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Category: Biotechnology News

  • ‘Selfish’ genes called introners proven to be a major source of genetic complexity

    ‘Selfish’ genes called introners proven to be a major source of genetic complexity

    DNA is the genetic code that provides the biological instructions for every living species, but not every bit of DNA helps the species survive. Some pieces of DNA are more like parasites, along for the ride and their own survival.

    To translate DNA into proteins, the building blocks of life, many of these selfish DNA elements have to be removed from the genetic code. Doing so enables the body to produce the wide diversity of proteins that allow for complex life, but the process can also lead to health problems, like some kinds of cancer.

    University of California, Santa Cruz researchers are studying the ways that these genetic elements hide and make copies of themselves, so they can propagate within a species’ DNA, or even hop from one species to an unrelated one in a process called “horizontal gene transfer.”

    A new study in the journal Proceedings of the National Academy of Sciences proves that a type of genetic element called “introners” are the cause of many of these selfish genes spreading within and between species. It provides evidence for eight instances in which introners have transferred between unrelated species, the first proven examples of this phenomenon.

    These results help us understand how genomes evolved to become so complex, and how we might take advantage of that complexity in human health research.

    “[Introners are] a way that genome architectures and complexity arise, but not necessarily because there is natural selection that favors this complexity,” said Russ Corbett-Detig, senior author on the study and professor of biomolecular engineering at the Baskin School of Engineering. “A few may ultimately benefit the host, but most are just cheaters that found a really good way to hide in the genome.”

    Investigating introners

    Corbett-Detig and his former undergraduate student Landen Gozashti, now a postdoctoral fellow at UC Berkeley following a stellar Harvard Ph.D., have spent years studying introns, the segments of noncoding DNA that must be removed before proteins can be produced.

    They wanted to figure out why these non protein-coding bits of DNA are seen in varying amounts across all animals, plants, fungi, and protists, and how they have managed to so successfully replicate themselves and survive. It’s long been a mystery of how all these introns first came to exist within DNA, as most don’t seem to serve an evolutionary function.

    Scientists are interested in this as a way to further understand genome evolution, but also because introns allow for a crucial process called “alternative splicing.” Introns must be spliced out of the DNA sequence to create proteins, but this process can have variation and error, which means that different versions of a protein can be created from the same gene. Ultimately this means that an organism can be more complex, but it also introduces the risk of health problems if splicing breaks a gene. Many researchers, including those at the UC Santa Cruz Genomics Institute, are studying how alternative splicing can be studied to better understand genetic disease. This research enhances the basic science of that health-related work.

    In this study, the researchers have proven that introners are one of the main ways that new introns appear with a species’ DNA. Introners are a kind of transposable element, a “jumping gene” that can move from part of a genome to another, that have found a way to successfully make copies of introns throughout a genome. The team’s past work has suggested this, but their advanced methods of searching the DNA of a wide range of species has now allowed them to definitively confirm their hypothesis.

    The researchers searched for introners in the DNA of thousands of species, something only recently made possible due to ongoing coordinated efforts to sequence a wide range of biodiversity and make the data publicly available, like the Earth BioGenome Project and the Sanger Tree of Life.

    They found evidence for 1,093 families of introners among the 8,716 genomes they analyzed, suggesting that there are many kinds of introners out there capable of spreading introns through the genomes of various species.

    “Because transposons are insanely diverse and present in basically every eukaryote, it implies that this really can be a very general way that new introns arise in different lineages,” Corbett-Detig said.

    These introners most commonly appeared in species of algae, fungi, and diverse single-celled eukaryotes, with an example found in a sea urchin and a tunicate, a tubular marine invertebrate.

    Transfer between species

    Among the many genomes they analyzed, the researchers found the first direct evidence for horizontal gene transfer of introners. They found eight examples of an introner hopping out of the genome of one species and settling into the genome of another unrelated species that mating could not explain.

    In one case, the researchers found horizontal gene transfer between two species so unrelated that their last common ancestor was 1.6 billion years ago. In looking at the genomes of the two species — a sea sponge and a marine protist called a dinoflagellate — they found evidence that around 40 million years ago, an introner jumped from one of these species to the other.

    The researchers hypothesize that the introners could be hitching a ride on giant viruses in order to transfer between species.

    “That virus itself is a selfish element as well, so this is like a selfish element shuttling around on another selfish element,” Corbett-Detig said.

    Although eight examples of horizontal gene transfer may not seem like many, the researchers believe there would be many more if they kept looking within the 8.74 million species of eukaryotes that exist.

    “Given how little of eukaryotic diversity we’ve sampled, I promise you that if we sampled the rest of them, we’d find many more,” Corbett-Detig said.

  • Researchers find CRISPR is capable of even more than we thought

    Researchers find CRISPR is capable of even more than we thought

    Every living creature on Earth needs to protect itself from things that would do it harm. Bacteria are no different. And despite their relative simplicity, they deploy remarkably savvy defensive strategies against viral invaders. The most well-known is CRISPR-Cas9, adapted for human use as the first FDA-approved genetic editing technique.

    In the past year, researchers at Rockefeller’s Laboratory of Bacteriology, headed by Luciano Marraffini, and at the MSKCC’s Structural Biology Laboratory, headed by Dinshaw Patel, have been studying key immune components of some CRISPR systems called CARF effectors. These newly discovered weapons take different approaches to achieving the same goal: arresting cellular activity, which prevents a virus from spreading through the rest of the bacterial population.

