GN/ New CRISPR-combo boosts genome editing power in plants
Genetics biweekly vol.29, 18th May — 1st June
TL;DR
- Scientists have developed CRISPR-Combo, a method to edit multiple genes in plants while simultaneously changing the expression of other genes. This new tool will enable genetic engineering combinations that work together to boost functionality and improve breeding of new crops.
- New research has big implications for genomic medicine. Scientists have defined with atomic precision a new genome editing tool that is less than half the size of CRISPR-Cas9 — currently the most reliable genome editing system. This new tool would allow scientists to fit genetic editors into smaller viral delivery systems to fix a variety of diseases.
- A 30-year-old genetic mystery has been solved. It has previously been established that touch can trigger stress reactions in plants. However, the molecular models for explaining this process have been quite spartan so far. Now researchers have found genetic keys that explain how plants respond so strongly to mechanical stimuli. Cracking this code could help lead to higher yields and improved stress resistance in crops in the future.
- Cystic fibrosis is a rare genetic disease which can cause very serious symptoms. It is caused by mutations in the CFTR gene, which regulates water movement across the cell membrane. Using a model reproducing a respiratory epithelium — a protective tissue composed of a monolayer of cells — scientists have discovered that a simple film of liquid is sufficient to restore the airways’ seal and reduce the risk of bacterial infection.
- Scientists have used single-particle cryogenic electron microscopy to determine the structure of the ribosome of Candida albicans. Their results reveal a potential target for new drugs.
- T cells, biology textbooks teach us, are the soldiers of the immune system, constantly on the ready to respond to a variety of threats, from viruses to tumors. However, without rest and maintenance T cells can die and leave their hosts more susceptible to pathogens, scientists report.
- A synthesized antibiotic derived from computer models of bacterial gene products appears to neutralize even drug-resistant bacteria. The compound, named cilagicin, works well in mice and employs a novel mechanism to attack MRSA, C. diff, and several other deadly pathogens.
- Researchers have identified the genetic causes of three mitochondrial diseases by figuring out what dozens of poorly understood mitochondrial proteins do. The functions of hundreds more mitochondrial proteins remain unknown, indicating that this approach could be a promising path to finding better ways to diagnose and treat the bewildering array of conditions linked to malfunctioning mitochondria.
- Many types of animals, including humans, successfully coexist with retroviruses, and ancient retrovirus viral elements can even be found within our genome. These endogenous retroviruses can be utilized for development and evolution. However, uncontrolled endogenous retroviruses may be a cause of disease in the human body. Now, researchers have discovered that endogenous retroviruses in our genome may pose a risk in regenerative medicine.
- We often think of biological arms races occurring between the immune system and pathogens, or predator and prey, but biologists have now discovered an example that plays out within a single genome. Their work in fruit flies may have implications for key biological processes in humans, including fertility and even cancer.
- And more!
Overview
Genetic technology is defined as the term which includes a range of activities concerned with the understanding of gene expression, advantages of natural genetic variation, modifying genes and transferring genes to new hosts. Genes are found in all living organisms and are transferred from one generation to the next. Gene technology encompasses several techniques including marker-assisted breeding, RNAi and genetic modification. Only some gene technologies produce genetically modified organisms.
Modern genetic technologies like genome editing would not be possible without all the previous generations of genetic technologies that have enabled scientists to discover what genes are, what they do and how DNA can be modified to add, remove or replace genes. You can find major genetic technologies development milestones via the link.
Gene Technology Market
- The valuation of the genetic engineering market is projected to escalate to USD 6.90 MN by the end of 2027.
- Global Genetic Engineering Market is projected to grow at 12.48% CAGR during the assessment period (2017–2027).
- North America holds the largest share in the global genetic engineering market, followed by Europe and the Asia Pacific, respectively.
- Another research provider, MarketsandMarkets, forecasts the genome editing, genome engineering market to grow from USD 3.19 billion in 2017 to USD 6.28 billion by 2022, at a compounded annual growth rate (CAGR) of 14.5% during the forecast period. The key factors propelling market growth are rising government funding and growth in the number of genomics projects, high prevalence of infectious diseases (like COVID-19) and cancer, technological advancements, increasing production of genetically modified (GM) crops, and growing application areas of genomics.
