Penn Engineers’ ‘LADL’ Uses Light to Serve Up On-demand Genome Folding

Every cell in your body has a copy of your genome, tightly coiled and packed into its nucleus. Since every copy is effectively identical, the difference between cell types and their biological functions comes down to which, how and when the individual genes in the genome are expressed, or translated into proteins.

Scientists are increasingly understanding the role that genome folding plays in this process. The way in which that linear sequence of genes are packed into the nucleus determines which genes come into physical contact with each other, which in turn influences gene expression.

LADL combines CRISPR/Cas9 and optogenetics to bring two distant points in a linear gene sequence into physical contact, forming a folding pattern known as a “loop.” Looping interactions influence gene expression, so the researchers envision LADL as being a powerful tool for studying these dynamics.

Jennifer Phillips-Cremins, assistant professor in Penn Engineering’s Department of Bioengineering, is a pioneer in this field, known as “3-D Epigenetics.” She and her colleagues have now demonstrated a new technique for quickly creating specific folding patterns on demand, using light as a trigger.

The technique, known as LADL or light-activated dynamic looping, combines aspects of two other powerful biotechnological tools: CRISPR/Cas9 and optogenetics. By using the former to target the ends of a specific genome fold, or loop, and then using the latter to snap the ends together like a magnet, the researchers can temporarily create loops between exact genomic segments in a matter of hours.

The ability to make these genome folds, and undo them, on such a short timeframe makes LADL a promising tool for studying 3D-epigenetic mechanisms in more detail. With previous research from the Phillips-Cremins lab implicating these mechanisms in a variety of neurodevelopmental diseases, they hope LADL will eventually play a role in future studies, or even treatments.

Jennifer Phillips-Cremins, Ji Hun Kim and Mayuri Rege

Alongside Phillips-Cremins, lab members Ji Hun Kim and Mayuri Rege led the study, and Jacqueline Valeri, Aryeh Metzger, Katelyn R. Titus, Thomas G. Gilgenast, Wanfeng Gong and Jonathan A. Beagan contributed to it. They collaborated with associate professor of Bioengineering Arjun Raj and Margaret C. Dunagin, a member of his lab.

The study was published in the journal Nature Methods.

“In recent years,” Phillips-Cremins says, “scientists in our fields have overcome technical and experimental challenges in order to create ultra-high resolution maps of how the DNA folds into intricate 3D patterns within the nucleus. Although we are now capable of visualizing the topological structures, such as loops, there is a critical gap in knowledge in how genome structure configurations contribute to genome function.”

In order to conduct experiments on these relationships, researchers studying these 3D patterns were in need of tools that could manipulate specific loops on command. Beyond the intrinsic physical challenges — putting two distant parts of the linear genome in physical contact is quite literally like threading a needle with a thread that is only a few atoms thick — such a technique would need to be rapid, reversible and work on the target regions with a minimum of disturbance to neighboring sequences.

The advent of CRISPR/Cas9 solved the targeting problem. A modification of the gene editing tool allowed researchers to home in on the desired sequences of DNA on either end of the loop they wanted to form. If those sequences could be engineered to seek one another out and snap together under the other necessary conditions, the loop could be formed on demand.

Cremins Lab members then sought out biological mechanisms that could bind the ends of the loops together, and found an ideal one in the toolkit of optogenetics. The proteins CIB1 and CRY2, found in Arabidopsis, a flowering plant that’s a common model organism for geneticists, are known to bind together when exposed to blue light.

“Once we turn the light on, these mechanisms begin working in a matter of milliseconds and make loops within four hours,” says Rege. “And when we turn the light off, the proteins disassociate, meaning that we expect the loop to fall apart.”

“There are tens of thousands of DNA loops formed in a cell,” Kim says. “Some are formed slowly, but many are fast, occurring within the span of a second. If we want to study those faster looping mechanisms, we need tools that can act on a comparable time scales.”

As shown in a 2013 Nature Methods paper by fellow Penn bioengineer Lukasz Bugaj, the optical response of the CRY2 protein is a key component of LADL. When the blue light is turned on, CRY2 proteins in cell immediately find one another and bind together into clumps large enough to be seen under magnification. When the light is turned off, the clumps begin to dissolve away.”