    In a recent publication in Science, the scientists announce the newest CARF effector they’ve discovered, which they coined Cat1. Thanks to an unusually complex molecular structure, this protein can deplete a metabolite essential for cellular function. Left without fuel, the viral invader’s plans for a further onslaught are brought to a grinding halt.

    “The collective work of our labs is revealing just how effective — and different — these CARF effectors are,” says Marraffini. “The range of their molecular activities is quite amazing.”

    Multiple defense systems

    CRISPR is a mechanism in the adaptive immune systems of bacteria and other certain single-cell organisms that offers protection against viruses, called phages. The six types of CRISPR systems work roughly the same way: A CRISPR RNA identifies foreign genetic code, which triggers a cas enzyme to mediate an immune response, often snipping off the invader material.

    But an increasing body of evidence indicates that CRISPR systems deploy a wide variety of defensive strategies beyond genetic scissors. Marraffini’s lab has led the way on much of this research. In particular, they have been studying a class of molecules in CRISPR-Cas10 systems called CARF effectors, which are proteins that are activated upon phage infection of a bacterium.

    CARF effector immunity is believed to work by creating an inhospitable environment for viral replication. For example, the Cam1 CARF effector causes membrane depolarization of an infected cell, while Cad1 triggers a sort of molecular fumigation, flooding an infected cell with toxic molecules.

    Metabolic freeze

    For the current study, the researchers wanted to try to identify additional CARF effectors. They used Foldseek, a powerful structural homology search tool, to find Cat1.

    They found that Cat1 is alerted to the presence of a virus by the binding of secondary messenger molecules called cyclic tetra-adenylate, or cA4, which stimulate the enzyme to cleave an essential metabolite in the cell called NAD+.

    “Once a sufficient amount of NAD+ is cleaved, the cell enters a growth-arrest state,” says co-first author Christian Baca, a TPCB graduate student in the Marraffini lab. “With cellular function on pause, the phage can no longer propagate and spread to the rest of the bacterial population. In this way, Cat1 is similar to Cam1 and Cad1 in that they all provide population-level bacterial immunity.”

    Unique complexity

    But while its immune strategy may be similar to these other CARF effectors, its form is not, as co-first author Puja Majumder, a postdoctoral research scholar in the Patel Lab, revealed through detailed structural analysis using cryo-EM.

    She found that the Cat1 protein has a surprisingly complex structure in which Cat1 dimers are glued by cA4 signal molecule, forming long filaments upon viral infection, and trap the NAD+ metabolites within sticky molecular pockets. “Once the NAD+ metabolite is cleaved by Cat1 filaments, it’s not available for the cell to use,” Majumder explains.

    But the protein’s singular structural complexity doesn’t stop there, she adds. “The filaments interact with each other to form trigonal spiral bundles, and these bundles can then expand to form pentagonal spiral bundles,” she says. The purpose of these structural components remains to be investigated.

    Also unusual is the fact Cat1 often seems to work alone. “Normally in type III CRISPR systems, you have two activities that contribute to the immunity effect,” Baca says. “However, most of the bacteria that encode Cat1 seem to primarily rely on Cat1 for their immunity effect.”

    Marraffini says these findings pose intriguing new questions. “While I think we’ve proven the big picture — that CARF effectors are great at preventing phage replication — we still have a lot to learn about the details of how they do it. It will be fascinating to see where this work leads us next.”

  • Artificial intelligence and genetics can help farmers grow corn with less fertilizer

    Artificial intelligence and genetics can help farmers grow corn with less fertilizer

    New York University scientists are using artificial intelligence to determine which genes collectively govern nitrogen use efficiency in plants such as corn, with the goal of helping farmers improve their crop yields and minimize the cost of nitrogen fertilizers.

    “By identifying genes-of-importance to nitrogen utilization, we can select for or even modify certain genes to enhance nitrogen use efficiency in major US crops like corn,” said Gloria Coruzzi, the Carroll & Milton Petrie Professor in NYU’s Department of Biology and Center for Genomics and Systems Biology and the senior author of the study, which appears in the journal The Plant Cell.

    In the last 50 years, farmers have been able to grow larger crop yields thanks to major improvements in plant breeding and fertilizers, including how efficiently crops uptake and use nitrogen, the key component of fertilizers.

    Still, most crops only use roughly 55 percent of the nitrogen in fertilizer that farmers apply to their fields, while the remainder ends up in the surrounding soil. When nitrogen seeps into groundwater, it can contaminate drinking water and cause harmful algae blooms in lakes, rivers, reservoirs, and warm ocean waters. Furthermore, the unused nitrogen that remains in the soil is converted by bacteria into nitrous oxide, a potent greenhouse gas that is 265 times more effective at trapping heat over a 100-year period than is carbon dioxide.

    The United States is the world’s leading producer of corn. This major cash crop requires large amounts of nitrogen to grow, but much of the fertilizer fed to corn is not taken up or used. Corn’s low nitrogen use efficiency presents a financial challenge for farmers, given the increasing costs of fertilizer — the majority of which is imported — and also risks harming the soil, water, air, and climate.

    To address this challenge in corn and other crops, NYU researchers have developed a novel process to improve nitrogen use efficiency that integrates plant genetics with machine learning, a type of artificial intelligence that detects patterns in data — in this case, to associate genes with a trait (nitrogen use efficiency).