Latest News & Research
Boosting plant genome editing with a versatile CRISPR-Combo system
by Changtian Pan, Gen Li, Aimee A. Malzahn, Yanhao Cheng, Benjamin Leyson, Simon Sretenovic, Filiz Gurel, Gary D. Coleman, Yiping Qi inNature Plants
Ten years ago, a new technology called CRISPR-CAS9, made it possible for scientists to change the genetic code of living organisms. As revolutionary as it was, the tool had its limitations. Like the first cell phones that could only perform one function, the original CRISPR method can perform one function: removing or replacing genes in a genetic sequence. Later iterations of CRISPR were developed for another function that allowed scientists to change gene expression by turning them on or off, without removing them from the genome. But each of these functions could only be performed independently in plants.
Now, scientists from the University of Maryland College of Agriculture and Natural Resources, have developed CRISPR-Combo, a method to edit multiple genes in plants while simultaneously changing the expression of other genes. This new tool will enable genetic engineering combinations that work together to boost functionality and improve breeding of new crops.
“The possibilities are really limitless in terms of the traits that can be combined,” said Yiping Qi, an associate professor in the Department of Plant Science and Landscape Architecture and co-author of the study. “But what is really exciting is that CRISPR-Combo introduces a level of sophistication to genetic engineering in plants that we haven’t had before.”
The benefits of manipulating more than one gene at a time can far outweigh the benefits of any one manipulation on its own. For example, imagine a blight raging through wheat fields, threatening farmer livelihoods and food security. If scientists could remove a gene from the wheat that makes it susceptible to the blight and simultaneously turn on genes that shorten the plant’s life cycle and increase seed production, they could rapidly produce blight-resistant wheat before the disease had the chance to do too much damage. That’s the type of engineering Qi and his team demonstrated in four different phases of experimentation.
Qi and his team had previously developed new CRISPR methods to regulate gene expression in plants, and to edit multiple genes at the same time. But to develop CRISPR-Combo, they had to establish that they could perform both of those genetic engineering functions in parallel without negative consequences. In this new paper, they demonstrated that using tomato and rice cells.
“As a proof of concept, we showed that we could knock out gene A and upregulate, or activate, gene B successfully, without accidentally crossing over and knocking out gene B or upregulating Gene A,” Qi said.
Then Qi and his colleagues tested CRISPR-Combo on a flowering plant called rockcress (ArabidopsisI), which is often used by researchers as a model for staple crops like corn and wheat. The researchers edited a gene that makes the plant more resistant to herbicides while activating a gene that causes early flowering, which produces seeds more quickly. The result was an herbicide-resistant rockcress plant that yielded eight generations in one year rather than the ordinary four.
For their third experiment, the team demonstrated how CRISPR-Combo could improve efficiency in plant breeding using tissue cultures from poplar trees. Breeding programs to develop new varieties of plants generally use tissue cultures rather than seeds — consider how a plant can regrow roots and leaves from a single stalk planted in the soil. Scientists genetically modify stem cells that have the ability to grow into full plants, and when those plants mature and produce seeds, the seeds will carry on the genetic modifications made to the stem cells.
Some plants are better at regenerating from tissue cultures than others, which makes this step the single largest bottleneck in genetic engineering of crops. For some plants the success rate is just 1%. Qi and his team addressed the bottleneck by first editing a few traits in poplar cells, then activating three genes that promote plant tissue regeneration.
“We showed in poplars that our new method could offer a solution to the tissue regeneration bottleneck, dramatically increasing the efficiency of genetic engineering,” Qi said.
Currently, growing genetically engineered plants from tissue cultures requires the addition of growth hormones, which activate growth promoting genes. The research team shortcut this process in rice by directly activating these genes with CRISPR-Combo. The result was gene-edited rice from tissue cultures that did not require hormone supplementation. Qi and his colleagues found that tissue cultures grown with their method expressed more of the edited gene than tissue grown using hormones.
“This method results in a highly efficient genome editing process,” Qi said.
Now that the team has demonstrated their CRISPR-Combo method works in a variety of plants for multiple purposes, they intend to conduct experiments in citrus, carrots and potatoes to test its viability in a fruit, vegetable and staple crop. They are also working to create an herbicide resistant golden rice with enhanced nutritional content and red rice with increased antioxidants.
Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9
by Gabriel Schuler, Chunyi Hu, Ailong Ke in Science
CRISPR has ushered in the era of genomic medicine. A line of powerful tools has been developed from the popular CRISPR-Cas9 to cure genetic diseases. However, there is a last-mile problem — these tools need to be effectively delivered into every cell of the patient, and most Cas9s are too big to be fitted into popular genome therapy vectors, such as the adenovirus-associated virus (AAV).