Fast acting folding mechanisms also have an advantage in that they lead to fewer perturbations of the surrounding genome, reducing the potential for unintended effects that would add noise to an experiment’s results.

The researchers tested LADL’s ability to create the desired loops using their high-definition 3D genome mapping techniques. With the help of Arjun Raj, an expert in measuring the activity of transcriptional RNA sequences, they also were able to demonstrate that the newly created loops were impacting gene expression.

The promise of the field of 3D-epigenetics is in investigating the relationships between these long-range loops and mechanisms that determine the timing and quantity of the proteins they code for. Being able to engineer those loops means researchers will be able to mimic those mechanisms in experimental conditions, making LADL a critical tool for studying the role of genome folding on a variety of diseases and disorders.

“It is critical to understand the genome structure-function relationship on short timescales because the spatiotemporal regulation of gene expression is essential to faithful human development and because the mis-expression of genes often goes wrong in human disease,” Phillips-Cremins says. “The engineering of genome topology with light opens up new possibilities to understanding the cause-and-effect of this relationship. Moreover we anticipate that, over the long term, the use of light will allow us to target specific human tissues and even to control looping in specific neuron subtypes in the brain.”

The research was supported by the New York Stem Cell Foundation; Alfred P. Sloan Foundation; the National Institutes of Health through its Director’s New Innovator Award from the National Institute of Mental Health, grant no. 1DP2MH11024701, and a 4D Nucleome Common Fund, grant no. 1U01HL1299980; and the National Science Foundation through a joint NSF-National Institute of General Medical Sciences grant to support research at the interface of the biological and mathematical sciences, grant no. 1562665, and a Graduate Research Fellowship, grant no. DGE-1321851.

Originally published on the Penn Engineering Medium blog.

Week in BioE: April 5, 2019

by Sophie Burkholder

Tulane Researchers Use Cancer Imaging Technique to Help Detect Preeclampsia

Preeclampsia is potentially life-threatening pregnancy disorder that typically occurs in about 200,000 expectant mothers every year. With symptoms of high blood pressure, swelling of the hands and feet, and protein presence in urine, preeclampsia is usually treatable if diagnosed early enough. However, current methods for diagnosis involve invasive procedures like cordocentesis, a procedure which takes a sample of fetal blood.

Researchers at Tulane School of Medicine led by assistant professor of bioengineering Carolyn Bayer, Ph.D., hope to improve diagnostics for preeclampsia with the use of spectral photoacoustic imaging. Using this technique, Bayer’s team noticed a nearly 12 percent decrease in placental oxygenation in rats with induced preeclampsia when compared to normal pregnant rats after only two days. If success in using this imaging technology continues at the clinical level, Bayer plans to find more applications of it in the detection and diagnosis of breast and ovarian cancers as well.

New CRISPR-powered device detects genetic mutations in minutes 

Two groups of researchers from the University of California, Berkeley and the Keck Graduate Institute of the Claremont Colleges recently collaborated to design what they call a “CRISPR-Chip” –  a combination of the CRISPR-Cas9 System with a graphene transistor to sequence DNA for the purpose of genetic mutation diagnosis. While companies like 23andMe made genetic testing and analysis more common and accessible for the general public in recent years, the CRISPR-Chip looks to streamline the technology even more.

This new chip eliminates the long and expensive amplification process involved in the typical polymerase chain reaction (PCR) used to read DNA sequences. In doing so, the CRISPR-Chip is much more of a point-of-care diagnostic, having the ability to quickly detect a given mutation or sequence when given a pure DNA sample. Led by Kiana Aran, Ph.D., the research team behind the CRISPR-Chip hopes that this new combination of nanoelectronics and modern biology will allow for a new world of possibilities in personalized medicine.

New Method of Brain Stimulation Might Alleviate Symptoms of Depression

Depression is one of the most common mental health disorders in the United States, with nearly 3 million cases every year. For most patients suffering from depression, treatment involves prolonged psychotherapy, antidepressant medication, or even electroconvulsive therapy in extreme cases. Now, scientists at the University of North Carolina School of Medicine study the use of transcranial alternating current stimulation (tACS) to alleviate symptoms of depression.