    Using a model-to-crop approach, NYU researchers tracked the evolutionary history of corn genes that are shared with Arabidopsis, a small flowering weed often used as a model organism in plant biology due to the ease of studying it in the lab using the power of molecular genetic approaches. In a previous study published in Nature Communications, Coruzzi’s team identified genes whose responsiveness to nitrogen was conserved between corn and Arabidopsis and validated their role in plants.

    In The Plant Cell study, their most recent on this topic, the NYU researchers built upon their work in corn and Arabidopsis to identify how nitrogen use efficiency is governed by groups of genes — also known as “regulons” — that are activated or repressed by the same transcription factor (a regulatory protein).

    “Traits like nitrogen use efficiency or photosynthesis are never controlled by one single gene. The beauty of the machine learning process is it learns sets of genes that are collectively responsible for a trait, and can also identify the transcription factor or factors that control these sets of genes,” said Coruzzi.

    The researchers first used RNA sequencing to measure how genes in corn and Arabidopsis respond to nitrogen treatment. Using these data, they trained machine learning models to identify nitrogen-responsive genes conserved across corn and Arabidopsis varieties, as well as the transcription factors that regulate the genes-of-importance to nitrogen use efficiency (NUE). For each “NUE Regulon” — the transcription factor and corresponding set of regulated NUE genes — the researchers calculated a collective machine learning score and then ranked the top performers based on how well the combined expression levels could accurately predict how efficiently nitrogen is used in field-grown varieties of corn.

    For the top-ranked NUE Regulons, the researchers used cell-based studies in both corn and Arabidopsis to validate the machine learning predictions for the set of genes in the genome that are regulated by each transcription factor. These experiments confirmed NUE Regulons for two corn transcription factors (ZmMYB34/R3) that regulate 24 genes controlling nitrogen use as well as for a closely related transcription factor in Arabidopsis (AtDIV1), which regulates 23 target genes sharing a genetic history with corn that also control nitrogen use. When fed back into the machine learning models, these model-to-crop conserved NUE Regulons significantly enhanced the ability of AI to predict nitrogen use efficiency across field-grown corn varieties.

    Identifying NUE Regulons of collective genes and related transcription factors that govern nitrogen use will enable crop scientists to breed or engineer corn that needs less fertilizer.

    “By looking at corn hybrids at the seedling stage to see if expression of the identified genes-of-importance to nitrogen use efficiency is high, rather than planting them in the field and measuring their nitrogen use, we can use molecular markers to select the hybrids at the seedling stage that are most efficient in nitrogen use, and then plant those varieties,” said Coruzzi. “This will not only result in a cost savings for farmers, but also reduce the harmful effects of nitrogen pollution of groundwaters and nitrous oxide greenhouse gas emissions.”

    New York University has filed a patent application covering the research and findings described in this paper. Additional study authors include Ji Huang, Tim Jeffers, Nathan Doner, Hung-Jui Shih, Samantha Frangos, and Manpreet Singh Katari of NYU; Chia-Yi Cheng of NYU and National Taiwan University, and Matthew Brooks of the US Department of Agriculture’s Agricultural Research Service. The research was supported by the National Science Foundation Plant Genome Research Program (IOS-1339362) and the National Institutes of Health (R01-GM121753, F32GM116347).

  • Designer microbe shows promise for reducing mercury absorption from seafood

    Designer microbe shows promise for reducing mercury absorption from seafood

    UCLA and UCSD scientists inserted DNA-encoding methylmercury detoxification enzymes into the genome of an abundant human gut bacterium. The engineered bacterium detoxified methylmercury in the gut of mice and dramatically reduced the amount that reached other tissues, such as the brain and liver.

    Mice given an oral probiotic containing the engineered microbe and fed a diet high in bluefin tuna had much lower methylmercury levels than expected, suggesting that a probiotic might eventually make it safer for people to consume fish.

    UCLA and UCSD scientists inserted DNA-encoding methylmercury detoxification enzymes into the genome of an abundant human gut bacterium. The engineered bacterium detoxified methylmercury in the gut of mice and dramatically reduced the amount that reached other tissues, such as the brain and liver.

    Mice given an oral probiotic containing the engineered microbe and fed a diet high in bluefin tuna had much lower methylmercury levels than expected, suggesting that a probiotic might eventually make it safer for people to consume fish.

    Researchers performed the tests using pregnant mice and found lower levels of methylmercury in both maternal and fetal tissues, and lower signs of mercury toxicity in the fetal brain.

    An engineered gut microbe can detoxify methylmercury, reducing the amount that passes into the brain and developing fetuses of mice fed a diet rich in fish, UCLA and UC San Diego’s Scripps Institution of Oceanography scientists have discovered.

    “We envision the possibility that people could take a probiotic to offset the risk of consuming too much methylmercury, especially when pregnant,” said UCLA associate professor and director of the UCLA Goodman-Luskin Microbiome Center Elaine Hsiao, who is the senior author of a paper describing the research in the journal Cell Host & Microbe.

    Mercury is a pollutant that enters water from several sources, the largest of which are human activities such as coal burning, artisanal gold mining and smelting, and wastes from consumer products. In the ocean, mercury transforms into a toxic form called methylmercury. It also biomagnifies, meaning that methylmercury concentrations in animal tissues increase up the food chain from algae-eaters to top predators like humans.

