molecule to either increase (upregulate) or decrease (downregulate) a target gene's activity, or turn it on/off entirely.

Additionally, they designed it so that it could affect two different genes at once. In a cell, the order or degree in which multiple genes are activated can produce different metabolic products.

"It's like driving a car. You control the wheel to control direction, and the engine to control the speed, and how you balance the two determines how the car moves," Qi said. "We can do the same thing in the cell by up- or downregulating genes, and produce different outcomes."

As a proof of principle, the scientists used the technique to take control of a yeast metabolic pathway, turning genes on and off in various orders to produce four different end products. They then tested it on two mammalian genes that are important in cell mobility, and were able to control the cell's direction and how fast it moved.

Future therapies
The ability to control genes is an attractive approach in designing genetic therapies for complex diseases that involve multiple genes, Qi said, and the new system may overcome several of the challenges of existing experimental therapies.

"Our technique allows us to directly control multiple specific genes and pathways in the genome without expressing new transgenes or uncontrolled behaviors, such as producing too much of a protein, or doing so in the wrong cells," Qi said. "We could eventually synthesize tens of thousands of RNA molecules to control the genome over a whole organism."

Next, Qi plans to test the technique in mice and refine the delivery method. Currently the scientists use a virus to insert the molecule into a cell, but he would eventually like to simply inject the molecules into an organism's blood.

"That is what is so exciting about working at Stanford, because the School of Medicine's immunology group is just around the corner, and working with them will help us address how to do this without triggering an immune response," said Qi, who is a member of the interdisciplinary Stanford ChEM-H institute. "I'm optimistic because everything about this system comes naturally from cells, and should be compatible with any organism."

Source: Stanford university

An Advanced Method of DNA Nanostructure Formation Developed

Figure 1: Uni-molecular magnetic tweezers orchestrating the DNA nanostructure formation

Professor Tae-Young Yoon’s research team from the Department of Physics at KAIST has developed a new method to form DNA nanostructures by using magnetic tweezers to observe and to induce the formation of the structure in real time.

Unlike traditional designs of "DNA origami" which relies on thermal or chemical annealing methods, the new technology utilizes a completely different dynamic in DNA folding. This allows the folding to be done within only ten minutes.

Developed in 2006, the "DNA origami" allows a long skeleton of DNA to be folded into an arbitrary structure by using small stapler DNA pieces. This has been a prominent method in DNA nanotechnology.
 

Figure 2: The evolution of DNA nanostructure formation using magnetic tweezers. The DNA nanostructure with a 21-nanometer size was formed in about eight minutes.

However, the traditional technology which adopts thermal processes could not control the DNA formation during the folding because every interaction among DNAs occurs simultaneously. Thus, the thermal processes, which take dozens of hours to complete, had to be repeated multiple times in order to find the optimal condition.

The research team designed a DNA folding using uni-molecular magnetic tweezers that applied force to a single DNA molecule while measuring the state of the DNA. Through this technology, they were able to induce the formation of DNA nanostructure and observe it at the same time.

During high temperature heat treatment, the first stage of conventional thermal processes, the internal structure of the long skeleton DNA untangles. To induce such state, after attaching one side of the skeleton DNA to the surface of glass and the other side to a magnetic material, the team unfolded the internal structure of the DNA by pulling the two sides apart with magnetic force.

Unlike the conventional thermal processes, this method lets the stapler DNA swiftly adhere to the skeleton DNA within a minute because the sites are revealed at room temperature.

After the stapler pieces connected to the skeleton, the team removed the magnetic force. Next, the structure folded through self-assembly as the stapler DNAs stuck to different sites on the skeleton DNA.

Professor Yoon said, “With the existing thermal methods, we could not differentiate the reactions of the DNA because the response of each DNA pieces mutually interacted with each other.” He added that “Using the magnetic tweezers, we were able to sort the process of DNA nanostructure formation into a series of reactions of DNA molecules that are well known, and shorten the time taken for formation in only ten minutes.”