Led by Flavio Frohlich, Ph.D., who has an adjunct appointment in biomedical engineering, this team of researchers based this new solution on information from each patient’s specific alpha oscillations, which are a kind of wave that can be detected by an electroencephalogram (EEG). Those who suffer from depression tend to have imbalanced alpha oscillations, so Frohlich and his team sought to use tACS to restore this balance in those patients. After seeing positive results from data collected two weeks after patients in a clinical trial receives the tACS treatment, Frohlich hopes that future applications will include treatment for even more mental health disorders and psychiatric illnesses.

University of Utah Researchers Receive Grant to Improve Hearing Devices for Deaf Patients

Engineers at the University of Utah are part of team that recently received a $9.7 million grant from the National Institute of Health (NIH) to design new implantable hearing devices for deaf patients, with the hope to improve beyond the sound quality of existing devices. The work will build upon a previous project at the University of Utah called the Utah Electrode Array, a brain-computer interface originally developed by Richard Normann, Ph.D., that can send and receive neural impulses to and from the brain. This new device will differ from a typical cochlear implant because the Utah Electrode Array assembly will be attached directly to the auditory nerve instead of the cochlea, providing the patient with a much higher resolution of sound.

People & Places

Vivek Shenoy, Eduardo D. Glandt President’s Distinguished Scholar in the Department of Materials Science and Engineering and Secondary Faculty in Bioengineering, has been named the recipient of the 2018–19 George H. Heilmeier Faculty Award for Excellence in Research for “for pioneering multi-scale models of nanomaterials and biological systems.”

The Heilmeier Award honors a Penn Engineering faculty member whose work is scientifically meritorious and has high technological impact and visibility. It is named for George H. Heilmeier, a Penn Engineering alumnus and overseer whose technological contributions include the development of liquid crystal displays and whose honors include the National Medal of Science and Kyoto Prize.

Read the rest of the story on Penn Engineering’s Medium blog.

We would also like to congratulate Jay Goldberg, Ph.D., from Marquette University on his election as a fellow to the National Academy of Inventors. Nominated largely for his six patents involving medical devices, Goldberg also brings this innovation to his courses. One in particular called Clinical Issues in Biomedical Engineering Design allows junior and senior undergraduates to observe the use of technology in clinical settings like the operating room, in an effort to get students thinking about how to improve the use of medical devices in these areas.

 

Week in BioE (March 22, 2019)

by Sophie Burkholder

A New Microscopy Technique Could Reduce the Risk of LASIK Surgery

Though over ten million Americans have undergone LASIK vision corrective surgery since the option became available about 20 years ago, the procedure still poses some risk to patients. In addition to the usual risks of any surgery however, LASIK has even more due to the lack of a precise way to measure the refractive properties of the eye, which forces surgeons to make approximations in their measurements during the procedure. In an effort to eliminate this risk, a University of Maryland team of researchers in the Optics Biotech Laboratory led by Giuliano Scarcelli, Ph. D., designed a microscopy technique that would allow for precise measurements of these properties.

Using a form of light-scattering technology called Brillouin spectroscopy, Scarcelli and his lab found a way to directly determine a patient’s refractive index – the quantity surgeons need to know to be able to measure and adjust the way light travels through the eye. Often used as a way to sense mechanical properties of tissues and cells, this technology holds promise for taking three-dimensional spatial observations of these structures around the eye. Scarcelli hopes to keep improving the resolution of the new technique, to further understanding of the eye, and reduce even more of the risks involved with LASIK surgery.

Taking Tissue Models to the Final Frontier

Space flight is likely to cause deleterious changes to the composition of bacterial flora, leading to an increased risk of infection. The environment may also affect the susceptibility of microorganisms within the spacecraft to antibiotics, key components of flown medical kits, and may modify the virulence of bacteria and other microorganisms that contaminate the fabric of the International Space Station and other flight platforms.