    This means that all the methylmercury in something an organism eats goes into its tissues and is passed on to whatever eats it. Organisms near the top of their food chains, like bluefin tuna and humans, acquire all the mercury accumulated by the previous links in the chain. Thus, people who primarily eat food containing high mercury levels are at higher risk of mercury poisoning and birth defects.

    “Despite global efforts to reduce mercury emissions and its accumulation in fish, methylmercury levels in seafood are not expected to decline anytime soon. Fish remains a major and culturally important part of the diet for many people around the world and we hope it continues to be,” said co-senior author and Scripps associate professor of marine biogeochemistry Amina Schartup.

    Researchers modified Bacteroides thetaiotaomicron, a naturally abundant bacterium in the human digestive tract, by inserting DNA-encoding mercury detoxification enzymes from a mercury-resistant soil bacterium. After confirming that the engineered bacteria could clear methylmercury in a test tube, they replaced the natural gut microbiomes of mice with the modified bacteria and orally administered a large amount of methylmercury all at once. The mice had lower levels of methylmercury in their intestines just three hours after, and the level continued to fall for four days, showing that the bacteria helped to effectively eliminate the methylmercury in the intestine.

    They next asked whether the engineered bacteria would be effective at decreasing tissue methylmercury levels when exposure occurs gradually through routine dietary intake. To test this, the researchers fed the mice diets containing bluefin tuna over a period of days and found that not only did the bacteria reduce mercury in the intestine just as well, but less entered the brain and liver, too.

    They then tested pregnant mice and found lower levels of methylmercury in both maternal and fetal tissues, and lower signs of mercury toxicity in the fetal brain.

    “By reducing dietary methylmercury in the intestine, the gut bacteria helped to eliminate it from the body before it could enter the maternal bloodstream and access the developing offspring,” said first author and UCLA research scientist Kristie Yu.

    Co-author and UCLA researcher Franciscus Chandra said that the lowered signs of toxicity in the fetal brain showed that the bacterium works at levels that are biologically meaningful.

    When the team repeated the experiments with salmon, which contains lower levels of methylmercury than in bluefin tuna, the bacterium was also effective.

    Finally, they fed the bacteria as an oral probiotic to mice with intact microbiomes and fed them the same bluefin tuna diet as the mice in the previous experiment. Under these conditions, the engineered bacteria were also remarkably effective at minimizing the amount of methylmercury that entered tissues, suggesting that one day, a probiotic could be developed to reduce the risks of a fish-based diet.

    The researchers were supported by funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development; the National Institute of Environmental Health Sciences; the National Science Foundation; the Research Corporation of Scientific Advancement; the Simons Foundation; and the Packard Foundation.

    Hsiao and Schartup are working on improving the efficacy of the bacterium and moving closer to translation to humans, an endeavor for which continued federal funding is critical.

  • Making magnetic biomaterials

    Making magnetic biomaterials

    What if doctors could guide life-saving treatments through the body using only a magnet?

    An interdisciplinary collaboration at the University of Pittsburgh’s Swanson School of Engineering is bringing that concept closer to reality with the development of silk iron microparticles (SIMPs) — tiny, magnetic, and biodegradable carriers designed to precisely deliver drugs and treatments to sites in the body like aneurysms or tumors.

    Led by Pitt alumna Ande Marini (BioE PhD ’25), now a postdoctoral scholar in cardiothoracic surgery at Stanford University, David Vorp, John A. Swanson Professor of bioengineering, and Justin Weinbaum, research assistant professor of bioengineering, the team’s results, “Chemical Conjugation of Iron Oxide Nanoparticles for the Development of Magnetically Directable Silk Particles”, were published in the February edition of ACS Applied Materials & Interfaces.

    Marini’s team was inspired to develop these particles as part of their lab’s mission to improve treatments for abdominal aortic aneurysms (AAA), which can be life-threatening if left untreated and lead to nearly 10,000 deaths per year. By enabling early stage, noninvasive delivery of regenerative therapies using extracellular vesicles — membrane capsules that can facilitate intercellular communication — they hope to ultimately reduce the need for surgical intervention for AAA.

    “We want to find a way to deliver extracellular vesicles to the site of an abdominal aortic aneurysm in the least invasive way possible.” Vorp said. “We envisioned that we could inject extracellular vesicles onto a carrier and then somehow guide the carrier to the outside of the aortic wall, so we came up with the idea of using magnetic attraction.”

    To create the magnetic particles, the team collaborated with Mostafa Bedewy, associate professor of mechanical engineering & materials science at the Swanson School, and Golnaz Tomaraei (IE PhD ’23), Pitt alumna and Bedewy’s former PhD student. The duo’s expertise in nanomaterials and nanofabrication helped the team create the magnetic nanoparticles, which are roughly one-hundred-thousandth the width of a human hair. At that extremely small scale, nanomaterials can be manipulated to take on unique properties, such as magnetic responsiveness.

    “Our role was to synthesize magnetic nanoparticles with the right properties and bond them to the silk so they’d stay attached during movement,” Bedewy said. “You can think of it like towing cargo — we created the particles to carry drugs, and the nanoparticles are the tow hook.”