He commented, “This nanostructure formation method will enable us to create more intricate and desirable DNA nanostructures by programming the folding of DNA origami structures.”

Conducted by Dr. Woori Bae under the guidance of Professor Yoon, the research findings were published online in the December 4th issue of Nature Communications.


Source: KAIST

Breathing in diesel exhaust leads to changes 'deep under the hood'

A student participates in the study while seated in a booth.
Credit: Image courtesy of University of British Columbia

Just two hours of exposure to diesel exhaust fumes can lead to fundamental health-related changes in biology by switching some genes on, while switching others off, according to researchers at the University of British Columbia and Vancouver Coastal Health.

The study involved putting volunteers in a polycarbonate-enclosed booth -- about the size of a standard bathroom -- while breathing in diluted and aged exhaust fumes that are about equal to the air quality along a Beijing highway, or a busy port in British Columbia.

The researchers examined how such exposure affected the chemical "coating" that attaches to many parts of a person's DNA. That carbon-hydrogen coating, called methylation, can silence or dampen a gene, preventing it from producing a protein -- sometimes to a person's benefit, sometimes not. Methylation is one of several mechanisms for controlling gene expression, which is the focus of a rapidly growing field of study called epigenetics.

The study, published this month in Particle and Fibre Toxicology, found that diesel exhaust caused changes in methylation at about 2,800 different points on people's DNA, affecting about 400 genes. In some places it led to more methylation; in more cases, it decreased methylation.

How these changes in gene expression translate to health is the next step for researchers. But this study shows how vulnerable our genetic machinery can be to air pollution, and that changes are taking place even if there are no obvious symptoms.

"Usually when we look at the effects of air pollution, we measure things that are clinically obvious -- air flow, blood pressure, heart rhythm," said senior author Dr. Chris Carlsten, an associate professor in the Division of Respiratory Medicine. "But asthma, higher blood pressure or arrhythmia might just be the gradual accumulation of epigenetic changes. So we've revealed a window into how these long-term problems arise. We're looking at changes 'deep under the hood.'"

The fact that DNA methylation was affected after only two hours of exposure has positive implications, said Carlsten, the AstraZeneca Chair in Occupational and Environmental Lung Disease.

"Any time you can show something happens that quickly, it means you can probably reverse it -- either through a therapy, a change in environment, or even diet," he said.

Carlsten's team, having catalogued the changes along the entire human genome, is now sharing its data with scientists who are further exploring the function of specific genes.

Source: University of British Columbia

Charged graphene gives DNA a stage to perform molecular gymnastics

DNA interacts with charged graphene and contorts into sequence-specific shapes when the charge is changed. Credit: Photo courtesy Alek Aksimentiev

When Illinois researchers set out to investigate a method to control how DNA moves through a tiny sequencing device, they did not know they were about to witness a display of molecular gymnastics.

Fast, accurate and affordable DNA sequencing is the first step toward personalized medicine. Threading a DNA molecule through a tiny hole, called a nanopore, in a sheet of graphene allows researchers to read the DNA sequence; however, they have limited control over how fast the DNA moves through the pore. In a new study published in the journal Nature Communications, University of Illinois physics professor Aleksei Aksimentiev and graduate student Manish Shankla applied an electric charge to the graphene sheet, hoping that the DNA would react to the charge in a way that would let them control its movement down to each individual link, or nucleotide, in the DNA chain.

"Ideally, you would want to step the DNA through the nanopore one nucleotide at a time," said Aksimentiev. "Take a measurement and then have another nucleotide in the sensing hole. That's the goal, and it hasn't been realized yet. We show that, to some degree, we can control the process by charging the graphene."

The researchers found that a positive charge in the graphene speeds up DNA movement through the nanopore, while a negative charge stops the DNA in its tracks. However, as they watched, the DNA seemed to dance across the graphene surface, pirouetting into shapes they had never seen, specific to the sequence of the DNA nucleotides.