In new research, Cornell scientists provide an explanation for how this problem is solved by nature: they define with atomic precision how a transposon-derived system edits DNA in RNA-guided fashion. Transposons are mobile genetic elements inside bacteria. A lineage of transposon encodes IscB, which is less than half the size of Cas9 but equally capable of DNA editing. Replacing Cas9 with IscB would definitively solve the size problem.
The researchers used cryo-electron microscopy (Cryo-EM) to visualize the IscB-ωRNA molecule from a transposon system in high resolution. They were able to capture snapshots of the system in different conformational states. They were even able to engineer slimmer IscB variants, by removing nonessential parts from IscB.
“Next-generation fancy applications require the gene editor to be fused with other enzymes and activities and most Cas9s are already too big for viral delivery. We are facing a traffic jam at the delivery end,” said corresponding author Ailong Ke, professor of molecular biology and genetics in the College of Arts and Sciences. “If Cas9s can be packaged into viral vectors that have been used for decades in the gene therapy field, like AAV, then we can be confident they can be delivered and we can focus research exclusively on the efficacy of the editing tool itself.”
CRISPR-Cas9 systems use an RNA as a guide to recognize a sequence of DNA. When a match is found, the Cas9 protein snips the target DNA at just the right place; it’s then possible to do surgery at the DNA level to fix genetic diseases. The cryo-EM data gathered by the Cornell team show that the IscB-ωRNA system works in a similar way, with its smaller size achieved by replacing parts of the Cas9 protein with a structured RNA (ωRNA) which is fused to the guide RNA. By replacing protein components of the larger Cas9 with RNA, the IscB protein is shrunken to the core chemical reaction centers which snip the target DNA.
“It’s about understanding the molecules’ structure and how they perform the chemical reactions,” said first author Gabriel Schuler, a doctoral student in the graduate field of microbiology. “Studying these transposons gives us a new starting point to generate more powerful and accessible gene editing tools.”
It is believed that transposons — mobile genetic elements — were the evolutionary precursors to CRISPR systems. They were discovered by Nobel Laureate Barbara McClintock ’23, M.A. ’25, Ph.D. ‘27.
“Transposons are specialized genetic hitchhikers, integrating into and splicing out of our genomes all the time,” Ke said. “The systems inside bacteria in particular are being selected constantly — nature has basically tossed the dice billions of times and come up with really powerful DNA surgical tools, CRISPR included. And now, by defining these enzymes in high resolution, we can tap into their powers.”
As small as IscB is compared to CRISPR Cas9, the researchers believe they will be able to shrink it even smaller. They’ve already removed 55 amino acids without affecting IscB’s activity; they hope to make future versions of this genome editor even smaller and hence even more useful.
Better understanding the function of the companion guide RNA was another motivation behind the study, said co-first author Chunyi Hu, a postdoctoral researcher in the Department of Molecular Biology and Genetics. “There’s still a lot of mystery — like why do transposons use an RNA-guided system? What other roles this RNA may be playing?”
One challenge that yet remains for the researchers is that while the IscB-ωRNA is extremely active in test tubes, it was not as efficient at altering DNA in human cells. The next step in their research will be to use the molecular structure to explore the possibilities they have identified for the cause of the low activity in human cells. “We have some ideas, a lot of them actually, that we are eager to test in the near future,” Schuler said.
Surface Hydration Protects Cystic Fibrosis Airways from Infection by Restoring Junctional Networks
by Juliette L. Simonin, Alexandre Luscher, Davide Losa, Mehdi Badaoui, Christian van Delden, Thilo Köhler, Marc Chanson in Cells
Cystic fibrosis is a rare genetic disease which can cause very serious symptoms. In particular, patients suffer from chronic bacterial infections that can lead to respiratory failure. It is caused by mutations in the CFTR gene, which regulates water movement across the cell membrane. Consequently, mucus quality is altered, it is no longer capable of capturing undesirable bacteria and expelling them. Using a model reproducing a respiratory epithelium — a protective tissue composed of a monolayer of cells — teams from the University of Geneva (UNIGE) have discovered that a simple film of liquid is sufficient to restore the airways’ seal and reduce the risk of bacterial infection.
These results open the way to new therapies based on mucus hydration. A promising alternative to current therapies that are often not widely enough effective.
