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Chemical and Biomolecular Engineering

A novel method developed by University of Illinois researchers could change the way metabolic engineering is done.

Researchers from the Carl R. Woese Institute for Genomic Biology’s Biosystems Design theme, including Huimin Zhao, the Steven L. Miller Chair of Chemical and Biomolecular Engineering, recently published a paper in Nature Communications outlining their new method, which could make the metabolic engineering process more efficient.

 

Huimin Zhao

Metabolic engineering involves engineering microorganisms to produce value-added products such as biofuels and chemicals. This is achieved by changing or deleting the expression of genes to modify the microorganism’s genome. In this process, several targets in the genome are modified in order to achieve specific goals.

“We can easily find several metabolic engineering targets to improve the desired phenotype,” said Jiazhang Lian, a visiting research associate at the IGB who is a co-author of the paper. “How to combine these beneficial genetic modifications is one of the biggest challenges in metabolic engineering.”

Traditionally, researchers test these targets individually in a series of time-consuming steps. These steps limit productivity and the yield of the final product — two crucial components in the metabolic engineering process.

The researchers decided to create a method that combines all of these steps and executes them simultaneously, making the process faster and easier.

They based this method on the CRISPR system, a method of genetic manipulation that uses a set of DNA sequences to modify genes within a cell.

This system uses three genetic manipulations that are frequently used in metabolic engineering: transcriptional activation, transcriptional interference, and gene deletion.

By using these manipulations simultaneously, scientists can explore different combinations of manipulations and discover which combination is best.

“We can now work with 20 targets,” Zhao said. “We can implement all of these (manipulations) for each target in a combinatorial manner to find out which combination actually will give us higher productivity or yield of the final product.”

The researchers tested the method in a species of yeast that is used in winemaking, baking, and the production of biofuels. They showed that using this method could improve the production of a specific product.

Their system, called CRISPR-AID, will allow researchers to easily explore all the possible target combinations. But the key is to find the optimal combination.

“If we compare metabolic engineering to a basketball team, we cannot build a strong team by simply putting the best players together,” Lian said. “Instead, we should try to find those who can collaborate and work synergistically.”

Their new system opens up thousands — even millions — of possibilities, which presents another logistical challenge.

They plan to find the best combinations by developing a high throughput screening method or using a robotic system such as the iBioFAB, a system located in the IGB that automatically produces synthetic biosystems.

“I believe the combination of CRISPR-AID with high throughput screening and iBioFAB will significantly advance the metabolic engineering field in the near future,” Lian said.

Zhao hopes to test their method on other organisms, using the same engineering principles but modifying the protocol for different organisms.

Eventually, they hope to extend to the genome scale — to be able to test all the genes in an organism at once — which would be a considerable leap in the field of metabolic engineering.

“If we can do that, we can make it truly modularized and also standardize the procedure,” Zhao said. “Then we really increase the throughput and the speed of metabolic engineering.”

Several research efforts aim to engineer microorganisms for the production of biofuels and chemicals, so any tools that can speed up the process are significant. Zhao believes this is true for their method.

“It’s not just an incremental improvement,” he said. “It’s a new way to do metabolic engineering.”

 

Congratulations to Huimin Zhao, who has been chosen to receive the 2018 Marvin Johnson Award from the Biochemical Technology Division of the American Chemical Society!

The Marvin J. Johnson Award in Microbial and Biochemical Technology recognizes outstanding research contributions toward the advancement of microbial and biochemical technology.

Zhao is the Steven L. Miller Chair of Chemical and Biomolecular Engineering, and a professor of chemistry, biochemistry, biophysics, and bioengineering at Illinois. His primary research interests are in the development and applications of synthetic biology tools to address society’s most daunting challenges in health, energy, and sustainability, and in the fundamental aspects of enzyme catalysis, cell metabolism, and gene regulation.

The award will be presented to Zhao at the 2018 Spring National Meeting of the ACS on March 18-22, 2018. At that meeting, Zhao will deliver a lecture on his research. Read more about Dr. Huimin Zhao.