“It has been known since the early days of human space flight that astronauts are more prone to infection,” says Dongeun (Dan) Huh, Wilf Family Term Assistant Professor in Bioengineering at Penn Engineering. “Infections can potentially be a serious threat to astronauts, but we don’t have a good fundamental understanding of how the microgravity environment changes the way our immune system reacts to pathogens.”

In collaboration with G. Scott Worthen, a physician-scientist in neonatology at the Children’s Hospital of Philadelphia, Huh will attempt to answer this question by sending tissues-on-chips to space. Last June, the Center for the Advancement of Science in Space (CASIS) and the National Center for Advancing Translational Sciences (NCATS), part of the National Institutes of Health (NIH), announced that the duo had received funding to study lung host defense in microgravity at the International Space Station.

Huh and Worthen aim to model respiratory infection, which accounts for more than 30 percent of all infections reported in astronauts. The project’s goals are to test engineered systems that model the airway and bone marrow, a critical organ in the immune system responsible for generating white blood cells, and to combine the models to emulate and understand the integrated immune responses of the human respiratory system in microgravity.

Read the rest of the article on Penn Engineering’s Medium Blog. Media contacts Evan Lerner and Janelle Weaver.

Sappi Limited Teams Up with the University of Maine to Develop Paper Microfluidics

At the Westbrook Technology Center of Sappi, a global pulp and paper company, researchers found ways to apply innovations in paper texture for medical use. So far, these include endeavors in medical test devices and patches for patient diagnostics. In collaboration with the Caitlin Howell, Ph.D., Assistant Professor of Chemical and Biomedical Engineering at the University of Maine, Sappi hopes to continue advances in these unconventional uses of their paper, especially as the business in paper for publishing purposes declines.

Sappi’s projects with the university focus on the development of paper microfluidics devices as what’s now becoming a widespread solution for obstacles in point-of-care diagnostics. One project in particular, called Sharklet, uses a paper that mimics shark skin as a way to impede unwanted microbial growth on the device – a key characteristic needed for its transition into commercial use. Beyond this example, Sappi’s work in developing paper microfluidics underscores the benefits of these devices in their mass producibility and adaptability.

New Observations of the WNT Pathway Deepen the Understanding of Protein Signaling in Cellular Development

Scientists at Rice University recently found that a protein signaling pathway called WNT, typically associated with its role in early organism development, can both listen for signals from a large amount of triggers and influence cell types throughout embryonic development. These new findings, published in PNAS, add to the already known functions of WNT, deepening our understanding of it and opening the doors to new potential applications of it in stem cell research.

Led by Aryeh Warmflash, Ph. D., researchers discovered that the WNT pathway is different between stem cells and differentiated cells, contrary to prior belief that it was the same for both. Using CRISPR-Cas9 gene editing technology, the Warmflash lab observed that the WNT signaling pathway is actually context-dependent throughout the process of cellular development. This research brings a whole new understanding to the way the WNT pathway operates, and could open the doors to new forms of gene therapy and treatments for diseases like cancers that involve genetic pathway mutations.

People and Places

In a recent article from Technical.ly Philly, named Group K Diagnostics on a list of ten promising startups in Philadelphia. Group K Diagnostics founder Brianna Wronko graduated with a B.S.E. from Penn’s Department of Bioengineering in 2017, and her point-of-care diagnostics company raised over $2 million in funding last year. Congratulations Brianna!

We would also like to congratulate Pamela K. Woodward, M.D., on her being named as the inaugural Hugh Monroe Wilson Professor of Radiology at the Washington University School of Medicine in St. Louis. Also a Professor of Biomedical Engineering at the university, Dr. Woodward leads a research lab with a focus on cardiovascular imaging, including work on new standards for diagnosis of pulmonary blood clots and on an atherosclerosis imaging agent.