    Magnetically directable materials have previously been utilized for a variety of medical applications, but the team’s unique approach was to create the SIMPs by chemically conjugating the magnetic nanoparticles to silk — an FDA approved, biocompatible material — using the chemical compound glutathione.

    “We bridged biomaterials and chemical conjugation to create particles that could be magnetically guided,” Marini said. “By chemically bonding iron oxide nanoparticles to the regenerated silk fibroin, we enhanced their magnetic movability so we can potentially localize them externally to a site of interest in the body. “

    This research opens the door to a wide range of future applications — from targeted cancer therapies to regenerative treatments for cardiovascular disease. With the ability to magnetically guide the particles, the next step is loading them with therapeutic cargo.

    “With this paper, we’re showing that we can create an empty carrier that can be magnetically moved,” Marini said. “The next step is figuring out what kind of cargo we can load — regenerative factors, drugs, or other materials people want to magnetically localize. Whether it’s delivering cancer drugs with fewer side effects or slowing down tissue degradation in aneurysms, this technology has broad potential for regenerative medicine.”

    At the nanoscale, these findings also help Bedewy’s team continue to tailor the particles’ molecular structure and control their drug release rates for enhanced biomedical applications.

    “We’re trying to create a toolbox of treatments, and in materials science, there’s a lot of room for making more tools that can be useful for medical doctors and bioengineers to help create different ways of resolving problems in the body,” Bedewy said. “This is an exciting project where people with very different sets of expertise came together to solve a problem and produce an outcome that could potentially have an immense impact on human lives.”

  • No more copy-pasting: DNA base editing for better Lactobacillus strains

    No more copy-pasting: DNA base editing for better Lactobacillus strains

    A Kobe University team was able to edit the DNA of Lactobacillus strains directly without a template from other organisms. This technique is indistinguishable from natural variation and enabled the researchers to create a strain that doesn’t produce diabetes-aggravating chemicals.

    Humans have improved the microorganisms we rely on for millennia, selecting variants that are better able to produce wine, yogurt, natto and many other products. More recently, direct genetic modification has emerged as a tool to exert more precise and efficient control over the improvement, but also has drawn much public criticism for often using DNA from unrelated organisms in these modifications. Kobe University bioengineer NISHIDA Keiji says, “As a consequence, using such transgenic techniques is not favorable for food products due to legislations being restrictive and social acceptance being low.”

    Nishida and his team have developed a technique that gives even more precise control over the genetic content of a microorganism that does not rely on template DNA from other organisms. He says: “We have invented a DNA base editing technology named ‘Target-AID,’ which is superior to conventional techniques such as ‘CRISPR-Cas9’ in several aspects. For example, CRISPR-Cas9 induces DNA breaks and often causes cell death, while our Target-AID inserts precise point mutations without such breaks.”

    In the journal Applied Microbiology and Biotechnology, Nishida and his team now report that they teamed up with the Kobe University venture Bio Palette Co. Ltd. and successfully applied their technique to industrially important Lactobacillus strains used in manufacturing dairy products and pickles. “We edited individual, targeted locations in the genome at almost 100% efficiency in two major but fairly different Lactobacillus species. This shows that the technique can be readily applied to a wide range of strains,” says Nishida.

    To further showcase the capabilities of their approach, they focused on a gene that is involved in the production of a chemical that aggravates type 2 diabetes. By using Target-AID to mutate that gene, they engineered a Lactobacillus strain that could produce yogurt with less than a tenth of that chemical, making it safer to consume for people with type 2 diabetes. Nishida says: “The bacteria we produced are not subject to regulations concerning genetically modified organisms when used as foods, supplements or medicines. We thus expect that they can be readily commercialized after appropriate safety confirmation.”

    In their paper, the Kobe University team also showed that they could modify multiple genes at once and that they could even use their technique as a novel approach for basic research. For the future, Nishida has a broad outlook, saying, “We think our research will enable probiotic products with various health benefits, such as alleviating the effects on lifestyle-related diseases, strengthening immunity, improving allergic tolerance, and other benefits.”

    This research was funded by the Japan Science and Technology Agency (grants JPMJOP1851 and JPMJGX23B4), the New Energy and Industrial Technology Development Organization, and the Japan Agency for Medical Research and Development (grants 21ek0109448h0002 and 24bk0104169s0201). It was conducted in collaboration with researchers from Bio Palette Co. Ltd.

  • New tool for cutting DNA: Promising prospects for biotechnology

    New tool for cutting DNA: Promising prospects for biotechnology

    An INRS team discovers a new family of enzymes capable of inducing targeted cuts in single-stranded DNA.

    A few years ago, the advent of technology known as CRISPR was a major breakthrough in the scientific world. Developed from a derivative of the immune system of bacteria, CRISPR enables double strands of nucleotides in deoxyribonucleic acid (DNA) to be cut. This makes it possible to specifically modify a targeted gene in plant, animal and human cells. Ultimately, CRISPR became a preferred method in the search for treatments for acquired or hereditary diseases.

    Recently, Professor Frédéric Veyrier at the Institut national de la recherche scientifique (INRS) and his team developed a new genetic tool based on a family of specific enzymes called Ssn that allows targeted cuts to be induced exclusively in single-stranded DNA.

    The results of their work were recently published in the journal Nature Communications. This major breakthrough sheds light on a crucial genetic mechanism that could revolutionize a multitude of biotechnology applications.