 

A new, multimillion dollar bioenergy research center at the University of Illinois that promises to be a catalyst for the development of sustainable, cost-effective biofuels and bioproducts will involve several faculty from Chemical and Biomolecular Engineering.

The Department of Energy announced earlier this summer it has awarded the University of Illinois $104 million for the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI). The center is a collaboration between Illinois’ Institute for Sustainability, Energy, and Environment (iSEE) and the Carl R. Woese Institute for Genomic Biology (IGB), and will include 17 partner institutions.

“As the United States seeks energy independence, we need to look at the most efficient ways to grow, transform, and market biofuels,” said Evan H. DeLucia, the G. William Arends Professor of Plant Biology and Baum Family Director of iSEE. DeLucia will serve as CABBI Director. “This grant is a game-changer, and CABBI will be at the forefront as we press toward a new bio-based economy. Our center’s holistic approach will generate new products directly from biomass, reducing our nation’s dependence on fossil fuels and making us more secure.”

CABBI is one of four Department of Energy Bioenergy Research Centers, joining the Great Lakes Bioenergy Research Center led by the University of Wisconsin, the Center for Bioenergy Innovation led by the DOE’s Oak Ridge National Laboratory, and the Joint Bioenergy Institute led by the DOE’s Lawrence Berkeley National Lab.

At Illinois, researchers will develop fuels and products by integrating three interconnected priority areas: Growing the Right Crops (feedstock development), Turning Plants into Fuel (conversion), and Determining the Environmental and Economic Bottom Line (sustainability). Crop Sciences professor Stephen Moose will lead the feedstock development theme, in which scientists will integrate recent advances in genomics, synthetic biology, and computational biology to increase the value of biomass crops. Madhu Khanna, ACES Distinguished Professor in Environmental Economics in the Department of Agricultural and Consumer Economics, will lead the sustainability theme in which researchers will provide an overarching framework for viewing outcomes from the feedstocks and conversion themes through an environmental and economic lens.

Dr. Huimin Zhao in his lab at the Carl R. Woese Institute for Genomic Biology

Dr. Huimin Zhao, the Steven L. Miller Chair in Chemical Engineering at Illinois, will lead the conversion area. His team of about 19 principal investigators, which include two other ChBE faculty at Illinois—Professor Chris Rao and Assistant Professor David Flaherty—will further develop a versatile, automated “biofoundry” for rapidly engineering microbial strains that can efficiently produce diverse, high-value molecules such as biodiesel, organic acids, jet fuels, lubricants, and alcohols. Using the design-build-test-learn framework, research in the conversion theme will overcome the challenges associated with driving biological systems to produce non-natural compounds.

“It’s a unique vision. We want to use the plants as the factories to produce lipid-based chemicals and then we’ll couple that with the microbial conversion platform to make more high-value added products. Furthermore, we will use the lignocellulose from those plants as the feedstocks to make a wide variety of chemicals,” Zhao said.

This approach is different from the other three existing bioenergy centers funded by the Department of Energy, he said. The other centers focus their work on developing technologies to deconstruct the lignocellulose to generate fermentable sugars like glucose and xylose and using those sugars to make a variety of fuels and chemicals.

He and his team of researchers also want to understand what constrains the production of those biofuels or chemicals in the microorganisms. And they want to produce more chemicals and biofuels as well. That’s why Zhao has brought in experts in catalysis like Dr. David Flaherty who will develop chemical catalysts to upgrade the fuels and chemicals produced by the microorganisms.

Dr. David Flaherty

Flaherty’s group will be collaborating with others in CABBI to develop catalysts to convert advantaged molecules produced from microorganisms, such as butanol and unsaturated fatty acids, into clean burning fuels. They’ll also be coproducing high value chemicals to help increase the economic viability of the overall process.

“The networks of catalytic reactions in these systems are incredibly complex, and to be successful, we will need to develop maps of the reaction pathways that exist and use that information to identify opportunities to control the selectivity to specific desired products,” Flaherty said.

Dr. Christopher Rao

Dr. Chris Rao will focus on the engineering of oleaginous yeast to produce biofuels and chemicals and to understand what constrains the production of those products in the yeast.