"It reminds me of Swan Lake," Aksimentiev said. "It's very acrobatic. We were very surprised by the variety of DNA conformations that we can observe at the surface of graphene when we charge it. There is one sequence that starts out laying down on the surface, and when we change the charge, they all tilt on the side like they are doing a one-armed push-up. Then we also have nucleotides that would lay back, or go up like a ballerina en pointe."

Aksimentiev hypothesizes that the conformations are so different and so specific to the sequence because each nucleotide has a slightly different distribution of electrons, the negatively charged parts of the atoms. There is even a visible difference when a nucleotide is methylated, a tiny chemical change that can turn a gene on or off.

By switching the charge in the graphene, the researchers can control not only the DNA's motion through the pore, but also the shape the DNA contorts into.

"Because it's reversible, we can force it to adopt one conformation and then force it to go back. That's why we call it gymnastics," Aksimentiev said.

The researchers extensively used the Blue Waters supercomputer at the National Center for Supercomputing Applications, housed at the University of Illinois. They mapped each individual atom in the complex DNA molecule and ran numerous simulations of many different DNA sequences. Supercomputing power was essential to carrying out the work, Aksimentiev said.

"This is a really computationally intensive project," he said. "Having access to Blue Waters was essential because with the sheer number of simulations, we would not have been able to finish them. It would have taken too long."

The next step is to combine a charged nanopore setup with a sensor to build a DNA sequencing device that would incorporate both motion control and nucleotide recognition. The researchers also hope to explore the unexpected conformational changes for insights into epigenetics, the field that studies how genes are expressed and moderated.

"DNA is much more complicated than just a double helix. It's a complex molecule that has many properties, and we are still uncovering them," Aksimentiev said.

Video animation of DNA dancing as the graphene charge changes:


Source: University of Illinois at Urbana-Champaign

A lab in your pocket: Using CAD to load dozens of tests on a lab-on-a-chip

Two computer-generated configurations for routing a droplet through multiple lab-on-a-chip diagnostic tests, many more than are currently possible using manual methods. The software was developed by Michigan Tech's Shiyan Hu and Chen Liao. The figure is reproduced with permission of IEEE Transactions on NanoBioscience. Credit: Chen Liao and Shiyan Hu

When you get sick, your physician may take a sample of your blood, send it to the lab and wait for results. In the near future, however, doctors may be able to run those tests almost instantly on a piece of plastic about the size of credit card.

These labs-on-a-chip would not only be quick -- results are available in minutes -- but also inexpensive and portable. They could be used miles from the nearest medical clinic to test for anything from HIV to diabetes. But as powerful as they may be, they could be far better, says Shiyan Hu, an associate professor of electrical and computer engineering at Michigan Technological University.

Generally, a lab-on-a-chip (LOC) can run no more than a test or two. That's because the chips are designed manually, says Hu. If the LOC were made using computer-aided design, you could run dozens of tests with a single drop of blood.

"In a very short time, you could test for many conditions," he said. "This really would be an entire lab on a chip."

With PhD student Chen Liao, Hu has taken the first step. "We have developed software to design the hardware," he said. Their work focuses on routing the droplet of blood or other fluid through each test on the chip efficiently while avoiding any chip contamination.

"It has taken us four years to do the software, but to manufacture the LOC would be inexpensive," Hu said. "The materials are very cheap, and the results are more accurate than a conventional lab's."

Ultimately, Hu aims to fabricate their own biochip using their software.

Their work was featured on the cover of the March edition of IEEE Transactions on Nanobiosciences and described in the article "Physical-Level Synthesis for Digital Lab-On-a-Chip Considering Variation, Contamination, and Defect." Liao was partially supported by an A. Richard Newton Graduate Scholarship, awarded by the Design Automation Conference.

Source: Michigan Technological University

Identifying gene-enhancers: New technique

Diane Dickel is the lead author of Nature Methods paper describing a new technique for identifying gene enhancers in the genomes of humans and other mammals. Credit: Roy Kaltschmidt

An international team led by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) has developed a new technique for identifying gene enhancers -- sequences of DNA that act to amplify the expression of a specific gene -- in the genomes of humans and other mammals. Called SIF-seq, for site-specific integration fluorescence-activated cell sorting followed by sequencing, this new technique complements existing genomic tools, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), and offers some additional benefits.