Despite recent therapeutic advances, people with cystic fibrosis — one in every 2,500 births in Europe — have a life expectancy of no more than 46 years and altered quality of life. The disease is caused by one or more mutations in the CFTR gene, which affects the proper functioning of an essential protective barrier. The epithelial cells that line the airways are usually sealed together and thus protect the airways from bacterial colonisation. They are also lined with a fluid, a slippery mucus that traps unwanted germs and carries them away. When the CFTR protein is altered, the junctions between the cells loosen and the dehydrated mucus tends to stagnate, both of which promote the development of respiratory infections.
“While it was already known that mucus hydration and the presence of sufficiently tight junctions preserved the integrity of the airways, the mechanisms involved and the links between these two mechanisms remained mysterious, which hindered the development of new therapies,” explains Marc Chanson, a professor in the Department of Cell Physiology and Metabolism and the Geneva Centre for Inflammation Research at the UNIGE Faculty of Medicine, who led this research.
The scientists first developed an in vitro model using human lung cells. This model, which was awarded the UNIGE 3R Prize in 2021 for reducing animal experimentation, reproduces airways epithelium of healthy and cystic fibrosis patients in a way that is both accurate and close to clinical reality. In collaboration with the team of Christian van Delden and Thilo Köhler from the Departments of Medicine and of Microbiology and Molecular Medicine at the UNIGE Faculty of Medicine, Marc Chanson and his team compared the response of epithelial cells invalidated for CFTR to bacterial infection, to which either hydrated, healthy mucus or physiological saline solution had been added.
“We observed a similar response in both cases: the presence of liquid, whatever its composition, restored the airways and protected them from infection,” explains Juliette Simonin, post-doctoral fellow in Marc Chanson’s laboratory and first author of the study. “Surface hydration is sufficient to tighten the junctions between cells and protects the epithelium integrity from bacterial colonisation, even when CFTR is not functioning.”
A triple therapy pharmacologically targeting the CFTR protein has recently become available on the market. However, it only targets certain mutations of the CFTR gene and is only prescribed for a specific population of people with cystic fibrosis. More widely effective and safe treatments are still sorely lacking.
“Our results provide evidence that rehydration of the airway surface is beneficial. The challenge now is to find a simple way of doing this in all people with the disease, whatever the mutation involved,” concludes Marc Chanson.
Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity
by Cara L. Brand, Mia T. Levine in Current Biology
Biological arms races are commonplace in nature. Cheetahs, for example, have evolved a sleek body form that lends itself to rapid running, enabling them to feast upon similarly speedy gazelles, the fastest of which may evade predation. On the molecular level, immune cells produce proteins to conquer pathogens, which may in turn evolve mutations to evade detection.
Though less well known, other games of one-upmanship unfold within the genome. In a new study, biologists at the University of Pennsylvania show, for the first time, evidence of a two-sided genomic arms race involving stretches of repetitive DNA called satellites. “Opposing” the rapidly evolving satellites in the arms race are similarly fast-evolving proteins that bind those satellites.
While satellite DNA does not encode genes, it can contribute to essential biological functions, such as formation of molecular machines that process and maintain chromosomes. When satellite repeats are improperly regulated, impairments to these crucial processes can result. Such disruptions are hallmarks of cancer and infertility.
Using two closely related species of fruit flies, researchers probed this arms race by purposefully introducing a species mismatch, pitting, for example, one species’ satellite DNA against the other species’ satellite-binding protein. Severe impairments to fertility were a result, underscoring evolution’s delicate balance, even at the level of a single genome.
“We typically think of our genome as a cohesive community of elements that make or regulate proteins to build a fertile and viable individual,” says Mia Levine, an assistant professor of biology in Penn’s School of Arts & Sciences and the senior author on the work. “This evokes the idea of a collaboration between our genomic elements, and that’s largely true.
“But some of these elements, we think, actually harm us,” she says. “This disquieting idea suggests that there needs to be a mechanism to keep them in check.”
The researchers’ findings, likely to also be relevant in humans, suggest that when satellite DNA occasionally escapes the management of satellite-binding proteins, significant costs to fitness can occur, including impacts on molecular pathways required for fertility and perhaps even those relevant in the development of cancer.
“These findings indicate that there is antagonistic evolution between these elements that can impact these seemingly conserved and essential molecular pathways,” says Cara Brand, a postdoc in Levine’s lab and first author on the work. “It means that, over evolutionary time, constant innovation is required to maintain the status quo.”