Lastly, we would like to congratulate all of the following researchers on their election to the National Academy of Engineering:

  • David Bishop, Ph. D., a professor at the College of Engineering at Boston University whose current research involves the development of personalized heart tissue as an all-encompassing treatment for patients with heart disease.
  • Joanna Aizenberg, Ph. D., a professor of chemistry and chemical biology at Harvard University who leads research in the synthesis of biomimetic inorganic materials
  • Gilda Barabino, Ph. D., the dean of the City College of New York’s Grove School for Engineering whose lab focuses on cartilage tissue engineering and treatments for sickle cell disease.
  • Karl Deisseroth, M.D., Ph. D., a professor of bioengineering at Stanford University whose research involves the re-engineering of brain circuits through novel electromagnetic brain stimulation techniques.
  • Rosalind Picard, Ph.D., the founder and director of the Affective Computing Research Group at the Massachusetts Institute of Technology’s Media Lab whose research focuses on the development of technology that can measure and understand human emotion.
  • And finally, Molly Stevens, Ph. D., the Research Director for Biomedical Material Sciences at the Imperial College of London with research in understanding biomaterial interfaces for biosensing and regenerative medicine.

Bioengineering Welcomes New Faculty Member Sydney Shaffer

Sydney Shaffer, MD, PhD

The Department of Bioengineering at the University of Pennsylvania is proud to announce the appointment of Sydney Shaffer, Ph.D., as an Assistant Professor of Bioengineering. She shares a joint appointment with Pathology and Laboratory Medicine. Originally from Atlanta, GA, Dr. Shaffer received her B.S. in Chemical Engineering from Georgia Institute of Technology, coming to Penn to complete her M.D./Ph.D. work in Bioengineering and the Perelman School of Medicine. After graduating in 2018, she conducted her postdoctoral work at Penn in Cancer Biology with Dr. Junwei Shi.

Dr. Shaffer’s research is is focused on understanding how differences present in single-cells can generate phenotypes such as drug resistance in cancer, oncogenesis, differentiation, and invasion. Our approach leverages cutting-edge technologies including high-throughput imaging, single-molecule RNA FISH, fluorescent protein tagging, CRISPR/Cas9 screening, and flow cytometry to investigate rare single-cell phenomena. Further information can be found at www.sydshafferlab.com.

In addition to her exciting research, Dr. Shaffer will be an enthusiastic new member of the Bioengineering Department community. In the short term, she will be taking over the popular class BE 400 (Preceptorships in Bioengineering) which gives undergraduates the rare chance to shadow renowned physicians over a period of ten weeks. She will also serve as a faculty advisor as well as a mentor to the lucky students in her classes and lab.

Dr. Shaffer says that, “With my research interests and training at the interface of engineering and medicine, I am thrilled to be part of the highly interdisciplinary community of Penn Bioengineering.”

“Sydney has a unique combination of creativity and impact in her work,” says Solomon R. Pollack Professor and Chair Dr. David Meaney. “Her work to untangle the secrets of how single cancer cells can develop resistance to a cancer drug  therefore leading to a return of the cancer  is nothing short of stunning. We are incredibly fortunate to have her on our faculty. ”

Week in BioE (August 3, 2017)

There’s news in bioengineering every week, to be sure, but the big story this past week is one that’s sure to continue appearing in headlines for days, weeks, and months — if not years — to come. This story is CRISPR-Cas9, or CRISPR for short, the gene-editing technology that many geneticists are viewing as the wave of the future in terms of the diagnosis and treatment of genetic disorders.

Standing for clustered regularly interspaced short palindromic repeats, CRISPR offers the ability to cut a cell’s genome at a predetermined location and remove and replace genes at this location. As a result, if the location is one at which the genes code for a particular disease, these genes can be edited out and replaced with healthy ones. Obviously, the implCRISPRications for this technology are enormous.

This week, it was reported that, for the first time, CRISPR was successfully used by scientists to edit the genomes of human embryos. As detailed in a paper published in Nature, these scientists edited the genomes of 50 single-cell embryos, which were subsequently allowed to undergo division until the three-day mark, at which point the multiple cells in the embryos were assessed to see whether the edits had been replicated in the new cells.  In 72% of them, they had been.

In this particular case, the gene edited out was one for a type of congenital heart defect, and the embryos were created from the eggs of healthy women and the sperm of men carrying the gene for the defect. However, the experiments prove that the technology could now be applied in other disorders.

Needless to say, the coverage of this science story has been enormous, so here is a collection of links to coverage on the topic. Enjoy!