"While ChIP-seq is very powerful in that it can query an entire genome for characteristics associated with enhancer activity in a single experiment, it can fail to identify some enhancers and identify some sites as being enhancers when they really aren't," says Diane Dickel, a geneticist with Berkeley Lab's Genomics Division and member of the SIF-seq development team. "SIF-seq is currently capable of testing only hundreds to a few thousand sites for enhancer activity in a single experiment, but can determine enhancer activity more accurately than ChIP-seq and is therefore a very good validation assay for assessing ChIP-seq results."

Dickel is the lead author of a paper in Nature Methods describing this new technique. The paper is titled "Function-based identification of mammalian enhancers using site-specific integration." The corresponding authors are Axel Visel and Len Pennacchio, also geneticists with Berkeley Lab's Genomics Division.

With the increasing awareness of the important role that gene enhancers play in normal cell development as well as in disease, there is strong scientific interest in identifying and characterizing these enhancers. This is a challenging task because an enhancer does not have to be located directly adjacent to the gene whose expression it regulates, but can instead be located hundreds of thousands of DNA base pairs away. The challenge is made even more difficult because the activity of many enhancers is restricted to specific tissues or cell types.

"For example, brain enhancers will not typically work in heart cells, which means that you must test your enhancer sequence in the correct cell type," Dickel says.

Currently, enhancers can be identified through chroma­tin-based assays, such as ChIP-seq, which predict enhancer elements indirectly based on the enhancer's association with specific epigenomic marks, such as transcription factors or molecular tags on DNA-associated histone proteins. Visel, Pennacchio, Dickel and their colleagues developed SIF-seq in response to the need for a higher-throughput functional enhancer assay that can be used in a wide variety of cell types and devel­opmental contexts.

"We've shown that SIF-seq can be used to identify enhancers active in cardiomyocytes, neural progenitor cells, and embryonic stem cells, and we think that it has the potential to be expanded for use in a much wider variety of cell types," Dickel says. "This means that many more types of enhancers could potentially be tested in vitro in cell culture."

In SIF-seq, hundreds or thousands of DNA fragments to be tested for enhancer activity are coupled to a reporter gene and targeted into a single, reproducible site in embryonic cell genomes. Every embryonic cell will have exactly one potential enhancer-reporter. 

Fluorescence-activated sorting is then used to identify and retrieve from this mix only those cells that display strong reporter gene expression, which represent the cells with the most active enhancers.

"Unlike previous enhancer assays for mammals, SIF-seq includes the integration of putative enhancers into a single genomic locus," says Visel. "Therefore, the activity of enhancers is assessed in a reproducible chromosomal context rather than from a transiently expressed plasmid. Furthermore, by making use of embryonic stem cells and in vitro differentia­tion, SIF-seq can be used to assess enhancer activity in a wide variety of disease-relevant cell types."

Adds Pennacchio, "The range of biologically or disease-relevant enhancers that SIF-seq can be used to identify is limited only by currently available stem cell differentiation methods. Although we did not explicitly test the activity of species-specific enhancers, such as those derived from certain classes of repetitive elements, our results strongly suggest that SIF-seq can be used to identify enhancers from other mammalian genomes where desired cell types are difficult or impossible to obtain."

The ability of SIF-seq to use reporter assays in mouse embryonic stem cells to identify human embryonic stem cell enhancers that are not present in the mouse genome opens the door to intriguing research possibilities as Dickel explains.

"Human and chimpanzee genes differ very little, so one hypothesis in evolutionary genomics holds that humans and chimpanzees are so phenotypically different because of differences in the way they regulate gene expression. It is very difficult to carry out enhancer identification through ChIP-seq that would be useful in studying this hypothesis," she says. 