It’s long been known that the genome is not composed solely of genes. In between genes that give rise to proteins one can find long stretches of what Levine calls “gobbledygook.”
“If genes are words and you were to read the story of our genome, these other parts are incoherent,” she says. “For a long time, it was ignored as genomic junk.”
Satellite DNA is part of this so-called “junk.” In Drosophila melanogaster, the fruit fly species often used as a scientific model organism, satellite repeats make up roughly half the genome. Because they evolve so rapidly without any apparent functional consequence, however, scientists used to believe satellite repeats were unlikely to be doing anything useful in the body. But more recent work has revised this “junk DNA” theory, revealing that the “gobbledygook,” satellite repeats included, plays a variety of roles, many related to maintaining genome integrity and structure in the nucleus.
“So this presents a paradox,” Levine says. “If these regions of the genome that are highly repetitive actually do important jobs, or, if not managed properly, can be harmful, it suggests that we need keep them in check.”
In 2001, a group of scientists put forward a theory, suggesting that coevolution was taking place, with the satellites rapidly evolving and satellite binding proteins evolving to keep up. In the two decades since, scientists have offered support to the theory. With genetic manipulation, these studies have introduced a satellite-binding protein from one species into the genome of a closely related species and observed what happens as a result of the mismatch.
“Often these gene swaps cause dysfunction,” says Brand, “particularly disrupting a process that is usually mediated by regions of the genome that are enriched with repetitive DNA.”
These investigations lent support to the coevolution theory. But until researchers could experimentally manipulate both the satellite-binding protein and the satellite DNA, it would be impossible to prove that the disruption they observed arose because of an interaction between the two elements. In the current work, Levine and Brand found a way to do just that. Another fruit fly species, Drosophila simulans, lacks a satellite repeat that spans a whopping 11 million nucleotide base pairs found in its close relative, D. melanogaster. This satellite was known to occupy the same cellular location as a protein called Maternal Haploid (MH). The researchers also had access to a mutant strain of D. melanogaster that lack the 11 million base pair repeat.
“It turns out the fly can live and reproduce just fine without this repeat,” Levine says. “So it gave us a unique opportunity to manipulate both sides of the arms race.”
To first investigate the satellite-binding protein side, the researchers used the CRISPR/Cas9 gene editing system to remove the original MH gene from D. melanogaster and add back the D. simulans version of the gene. Compared to control females, female flies with the D. simulans MH gene had significantly reduced fertility, producing substantially fewer eggs. Flies that lacked MH altogether, however, were unable to produce any offspring; the embryos were not viable.
“This was interesting because it showed that these satellite-binding proteins are essential, even though they’re rapidly evolving,” says Brand. “Doing the gene swap showed us that we could rescue the ability to make embryos. But another function, related to the ovary and egg production, was impaired.”
Looking closely at the ovaries, Brand and Levine discovered that the apparent cause of reduced egg formation and atrophied ovaries was DNA damage. Such damage often triggers a checkpoint protein to stop developmental pathways. When the researchers repeated the experiments in a fly with a broken checkpoint protein, egg production levels were restored to a higher level.
Levine and Brand were then ready to test the other side of the coevolutionary arms race, to find evidence that the problems that arose with the swapped MH protein were due to an incompatibility with the 11 million base pair satellite, or if they were acting on a different genetic element. Here they relied on the D. melanogaster strain that was missing the repeat and found that the gene swap now had no effect on these flies. DNA damage levels, egg production, and ovary size were all normal.
Looking to the closest relative of the MH protein in humans, a protein called Spartan, gave the scientists a clue as to the mechanism behind these results. In humans, Spartan is understood to digest proteins that can get stuck on DNA, posing an obstacle to various processes and packaging that DNA must undergo. “After everything we’d discovered thus far,” Levine says, “we thought, maybe this wrong species version of the protein is chewing up something it shouldn’t.”
One of the proteins often targeted by Spartan is Topoisomerase II, or Top2, an enzyme that can help resolve tangles in tightly wound and entangled DNA. To see whether the negative effects of the MH gene mismatch owed to inappropriate degradation of Top2, they overexpressed Top2 and found fertility was restored. Reducing Top2, on the other hand, exacerbated the reduction in fertility.