“If you think from the organisms’ point of view, they don’t want to produce the product we want at large amounts because it will not benefit their survival and growth. … So we have to hijack the native metabolism to make the organisms themselves produce the product we want,” Zhao said.

The goal of metabolic engineering is to essentially engineer microorganisms to produce useful chemicals, but if they produce at low levels, that would not be economical, Zhao said. How does one make the organism produce a lot of product in a very short period of time? That’s another challenge researchers will address.

“In the traditional chemical industry, what a chemist or chemical engineer often does is to develop chemical catalysts and use them to convert non-renewable petroleum oils into fuels and chemicals. Now we want to develop biological catalysts such as microorganisms and enzymes and use them to convert renewable plant biomass into fuels and chemicals, which represents a paradigm shift in the chemical industry,” Zhao said.

Zhao, Rao and Flaherty have collaborated before. All three were involved with the Energy Biosciences Institute, which was established in 2007 as a partnership between Illinois and the University of California at Berkeley and Lawrence Berkeley National Lab, with initial funding from BP. Because of their work with EBI, Illinois had a strong track record of collaboration and accomplishments in bioenergy research and an infrastructure in place, Zhao said.

At the Carl R. Woese Institute for Genomic Biology, Zhao leads the Biosystems Design theme and he has been involved in development of the Illinois Biological Foundry for Advanced Biomanufacturing, or iBioFAB. Housed at IGB, iBioFAB is a computational and physical infrastructure that supports rapid design, fabrication, validation/quality control, and analysis of genetic constructs and organisms.

IGB will oversee and integrate CABBI’s core science team under one roof.

“The IGB, now with over a decade of experience in successfully addressing grand challenges by transdisciplinary integration of the life sciences, physical sciences, and engineering, will provide an outstanding environment for the talented CABBI team,” said director Gene Robinson.

The Institute for Sustainability, Energy, and Environment will coordinate and integrate field work off campus and at the Illinois Energy Farm, a 320-acre site that enables researchers to trial promising biofuel feedstocks at scale, and it will utilize the nearly complete, $32 million Integrated Bioprocessing Research Laboratory.

“We are very excited about this project because this will further build on our strengths in this area. This is definitely the future. I’m pleased the Department of Energy is committed to this direction,” Zhao said.

The center is expected to receive $4 million in fiscal year 2018, then $25 million a year in 2019-22.

Partner institutions include Brookhaven (N.Y.) National Laboratory; the Lawrence Berkeley National Laboratory’s Joint Genome Institute in Berkeley, Calif.; the U.S. Department of Agriculture’s (USDA) Agricultural Research Service (ARS) in Houma, La., the USDA ARS in Peoria, Ill.; Iowa State University; Princeton University; Mississippi State University; the University of California-Berkeley; West Virginia University; Boston University; the University of Wisconsin-Madison; Colorado State University; the University of Idaho; the University of Florida; the University of Nebraska; the Institute for Systems Biology in Seattle; and the HudsonAlpha Institute for Biotechnology in Huntsville, Ala.

The Department of Energy’s Bioenergy Research Program was established in 2007 and has led to 2,630 peer-reviewed publications, 607 invention disclosures, 378 patent applications, 191 licenses or options, 92 patents, and 14 start-up companies.

Congratulations to Dr. Huimin Zhao, recipient of the 2017 Award for Excellence in Biological Engineering Publication!

Huimin Zhao
Huimin Zhao

The Biotechnology Progress Award for Excellence in Biological Engineering Publication recognizes outstanding contributions to the literature in biomedical engineering, biological engineering, biotechnology, biochemical engineering and related fields. The award, which is underwritten by John Wiley & Sons, celebrates excellence and foundational contributions to biotechnology and biological engineering through a body of work: a seminal paper, a review, a research report, or other material of significant interest and importance.

The award will be presented a the annual meeting of the American Institute of Chemical Engineers in November.