"However, because SIF-seq only requires DNA sequence from a mammal and can be used in a variety of cell types, it should be possible to compare the neuronal enhancers present in a large genomic region from human to the neuronal enhancers present in the orthologous chimpanzee region. This could potentially tell us interesting things about the genetic differences that differentiate human brain development from that of other primates."

Source DOE/Lawrence Berkeley National Laboratory

Ancient Europeans intolerant to lactose for 5,000 years after they adopted agriculture

Milk

By analysing DNA extracted from the petrous bones of skulls of ancient Europeans, scientists have identified that these peoples remained intolerant to lactose (natural sugar in the milk of mammals) for 5,000 years after they adopted agricultural practices and 4,000 years after the onset of cheese-making among Central European Neolithic farmers.

The findings published online in the scientific journal Nature Communications (21 Oct) also suggest that major technological transitions in Central Europe between the Neolithic, Bronze Age and Iron Age were also associated with major changes in the genetics of these populations.

For the study, the international team of scientists examined nuclear ancient DNA extracted from thirteen individuals from burials from archaeological sites located in the Great Hungarian Plain, an area known to have been at the crossroads of major cultural transformations that shaped European prehistory. The skeletons sampled date from 5,700 BC (Early Neolithic) to 800 BC (Iron Age).

It took several years of experimentation with different bones of varying density and DNA preservation for the scientists to discover that the inner ear region of the petrous bone in the skull, which is the hardest bone and well protected from damage, is ideal for ancient DNA analysis in humans and any other mammals.

According to Professor Ron Pinhasi from the UCD Earth Institute and UCD School of Archaeology, University College Dublin, the joint senior author on the paper, "the high percentage DNA yield from the petrous bones exceeded those from other bones by up to 183-fold. This gave us anywhere between 12% and almost 90% human DNA in our samples compared to somewhere between 0% and 20% obtained from teeth, fingers and rib bones."

For the first time, these exceptionally high percentage DNA yields from ancient remains made it possible for scientists to systematically analyse a series of skeletons from the same region and check for known genetic markers including lactose intolerance.

"Our findings show progression towards lighter skin pigmentation as hunter and gatherers and non-local farmers intermarried, but surprisingly no presence of increased lactose persistence or tolerance to lactose" adds Professor Pinhasi.

"This means that these ancient Europeans would have had domesticated animals like cows, goats and sheep, but they would not yet have genetically developed a tolerance for drinking large quantities of milk from mammals," he says.

According to Professor Dan Bradley from the Smurfit Institute of Genetics, Trinity College Dublin, co-senior author on the paper, "our results also imply that the great changes in prehistoric technology including the adoption of farming, followed by the first use of the hard metals, bronze and then iron, were each associated with the substantial influx of new people. We can no longer believe these fundamental innovations were simply absorbed by existing populations in a sort of cultural osmosis."

Source: University College Dublin

Visualizing DNA double-strand break process for the first time

The enzyme I-DmoI (purple) is specifically associated to the double strand of DNA (yellow and green). Credit: CNIO

Scientists from the Spanish National Cancer Research Centre (CNIO), led by Guillermo Montoya, have developed a method for producing biological crystals that has allowed scientists to observe --for the first time-- DNA double chain breaks. They have also developed a computer simulation that makes this process, which lasts in the order of millionths of a second, visible to the human eye. The study is published today by the journal Nature Structural & Molecular Biology.

"We knew that enzymes, or proteins, endonucleases, are responsible for these double strand breaks, but we didn't know exactly how it worked until now," said Montoya. "In our study, we describe in detail the dynamics of this basic biological reaction mediated by the enzyme I-Dmol. Our observations can be extrapolated to many other families of endonucleases that behave identically."

DNA breaks occur in several natural processes that are vital for life: mutagenesis, synthesis, recombination and repair. In the molecular biology field, they can also be generated synthetically. Once the exact mechanism that produces these breaks has been uncovered, this knowledge can be used in multiple biotechnological applications: from the correction of mutations to treat rare and genetic diseases, to the development of genetically modified organisms.