“This repair process that MH is involved in happens in yeast, in flies, in humans, across the tree of life,” says Brand. “Yet we’re seeing rapid or adaptive evolution of these proteins involved. That suggests that this seemingly conserved and essential pathway requires evolutionary innovation.” In other words, coevolution must proceed apace, just to maintain this essential pathway.
In future work, Brand and Levine will be looking to see if segments of the genome beyond satellites are involved and will be looking in other organisms, including mammals, to drill down into the molecular players of these evolutionary arms races.
“There’s no reason to believe that these arms races are playing out only in flies,” Levine says. “The same types of proteins and satellites in primates also evolve rapidly and that tells us that what we are studying is broadly relevant.”
The focal genes involved in this study have important roles in human health. Spartan mutations have been associated with cancer and ineffective regulation of satellite DNA could shed light on infertility and miscarriage.
“The number of miscarriages is remarkably high, and certainly satellite DNA is an unprobed source of aneuploidy and genome instability,” Levine says.
The CD8α–PILRα interaction maintains CD8 + T cell quiescence
by Linghua Zheng, Xue Han, Sheng Yao, Yuwen Zhu, John Klement, Shirley Wu, Lan Ji, Gefeng Zhu, Xiaoxiao Cheng, Zuzana Tobiasova, Weiwei Yu, Baozhu Huang, Matthew D. Vesely, Jun Wang, Jianping Zhang, Edward Quinlan, Lieping Chen in Science
T cells, biology textbooks teach us, are the soldiers of the immune system, constantly on the ready to respond to a variety of threats, from viruses to tumors. However, without rest and maintenance T cells can die and leave their hosts more susceptible to pathogens, Yale scientists report.
“We may have to change how we teach T cell biology,” said Lieping Chen, the United Technologies Corporation Professor in Cancer Research at Yale and professor of immunobiology, of dermatology, and of medicine and senior author of the study.
Until pathogens are detected, T cells remain in a quiescent state. However, the molecular mechanisms that keep T cells inactive were previously unknown.
In the new study, Yale researchers show that a protein known as CD8a — which is found in a subset of T cells called CD8 cells — is crucial to keeping the cells in this dormant state. When scientists deleted this protein in mice, the protective CD8 cells were unable to enter a quiescent state and died, leaving the host vulnerable to infections.
Further, they identified another protein, PILRa, that provides a biochemical signal to CD8a. By disrupting this protein pair, both “memory” CD8 cells — cells that previously had been exposed to pathogens — and naïve cells died because they lacked the ability to stay in a quiescent state.
The researchers hope that understanding why this resting state is crucial to maintenance and survival of T cells can lead to improved immune system function.
Chen noted that as people age they tend to lose both naïve and memory T cells, making older individuals more susceptible to infections. It is possible that the inability of T cells to remain in a quiescent state could lead to people becoming more susceptible to infections and cancer, the authors suggest.
E-site drug specificity of the human pathogen Candida albicans ribosome
by Yury Zgadzay, Olga Kolosova, Artem Stetsenko, Cheng Wu, David Bruchlen, Konstantin Usachev, Shamil Validov, Lasse Jenner, Andrey Rogachev, Gulnara Yusupova, Matthew S. Sachs, Albert Guskov, Marat Yusupov in Science Advances
Most people carry the fungus Candida albicans on their bodies, without this causing many problems. However, a systemic infection with this fungus is dangerous and difficult to treat. Few antimicrobials are effective and drug resistance is increasing. An international group of scientists, including Albert Guskov, associate professor at the University of Groningen, have used single-particle cryogenic electron microscopy to determine the structure of the fungal ribosome. Their results reveal a potential target for new drugs.
Candida albicans usually causes no problems or just an itchy skin infection that is easily treated. However, in rare cases, it may cause systemic infections that can be fatal. Existing antifungal drugs cause a lot of side effects and are expensive. Furthermore, C. albicans isbecoming more drug resistant, so there is a real need for new drug targets.
‘We noted that no antifungal drugs are targeting protein synthesis, while half of the antibacterial drugs interfere with this system,’ says Guskov. A reason for this is that fungal ribosomes, the cellular machineries that translate the genetic code into proteins, are very similar in humans and fungi. ‘So, you would need a very selective drug to avoid killing our own cells.’