Zhao is the Steven L. Miller Chair of Chemical and Biomolecular Engineering, and a professor of chemistry, biochemistry, biophysics, and bioengineering at Illinois. His primary research interests are in the development and applications of synthetic biology tools to address society’s most daunting challenges in health, energy, and sustainability, and in the fundamental aspects of enzyme catalysis, cell metabolism, and gene regulation. Read more about Dr. Zhao.

 

 

New approach combines synthetic biology, genome editing tools, and automation to quickly and effectively produce novel yeast strains

One of humankind’s oldest industrial partners is yeast, a familiar microbe that enabled early societies to brew beer and leaven bread and empowers modern ones to synthesize biofuels and conduct key biomedical research. Yeast remains a vital biological agent, yet our ability to explore and influence its genomic activity has lagged.

In a new article in Nature Communications (DOI: 10.1038/NCOMMS15187), University of Illinois researchers describe how their successful integration of several cutting-edge technologies—creation of standardized genetic components, implementation of customizable genome editing tools, and large-scale automation of molecular biology laboratory tasks—will enhance our ability to work with yeast. The results of their new method demonstrate its potential to produce valuable novel strains of yeast for industrial use, as well as to reveal a more sophisticated understanding of the yeast genome.

Zhao and Si
Dr. Huimin Zhao and IGB Research Fellow Tong Si

“The goal of the work was really to develop a genome-scale engineering tool for yeast . . . traditional metabolic engineering focused on just a few genes and the few existing genome-scale engineering tools are only applicable to bacteria, not eukaryotic organisms like yeast,” said Steven L. Miller Chair of Chemical and Biomolecular Engineering Huimin Zhao, who led the study. “A second innovation is the use of synthetic biology concepts, the modularization of the parts, and integration with a robotic system, so we can do it in high-throughput.”

The team focused on yeast in part because of its important modern-day applications; yeasts are used to convert the sugars of biomass feedstocks into biofuels such as ethanol and industrial chemicals such as lactic acid, or to break down organic pollutants. Because yeast and other fungi, like humans, are eukaryotes, organisms with a compartmentalized cellular structure and complex mechanisms for control of their gene activity, study of yeast genome function is also a key component of biomedical research.

“In basic science, a lot of fundamental eukaryotic biology is studied in yeast,” said Tong Si, a Carl R. Woese Institute for Genomic Biology Research Fellow. “People have a limited understanding of these complicated systems. Although there are approximately 6,000 genes in yeast, people probably know less than 1,000 by their functions; all the others, people do not know.”

The group took the first step toward their goal of a novel engineering strategy for yeast by creating what is known as a cDNA library: a collection of over 90% of the genes from the genome of baker’s yeast (Saccharomyces cerevisiae), arranged within a custom segment of DNA so that each gene will be, in one version, overactive within a yeast cell, and in a second version, reduced in activity.

Zhao and colleagues examined the ability of the CRISPR-Cas system, a set of molecules borrowed from a form of immune system in bacteria (CRISPR stands for clustered regularly interspaced short palindromic repeats, describing a feature of this system in bacterial genomes). This system allowed Zhao to make precise cuts in the yeast genome, into which the standardized genetic parts from their library could insert themselves.

“The first time we did this, in 2013, there was no CRISPR . . . the best we could get was 1% of the cells modified in one run,” said Si. “We struggled a little on that, and when CRISPR came out, that worked. We got it to 70% [cells modified], so that was very important.”

With gene activity-modulating parts integrating into the genome with such high efficiency, the researchers were able to randomly generate many different strains of yeast, each with its own unique set of modifications. These strains were subjected to artificial selection processes to identify those that had desirable traits, such as the ability to survive exposure to reagents used in the biofuel production process.

This selection process was greatly aided by the Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB), a robotic system that performs most of the laboratory work described above in an automated way, including selection of promising yeast strains. Use of iBioFAB greatly accelerated the work, enabling simultaneous creation and testing of many unique strains. The iBioFAB was conceived and developed by the Biosystems Design research theme at the Carl R. Woese Institute for Genomic Biology (IGB), which is led by Zhao.

With support from the High Performance Biological Computing Group at Illinois, Zhao, Si and their colleagues analyzed the modified genomes of their most promising yeast strains.  They identified combinations of genes whose altered activities contributed to desirable traits; the functions of some of these genes were previously unknown, demonstrating the technique’s ability to generate new biological knowledge.