Slow-motion reaction

Enzymes are highly specialised dynamic systems. Their nicking function could be compared, said Montoya, to a specially designed fabric-cutting machine that "it would only make a cut when a piece of clothing with a specific combination of colours passed under the blade."

In this case, researchers concentrated on observing the conformational changes that occurred in the I-Dmol active site; the area that contains the amino acids that act as a blade and produces DNA breaks.

By altering the temperature and pH balance, the CNIO team has managed to delay a chemical reaction that typically occurs in microseconds by up to ten days. Under those conditions, they have created a slow-motion film of the whole process.

"By introducing a magnesium cation we were able to trigger the enzyme reaction and subsequently to produce biological crystals and freeze them at -200ºC," said Montoya. "In that way, we were able to collect up to 185 crystal structures that represent all of the conformational changes taking place at each step of the reaction."

Finally, using computational analysis, the researchers illustrated the seven intermediate stages of the DNA chain separation process. "It is very exciting, because the elucidation of this mechanism will give us the information we need to redesign these enzymes and provide precise molecular scissors, which are essential tools for modifying the genome," he concluded.

Source: Centro Nacional de Investigaciones Oncologicas (CNIO)

Scientists produce cloned embryos of extinct frog

This is a gastric-brooding frog, Rheobatrachus silus, giving oral birth in the lab of Mike Tyler of the University of Adelaide. Credit: Mike Tyler, University of Adelaide

The genome of an extinct Australian frog has been revived and reactivated by a team of scientists using sophisticated cloning technology to implant a "dead" cell nucleus into a fresh egg from another frog species.

The bizarre gastric-brooding frog, Rheobatrachus silus -- which uniquely swallowed its eggs, brooded its young in its stomach and gave birth through its mouth -- became extinct in 1983.

But the Lazarus Project team has been able to recover cell nuclei from tissues collected in the 1970s and kept for 40 years in a conventional deep freezer. The "de-extinction" project aims to bring the frog back to life.

In repeated experiments over five years, the researchers used a laboratory technique known as somatic cell nuclear transfer. They took fresh donor eggs from the distantly related Great Barred Frog, Mixophyes fasciolatus, inactivated the egg nuclei and replaced them with dead nuclei from the extinct frog. Some of the eggs spontaneously began to divide and grow to early embryo stage -- a tiny ball of many living cells.

Although none of the embryos survived beyond a few days, genetic tests confirmed that the dividing cells contain the genetic material from the extinct frog.
The results are yet to be published.

"We are watching Lazarus arise from the dead, step by exciting step," says the leader of the Lazarus Project team, Professor Mike Archer, of the University of New South Wales, in Sydney. "We've reactivated dead cells into living ones and revived the extinct frog's genome in the process. Now we have fresh cryo-preserved cells of the extinct frog to use in future cloning experiments.

"We're increasingly confident that the hurdles ahead are technological and not biological and that we will succeed. Importantly, we've demonstrated already the great promise this technology has as a conservation tool when hundreds of the world's amphibian species are in catastrophic decline."

The technical work was led by Dr Andrew French and Dr Jitong Guo, formerly of Monash University, in a University of Newcastle laboratory led by frog expert, Professor Michael Mahony, along with Mr Simon Clulow and Dr John Clulow. The frozen specimens were preserved and provided by Professor Mike Tyler, of the University of Adelaide, who extensively studied both species of gastric-brooding frog -- R. silus and R. vitellinus -- before they vanished in the wild in 1979 and 1985 respectively.

UNSW's Professor Archer spoke publicly for the first time today about the Lazarus Project and also about his ongoing interest in cloning the extinct Australian thylacine, or Tasmanian tiger, at the TEDx DeExtinction event in Washington DC, hosted by Revive and Restore and the National Geographic Society.

Researchers from around the world are gathered there to discuss progress and plans to 'de-extinct' other extinct animals and plants. Possible candidate species include the woolly mammoth, dodo, Cuban red macaw and New Zealand's giant moa.

Source: University of New South Wales