    A form of DNA with a key role

    Single-stranded DNA is less common than double-stranded DNA. It is often found in some viruses and plays a key role in certain biological processes, such as cell replication or repair. Single-stranded DNA is also used in many technologies (sequencing, gene editing, molecular diagnostics, nanotechnology).

    To date, no endonuclease — enzyme that cuts DNA — has been described as exclusively targeting a single-stranded DNA sequence, which has constituted a barrier to the development of technologies based on this type of DNA.

    Now, for the first time in a laboratory, Professor Veyrier’s team has identified a family of enzymes capable of cutting a specific sequence in single-stranded DNA: the family of Ssn endonucleases.

    To achieve this, the research team at INRS’s Armand-Frappier Santé Biotechnologie Research Centre first characterized a new family of endonucleases part of the GIY-YIG superfamily called Ssn. More specifically, researchers focused on one of these enzymes in the bacterium Neisseria meningitidis, also known as the meningococcus. The enzyme targeted in the study is crucial to the exchange and alteration of genetic material, which influences evolution.

    “In studying it, we found that it recognizes a specific sequence that is found in many instances in its genome and plays a key role in the natural transformation of the bacterium. This interaction directly influences the dynamics of the bacterial genome,” explains Professor Veyrier, a specialist in genomic bacteriology and evolution.

    In addition to this fundamental discovery, INRS’s research scientists identified thousands of other similar enzymes. “We demonstrated that they are able to recognize and specifically cut their own single-stranded DNA sequence. Thousands of enzymes therefore have this property with their own specificity,” adds Alex Rivera-Millot, a postdoctoral fellow on Professor Veyrier’s team and co-first author of the study.

    An undeniable asset for health research

    These results, which represent a new tool for DNA recognition and exchange, are significant. They pave the way to many novel applications in biology and medicine. On the one hand, understanding this mechanism could help better control the bacteria in question and the associated infections.

    On the other, the discovery of enzymes specific to single-stranded DNA makes it possible to develop more precise and efficient genetic manipulation tools. This could namely improve methods of gene editing, DNA detection and molecular diagnosis. These enzymes could also be used to detect and manipulate DNA in various medical and industrial applications, such as pathogen detection or genetic manipulation for medical and therapeutic purposes.

    All of these avenues hold significant promise for addressing many health issues. Currently, there is a patent pending for the results of this work.

  • A new tool for parsing the ‘metabolic dialogue’ between microorganisms

    A new tool for parsing the ‘metabolic dialogue’ between microorganisms

    Scientists at the Max Planck Institute for Plant Breeding Research have developed an innovative system — called MetaFlowTrain — that allows the study of metabolic exchange and interactions within microbial communities under different environmental conditions. The study is now published in Nature Communications.

    Microbial communities consist of groups of microorganisms, such as bacteria, fungi, and other microscopic life forms, that coexist in specific environments. Although invisible to the naked eye, these communities are key for nutrient cycling, food web dynamics, and the degradation of pollutants in ecosystems. They are also essential for supporting the health of plants, animals, and humans by enhancing nutrient acquisition, bolstering the immune system, and protecting against harmful pathogens.

    The tiny organisms within microbial communities interact both with their environment and with each other not only via physical contacts — but also through the exchange of metabolites. These so-called exometabolites are small molecules secreted by microorganisms in the environment, such as amino acids, organic acids, alcohols, and secondary metabolites. They play crucial roles in shaping microbial communities, influencing interactions through cooperation and competition. In these highly dynamic systems, it has been challenging for scientists to identify which microorganisms produce specific metabolites and how these metabolites affect other members of the microbial community.

    Stéphane Hacquard and his team work on understanding the interactions between the members of plant-associated microbial communities. They realized that compartmentalizing the microorganisms would allow exclusion of the effects due to physical contact; thus, any remaining phenomena can be attributed solely to the exchange of metabolites and signals.

    The fruit of this realization is the MetaFlowTrain, a purpose-built fluidic system that allows scientists to introduce different microorganisms into special 3D-printed microchambers. These microchambers are surrounded by filters that permit metabolite exchange while preventing microorganism transfer. These microchambers can stand alone or be put in a series (e.g., such as a train with different wagons), each holding different groups of microorganisms. For instance, with bacteria in the first chamber and fungi in the second, scientists can now observe how bacteria affect fungi — and vice versa — by simply switching the chambers.

    The constant flow of fresh medium connecting these chambers allows different stress factors to be introduced to the system and prevents nutrient depletion in the microchambers. This represents a key innovation to identify novel microbial exometabolites with bioactive or signalling properties that shape microbial communities.

    MetaFlowTrain is easy to build and affordable, and holds enormous potential for identifying molecules that mediate microbe-microbe interactions and microbe-host associations. Specifically, scientists can now better ‘eavesdrop’ on the metabolic dialogues that drive cooperation or competition in microbial communities. Further, harnessing the system’s capabilities to discover novel antimicrobials that inhibit destructive plant pathogens presents new opportunities for sustainable agriculture and crop protection. In addition, the system could also be used to identify natural compounds for medicine and other fields.

    “The staggering diversity of molecules produced by microbes is a result of millions of years of evolution. These molecules serve different functions that help these communities to survive, adapt, and thrive in various environments. Understanding the functions and the mode of actions of some of these molecules will drive agricultural innovations,” says Stéphane Hacquard.