“I think the key difference between this method and the other existing metabolic engineering strategies in yeast is really the scale,” said Zhao. “The current metabolic engineering strategies are all focused on just a few genes, dozens of genes at most . . .  it’s very intuitive.  With this we can explore all the genes, we can identify a lot of targets that cannot be intuited.”

The work, which was funded by the Roy J. Carver Charitable Trust, IGB, Defense Advanced Research Program Agency, and National Academies Keck Futures Initiative on Synthetic Biology, paves the way for similar approaches to broad-scale, automated genome engineering of other eukaryotic species.

By Claudia Lutz, Carl R. Woese Institute for Genomic Biology

By Liz Ahlberg Touchstone, University of Illinois News Bureau

In the fight against disease, many weapons in the medicinal arsenal have been plundered from bacteria themselves. Using CRISPR-Cas9 gene-editing technology, researchers have now uncovered even more potential treasure hidden in silent genes.

A new study from researchers at the University of Illinois and colleagues at the Agency for Science, Technology and Research in Singapore used CRISPR technology to turn on unexpressed, or “silent,” gene clusters in Streptomyces, a common class of bacteria that naturally produce many compounds that have already been used as antibiotics, anti-cancer agents and other drugs. The study, led by chemical and biomolecular engineering professor Huimin Zhao, was published in the journal Nature Chemical Biology.

Clockwise from back middle: Behnam Enghiad, graduate student; Shangwen Luo, postdoc; Tajie Luo, grad student; and Huimin Zhao, professor of chemical and biomolecular engineering
Clockwise from back middle: Behnam Enghiad, graduate student; Shangwen Luo, postdoc; Tajie Luo, grad student; and Huimin Zhao, Steven L. Miller Chair in Chemical Engineering. Photo by L. Brian Stauffer.

“In the past, researchers just screened the natural products that bacteria made in the lab to search for new drugs,” Zhao said. “But once whole bacterial genomes were sequenced, we realized that we have only discovered a small fraction of the natural products coded in the genome.

“The vast majority of biosynthetic gene clusters are not expressed under laboratory conditions, or are expressed at very low levels. That’s why we call them silent. There are a lot of new drugs and new knowledge waiting to be discovered from these silent gene clusters. They are truly hidden treasures.”

To mine for undiscovered genomic treasure, the researchers first used computational tools to identify silent biosynthetic gene clusters – small groups of genes involved in making chemical products. Then they used CRISPR technology to insert a strong promoter sequence before each gene that they wanted to activate, prompting the cell to make the natural products that the genes clusters coded for.

“This is a less-explored direction with the CRISPR technology. Most CRISPR-related research focuses on biomedical applications, like treating genetic diseases, but we are using it for drug discovery,” Zhao said. His lab was the first to adapt the CRISPR system for Streptomyces. “In the past, it was very difficult to turn on or off a specific gene in Streptomyces species. With CRISPR, now we can target almost any gene with high efficiency.”

The Illinois team collaborated with a team from A*STAR in Singapore.

Zhao.CRISPR.AStar
Pictured: Wan Lin Yeo, Huimin Zhao, Ee Lui Ang, Mingzi M. Zhang, Fong Tian Wong, Yee Hwee Lim and Elena Heng. Photo courtesy of Huimin Zhao.

The team succeeded in activating a number of silent biosynthetic gene clusters. To look for drug candidates, each product needs to be isolated and studied to determine what it does. As a demonstration, the researchers isolated and determined the structure of one of the novel compounds produced from a silent biosynthetic gene cluster, and found that it has a fundamentally different structure from other Streptomyces-derived drugs – a potential diamond in the rough.

Zhao said such new compounds could lead to new classes of drugs that elude antibiotic resistance or fight cancer from a different angle.

“Antimicrobial resistance is a global challenge. We want to find new modes of action, new properties, so we can uncover new ways to attack cancer or pathogens. We want to identify new chemical scaffolds leading to new drugs, rather than modifying existing types of drugs,” he said.