Therefore, Guskov and his collaborators reasoned that obtaining the structure of the C. albicans ribosomes would be valuable in finding drug targets. The classical approach is to grow crystals from purified ribosomes and to determine their structure using X-ray crystallography; however, this is a laborious technique. Instead, they used single-particle cryogenic electron microscopy, where a large number of single particles are imaged at very low temperatures in an electron microscope. The images of single particles — seen from different angles — are subsequently combined to produce an atomic-resolution structure.
‘In this way, we solved the structures of vacant and inhibitor-bound fungal ribosomes and compared their functions to those of ribosomes from yeast and rabbit — the latter as a model for the human ribosome — and repeated this for ribosomes bound to different inhibitors,’ explains Guskov.
One of these inhibitors was the antimicrobial cycloheximide (CHX), to which C. albicans is known to be resistant. By comparing the structures, the scientists noted that a single mutation in the E-site, which plays a key part in protein synthesis, prevents CHX from binding to C. albicans ribosomes. ‘The mutation changed one amino acid in the structure of this E-site from proline to glutamine. This substitution reduces the size of the binding site, so the inhibitor can’t attach and is therefore ineffective.’ Another inhibitor, phyllanthoside, is not blocked by the mutation.
‘By comparing the structures of the E-sites in vacant ribosomes in C. albicans and humans and information on the way that different inhibitors bind to the site, we can develop a specific inhibitor that blocks fungal ribosomes but not those of humans. This would then be a selective drug to treat fungal infections.’ The scientists are currently screening libraries of molecules to find drug leads.
‘It is extremely challenging to develop a vaccine against C. albicans, like we did for the coronavirus. So, we need drugs to treat systemic infections,’ Guskov explains. ‘The increasing drug resistance of this fungus is a real threat. If this continues, we could be in serious trouble unless new drugs are developed.’
Bioinformatic prospecting and synthesis of a bifunctional lipopeptide antibiotic that evades resistance
by Zongqiang Wang, Bimal Koirala, Yozen Hernandez, Matthew Zimmerman, Sean F. Brady in Science
A new antibiotic, synthesized at The Rockefeller University and derived from computer models of bacterial gene products, appears to neutralize even drug-resistant bacteria. The compound, named cilagicin, works well in mice and employs a novel mechanism to attack MRSA, C. diff, and several other deadly pathogens, according to a study.
The results suggest that a new generation of antibiotics could be derived from computational models. “This isn’t just a cool new molecule, it’s a validation of a novel approach to drug discovery,” says Rockefeller’s Sean F. Brady. “This study is an example of computational biology, genetic sequencing, and synthetic chemistry coming together to unlock the secrets of bacterial evolution.”
Bacteria have spent billions of years evolving unique ways to kill one another, so it’s perhaps unsurprising that many of our most powerful antibiotics are derived from bacteria themselves. With the exceptions of penicillin and a few other notables derived from fungi, most antibiotics were first weaponized by bacteria to fight off fellow bacteria.
“Eons of evolution have given bacteria unique ways of engaging in warfare and killing other bacteria without their foes developing resistance,” says Brady, the Evnin Professor and head of the Laboratory of Genetically Encoded Small Molecules. Antibiotic drug discovery once largely consisted of scientists growing streptomyces or bacillus in the lab and bottling their secrets to treat human disease.
But with the rise of antibiotic-resistant bacteria, there is an urgent need for new active compounds — and we may be running out of bacteria that are easy to exploit. Untold numbers of antibiotics, however, are likely hidden within the genomes of stubborn bacteria that are tricky or impossible to study in the lab. “Many antibiotics come from bacteria, but most bacteria can’t be grown in the lab,” Brady says. “It follows that we’re probably missing out on most antibiotics.”
An alternative method, championed by the Brady lab for the past fifteen years, involves finding antibacterial genes in soil and growing them within more lab-friendly bacteria. But even this strategy has its limitations. Most antibiotics are derived from genetic sequences locked within clusters of bacterial genes, known as biosynthetic gene clusters, that function as a unit to collectively code for a series of proteins. But those clusters are often inaccessible with current technologies.
“Bacteria are complicated, and just because we can sequence a gene doesn’t mean we know how the bacteria would turn it on to produce proteins,” Brady says. “There are thousands and thousands of uncharacterized gene clusters, and we have only ever figured out how to activate a fraction of them.”
Frustrated with their inability to unlock many bacterial gene clusters, Brady and colleagues turned to algorithms. By teasing apart the genetic instructions within a DNA sequence, modern algorithms can predict the structure of the antibiotic like compounds that a bacterium with these sequences would produce. Organic chemists can then take that data and synthesize the predicted structure in the lab.