  • Further translation of the language of the genome

    Further translation of the language of the genome

    New research has uncovered more about the complexity of human gene regulation by identifying certain sequences of proteins called transcription factors that bind to DNA and regulate the expression of human genes.

    Published today (9 April) in Nature, researchers from the Wellcome Sanger Institute, the University of Cambridge and their collaborators explored how DNA-guided transcription factors interact with each other.

    This research adds to the groundwork of understanding the complex language of the gene regulatory code, and how DNA sequence patterns located close to our genes influence human development and disease risk.

    Each gene has a regulatory region that contains instructions on when and where the gene is expressed. This information is written in a code that is read by transcription factors, which bind to specific DNA sequences and either increase or decrease the gene’s expression.

    Previous research has explored the ‘language’ of the genome — the regulatory code that controls gene expression. It found that cooperation between multiple transcription factors is a key feature of transcription factor-DNA binding, with DNA actively facilitating interactions between various transcription factors.1 With the regulatory code being far more complex than the genetic code, which explains how DNA sequence determines the structure of proteins, researchers are aiming to understand the regulatory language in more detail, focusing on the ‘words’ and ‘grammar’ — such as the transcription factors — that influence when and where genes are expressed.

    This deeper understanding is crucial for uncovering how cells develop into specific types, how organs form and where they are located in the body during embryonic development, and for understanding what goes wrong in disease.

    The interactions between transcription factors guided by DNA are poorly understood. In a new study, researchers from the Sanger Institute and the University of Cambridge used two novel algorithms to analyse 58,000 pairs of transcription factors from human cells. They did this to identify how and where transcription factors interact with each other to bolster their understanding of the genomic language.2

    The researchers’ results reveal new patterns and preferences in how certain transcription factors interact with each other — also known as ‘motifs’. In this study, the researchers estimate that they identified between 18 and 47 per cent of all human transcription factor pair motifs, greatly adding to their understanding of the regulatory code.

    The team found that certain motifs they identified are present in developmental enhancers — DNA regulatory elements that activate transcription of a gene — that control important stages such as development of fingers. For example, the research notes that certain sequences of transcription factor motifs, or ‘words’ in the language, influence whether or not someone develops polydactyly — too many fingers — or syndactyly — a fusion of fingers.

    The findings also have implications for how scientists will use computational models — such as artificial intelligence — to predict protein structures in the future. Whilst these tools can predict the overall structure, they often cannot look into smaller details, such as how transcription factors interact with each other on DNA. These small interactions can have a big impact on human development, but computational models cannot always predict this. The researchers hope that future models will be able to incorporate the more minute transcription factor details to better predict protein structure and protein-DNA interactions.

    This research marks a step forward in studying the smaller ‘words’ in the language of gene expression. By identifying small but key motifs in the genome, this research will help scientists understand and interpret the mechanisms influenced by transcription factors, particularly in the non-coding regions of the genome. These regions — which make up 99 per cent of the genome — do not code for proteins but still play a significant role in regulation of gene expression, and risk for development of disease.

    Dr Ilya Sokolov, an author of the study at the Wellcome Sanger Institute, said: “By gaining a deeper understanding of how transcription factors interact when guided by DNA, we hope our research will shed light on the molecular basis of the regulatory code, particularly in the context of developmental disorders. These interactions are evolutionarily conserved across mammals and offer several advantages in development, from incorporating positional information to creating sharper gene expression responses. With advanced insights into the regulatory code, we are excited to help drive future research that will improve our understanding of human development and developmental disorders.”

    Professor Jussi Taipale, senior author of the study and Group Leader at the Wellcome Sanger Institute, said: “The human genome’s regulatory code is very complex, far more complex than the genetic code, and this research into transcription factor interactions unlocks deeper insights into the ‘language’ of the genome. Not only does our study provide more information into patterns of human development but it paves the way for future work with computational models that can hopefully incorporate these new data to better understand gene regulation.”

    Notes

    1. Arttu Jolma, Yimeng Yin, Kazuhiro R. Nitta, Kashyap Dave, Alexander Popov, Minna Taipale, Martin Enge, Teemu Kivioja, Ekaterina Morgunova, Jussi Taipale.(2015) ‘DNA-dependent formation of transcription factor pairs alters their binding specificity.’Nature. DOI: 10.1038/nature15518
    2. The researchers expressed a set of human transcription factors — enriched in proteins that are conserved in mammals — in Escherichia coli, combined them into a total of 58,754 transcription factor (TF) pairs and analysed their interactions by CAP-SELEX — consecutive affinity purification evolution of ligands by exponential enrichment. CAP-SELEX is a method which enables the discovery of TF-TF-DNA binding preferences.
  • From bacterial immunity to plant sex

    From bacterial immunity to plant sex

    Could it be that one of only three known markers directly targeting the DNA does not exist outside the realm of microbes? Now, researchers led by Xiaoqi Feng at the Institute of Science and Technology Austria (ISTA) have demonstrated that this marker — N4-methylcytosine (4mC) — is essential for sperm development and maturation in the liverwort Marchantia polymorpha, a key organism in plant evolution. The findings were published in Cell.