The U.S. National Institutes of Health and the National Research Foundation of Singapore supported this work.

To reach Huimin Zhao, call (217)333-2631; email: zhao5@illinois.edu.

The paper “CRISPR–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters” is available online. doi:10.1038/nchembio.2341

Research by Professor of Chemical and Biomolecular Engineering Huimin Zhao and graduate student Behnam Enghiad is pioneering a new method of genetic engineering for basic and applied biological research and medicine. Their work, reported in ACS Synthetic Biology, has the potential to open new doors in genomic research by improving the precision and adherence of sliced DNA.

“Using our technology, we can create highly active artificial restriction enzymes with virtually any sequence specificity and defined sticky ends of varying length,” said Zhao. “This is a rare example in biotechnology where a desired biological function or reagent can be readily and precisely designed in a rational manner.”

Zhao.SB.image

Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research, however have limited sequence specificity and availability. The Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) is capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length.

Restriction enzymes are an important tool in genomic research: by cutting DNA at a specific site, they create a space wherein foreign DNA can be introduced for gene-editing purposes. This process is not only achieved by naturally-occurring restriction enzymes; other artificial restriction enzymes, or AREs, have risen to prominence in recent years. CRISPR-Cas9, a bacterial immune system used for “cut-and-paste” gene editing, and TALENs, modified restriction enzymes, are two popular examples of such techniques.

Though useful in genetic engineering, no AREs generate defined “sticky ends”—an uneven break in the DNA ladder-structure that leaves complementary overhangs, improving adhesion when introducing new DNA. “If you can cleave two different DNA samples with the same restriction enzyme, the sticky ends that are generated are complementary,” explained Enghiad. “They will hybridize with each other, and if you use a ligase, you can stick them together.”

However, restriction enzymes themselves have a critical drawback: the recognition sequence which prompts them to cut is very short—usually only four to eight base pairs. Because the enzymes will cut anywhere that sequence appears, researchers rely on finding a restriction enzyme whose cut site appears only once in the genome of their organism or plasmid—an often difficult proposition when the DNA at hand might be thousands of base pairs long.

This problem has been partially solved simply by the sheer number of restriction enzymes discovered: more than 3600 have been characterized, and over 250 are commercially available. “Just in our freezer, for our other research, we have probably over 100 different restriction enzymes,” said Enghiad. “We look through them all whenever we want to assemble something … the chance of finding the unique restriction site is so low.

“Our new technology unifies all of those restriction enzymes into a single system consisting of one protein and two DNA guides. Not only have you replaced them, but you can now target sites that no available restriction enzymes can.”

Enghiad and Zhao’s new technique creates AREs through the use of an Argonaute protein (PfAgo) taken from Pyrococcus furiosus, an archeal species. Led by two DNA guides, PfAgo is able to recognize much longer sequences when finding its cut site, increasing specificity and removing much of the obstacles posed by restriction enzymes. Further, PfAgo can create longer sticky ends than even restriction enzymes, a substantial benefit as compared to other AREs.

“When we started, I was inspired by a paper about a related protein—TtAgo. It could use DNA guides to cleave DNA, but the protein is only active at temperatures up to 75 degrees,” explained Enghiad. “DNA strands start to separate at temperatures higher than 75 degrees, which could allow Ago proteins to cleave double stranded DNA. If there were a protein that was active at higher temperatures, I reasoned, that protein could be used as an artificial restriction enzyme.

“So I started looking for that, and what I found was PfAgo.”

In addition to replacing restriction enzymes in genetic engineering processes, Enghiad and Zhao believe their technology will have broad applications in the biological research. By creating arbitrary sticky ends, PfAgo could make assembly of large DNA molecules easier, and enables cloning of large DNA molecules such as biochemical pathways and large genes.

The application of these techniques is broad-reaching: ranging from discovery of new small molecule drugs to engineering of microbial cell factories for synthesis of fuels and chemicals to molecular diagnostics of genetic diseases and pathogens, which are the areas Zhao and Enghiad are currently exploring.

“Due to its unprecedented simplicity and programmability (a single protein plus DNA guides for targeting), as well as accessibility … we expect PfAgo-based AREs will become a powerful and indispensable tool in all restriction enzyme or nuclease-enabled biotechnological applications and fundamental biological research,” said Zhao.  “It is to molecular biology as the CRISPR technology is to cell biology.”

Written by Kathryne Metcalf of the Carl R. Woese Institute for Genomic Biology.

Searching a whole genome for one particular sequence is like trying to fish a specific piece from the box of a billion-piece puzzle. Using advanced imaging techniques, University of Illinois researchers have observed how one set of genome-editing proteins finds its specific targets, which could help them design better gene therapies to treat disease.

Illinois chemical and biomolecular engineering professors Charles Schroeder and Huimin Zhao, along with graduate students Luke Cuculis and Zhanar Abil, published their work in the journal Nature Communications.

Professors Huimin Zhao and Charles Schroeder, Professor Huimin Zhao, professor Charles Schroeder and graduate students Luke Cuculis and Zhanar Abil. Photo by L. Brian Stauffer.
Professors Huimin Zhao and Charles Schroeder, Professor Huimin Zhao, professor Charles Schroeder and graduate students Luke Cuculis and Zhanar Abil. Photo by L. Brian Stauffer.

TALE proteins, or transcription activator-like effectors, can be programmed to recognize and bind to specific regions of DNA. Researchers have been interested in using TALE proteins for synthetic biology, such as genome editing in plants or bacteria, or for gene therapy. For example, Zhao’s group explores using TALE proteins to treat sickle cell anemia, which is caused by a mutation in one link of the DNA chain.

“People have been using this technique, but nobody fully understood the mechanism before,” Schroeder said. “The main question is, how do these proteins find their target sites? They are designed to bind to a particular site, but there’s this big genome with billions of bases, so how does the protein find its site? If you understand the mechanism, you might be able to engineer better, more improved proteins.”

The researchers used imaging techniques that let them watch through a microscope how individual TALE proteins interact with a string of DNA. They observed that the proteins seek and find using a combination of sliding and hopping. The proteins bind to the DNA and slide along the helix, traveling down the DNA molecule like a highway. The researchers also observed that the proteins perform frequent, short hops along their paths, allowing them to move more efficiently but never straying far from the DNA.

“The combination of sliding and hopping means they can cover more ground and potentially move past obstacles that might be in their way,” said Cuculis, a co-first author of the paper.

“The combination of behaviors also would allow a TALE protein to switch strands of the DNA double helix and sample both strands, increasing the chance of finding its target site,” said Abil, the other co-first author.

They also analyzed which parts of the protein did the work, finding a division of labor among the domains within the protein: One part searches, while another part binds to the specific target sequence. This finding excited the researchers, because it gave them insight into where to tweak the protein design so that it binds even more selectively.

“The major goal would be to engineer improved proteins that have lower off-target binding. You don’t want them to bind to the wrong place,” Schroeder said. “If we engineer a protein in such a way where we don’t just naively change the specific binding domain, but design a new protein with distinct parts of the protein in different places, we might be able to increase the efficiency without increasing mistakes.”

A single-molecule imaging technique let the researchers observe how individual TALE proteins interacted with a strand of DNA. Graphic by Luke Cuculis
A single-molecule imaging technique let the researchers observe how individual TALE proteins interacted with a strand of DNA. Graphic by Luke Cuculis

Next, the researchers are watching the proteins work in live cells to see if the behavior changes when immersed in the bustling activity within the cell nucleus.

“It gives us a better understanding of the genome editing mechanism,” Zhao said. “When we understand it better, it provides new insights for the design of the protein. If you really talk about therapeutic applications, it needs to be a specific design.”

The Carl R. Woese Institute for Genomic Biology at the U. of I. and the David and Lucile Packard Foundation supported this work. Schroeder and Zhao also are affiliated with the department of chemistry, the Center for Biophysics and Quantitative Biology, and the Carl R. Woese Institute for Genomic Biology at the U. of I.

–Liz Ahlberg, University of Illinois News Bureau

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