    It sometimes takes a living fossil to make some of the most interesting findings about life and evolution. A relative of mosses, the liverwort Marchantia is one of the oldest existing plant forms and probably the oldest lineage of plants to colonize land. In addition to its central function in the plants’ transition from an aquatic to a terrestrial ecosystem, Marchantia still uses an ancestral form of reproduction that is no longer found in most plants. Whereas other plants have evolved sexual reproduction independently of water, Marchantia sperm is released into rainwater drops splashing on the plants’ surface and swims to fertilize the nearby female plants. However, many molecular aspects of Marchantia sperm function have remained in the dark to this day.

    The group of Xiaoqi Feng at the Institute of Science and Technology Austria (ISTA) uses this unique plant as a model to uncover yet unknown molecular principles that shed new light on the evolution of sexual reproduction. And their latest findings are no less than groundbreaking. “Our study provides conclusive evidence for a new DNA marker in plants or animals with a very important function: It is essential for sexual reproduction in Marchantia, specifically affecting sperm development in male plants,” says Feng. The findings could have applications in biotechnology, opening up perspectives for regulating gene expression without altering the underlying DNA sequence.

    A long search beyond the realm of microbes

    N4-methylcytosine (4mC) is a form of immunity in bacteria, camouflaging their genome from enzymes that degrade foreign sequences by adding a methyl chemical group to target locations. It is one of the three known DNA markers: 4mC, 5mC, and 6mA. Since these chemical markers only ‘mask’ the underlying genetic code without mutating it, they are called “epigenetic markers,” based on the Greek prefix epi, meaning ‘over,’ ‘outside of,’ or ‘around.’ While 4mC and 5mC are chemical modifications of the cytosine nucleotide — one of the building blocks of DNA — 6mA is the only one to target the adenine nucleotide. Since it was never conclusively documented in plants or animals, 4mC has interested the scientific community for a long time.

    The levels are “crazy” — but needed for agile sperm

    In their quest to decipher the molecular mechanisms of sperm function in Marchantia, Feng and her team distinguished two waves of extensive DNA methylation during sperm development. These two waves literally cover the sperm’s genome. They showed that the first wave corresponded to 5mC, a DNA modification already known in animals and plants that targets and silences so-called “jumping genes.” However, 5mC alone could not explain the second wave of extensive methylation, which targets short sequences of two nucleotides scattered all over coding genes, the so-called CG (cytosine-guanine) dinucleotides. In addition, the team showed that genes with a sequence similarity to N4-cytosine methyltransferases, the enzymes that catalyze 4mC methylation in bacteria, were expressed in the same window of the plant’s sperm development. Could the team just have stumbled upon the enzymes for 4mC outside of microbes?

    Motivated by this discovery, they used myriad quantitative techniques to show that 4mC was indeed the culprit. They found that 4mC alone was responsible for a whopping fifteen percent of methylated cytosines in the mature Marchantia sperm as opposed to less than one percent in bacteria. “Having used a battery of conclusive experimental approaches, we can safely say we are sure of our results,” says Feng. “The levels of 4mC we detected in Marchantia sperm are crazy to think of.” Thus, the team demonstrated that the extensive 5mC and 4mC methylation waves were orchestrated during the plant’s sperm development. In addition, they showed that these “crazy” levels of 4mC had key functions in the Marchantia sperm. Without 4mC, the plant’s sperm can no longer compete for fertilization: its swimming becomes slower and less directional, its fertility is drastically decreased, and even in the rare cases of successful fertilization, the plant embryo’s early development is affected.

    Horizontal gene transfer

    Another form of DNA methylation, 6mA, was erroneously reported by other researchers in several organisms before its detection was traced to bacterial contamination. This has left a bad taste in scientists’ mouths and led to increased scrutiny in the DNA methylation field. “It is very important to use multiple independent methods for the detection of DNA methylation, and we wanted to make absolutely sure that our claims were sound. But in a way, the high level of 4mC was helpful for our confidence. After all, no bacterium carries this much methylation,” says Feng. But how did 4mC arise in Marchantia in the first place? The answer lies in transferring genetic material between different species outside of sexual reproduction, an evolutionary phenomenon called horizontal gene transfer, or HGT.

    HGT has been reported to happen frequently — on an evolutionary scale — between bacteria and plants. This is most probably how 4mC reached Marchantia. HGT events from soil bacteria to plants have contributed to the plant’s transition from an aquatic to a terrestrial environment. “This might be one of nature’s ad-hoc events that boosted evolution. The horizontal transfer of genetic material from bacteria to Marchantia turned out quite useful for the plants, so the trait was kept and selected for,” explains Feng.

    More 4mC waiting to be discovered?

    The liverwort is the only plant group where 4mC has been conclusively documented to date. “However, our findings are probably not a coincidence,” says Feng. In fact, early developmental stages in mammals usually include extensive methylation reprogramming, completely erasing and re-establishing the DNA epigenetic modifications. “So, we might need to look at a specific window of development in other plants and animals. 4mC might also be there, waiting to be discovered.”

    The present study sheds light on a fundamental function of sexual reproduction that evolved in nature and could find applications in biotechnology. By dissecting the precise function of the 4mC epigenetic modification in Marchantia, this molecular mechanism could potentially be used as a tool for epigenetic genome editing, i.e., methods that target gene expression without altering the DNA sequence.

    The present study was started at the Department of Cell and Developmental Biology, John Innes Centre, Norwich, UK, before Xiaoqi Feng joined the Institute of Science and Technology Austria (ISTA) faculty.