It may not always be a perfect prediction. “The molecule that we end up with is presumably, but not necessarily, what those genes would produce in nature,” Brady says. “We aren’t concerned if it is not exactly right — we only need the synthetic molecule to be close enough that it acts similarly to the compound that evolved in nature.”
Postdoctoral associates Zonggiang Wang and Bimal Koirala from the Brady lab began by searching through an enormous genetic-sequence database for promising bacterial genes that were predicted to be involved in killing other bacteria and hadn’t been examined previously. The “cil” gene cluster, which had not yet been explored in this context, stood out for its proximity to other genes involved in making antibiotics. The researchers duly fed its relevant sequences into an algorithm, which proposed a handful of compounds that cil likely produces. One compound, aptly dubbed cilagicin, turned out to be an active antibiotic.
Cilagicin reliably killed Gram-positive bacteria in the lab, did not harm human cells, and (once chemically optimized for use in animals) successfully treated bacterial infections in mice. Of particular interest, cilagicin was potent against several drug-resistant bacteria and, even when pitted against bacteria grown specifically to resist cilagicin, the synthetic compound prevailed.
Brady, Wang, Koirala and colleagues determined that cilagicin works by binding two molecules, C55-P and C55-PP, both of which help maintain bacterial cell walls. Existing antibiotics such as bacitracin bind one of those two molecules but never both, and bacteria can often resist such drugs by cobbling together a cell wall with the remaining molecule. The team suspects that cilagicin’s ability to take both molecules offline may present an insurmountable barrier that prevents resistance.
Cilagicin is still far from human trials. In follow-up studies, the Brady lab will perform further syntheses to optimize the compound and test it in animal models against more diverse pathogens to determine which diseases it may be most effective in treating. Beyond the clinical implications of cilagicin, however, the study demonstrates a scalable method that researchers could use to discover and develop new antibiotics.
“This work is a prime example of what could be found hidden within a gene cluster,” Brady says. “We think that we can now unlock large numbers of novel natural compounds with this strategy, which we hope will provide an exciting new pool of drug candidates.”
Defining mitochondrial protein functions through deep multi-omic profiling
by Rensvold JW, Shishkova E, Sverchkov Y, et al in Nature
When something goes wrong in mitochondria, the tiny organelles that power cells, it can cause a bewildering variety of symptoms such as poor growth, fatigue and weakness, seizures, developmental and cognitive disabilities, and vision problems. The culprit could be a defect in any of the 1,300 or so proteins that make up mitochondria, but scientists have very little idea what many of those proteins do, making it difficult to identify the faulty protein and treat the condition.
Researchers at Washington University School of Medicine in St. Louis and the University of Wisconsin-Madison systematically analyzed dozens of mitochondrial proteins of unknown function and suggested functions for many of them. Using these data as a starting point, they identified the genetic causes of three mitochondrial diseases and proposed another 20 possibilities for further investigation. The findings indicate that understanding how mitochondria’s hundreds of proteins work together to generate power and perform the organelles’ other functions could be a promising path to finding better ways to diagnose and treat such conditions.
“We have a parts list for mitochondria, but we don’t know what many of the parts do,” said co-senior author David J. Pagliarini, PhD, the Hugo F. and Ina C. Urbauer Professor and a BJC Investigator at Washington University. “It’s similar to if you had a problem with your car, and you brought it to a mechanic, and upon opening the hood they said,
‘We’ve never seen half of these parts before.’ They wouldn’t know how to fix it. This study is an attempt to define the functions of as many of those mitochondrial parts as we can so we have a better understanding of what happens when they don’t work and, ultimately, a better chance at devising therapeutics to rectify those problems.”
Mitochondrial diseases are a group of rare genetic conditions that collectively affect one in every 4,300 people. Since mitochondria provide energy for almost all cells, people with defects in their mitochondria can have symptoms in any part of the body, although the symptoms tend to be most pronounced in the tissues that require the most energy, such as the heart, brain and muscles.
To better understand how mitochondria work, Pagliarini teamed up with colleagues, including co-senior author Joshua J. Coon, PhD, a UW-Madison professor of biomolecular chemistry & chemistry and an investigator with the Morgridge Institute for Research; and co-first authors Jarred W. Rensvold, PhD, a former staff scientist in Pagliarini’s lab, and Evgenia Shishkova, PhD, a staff scientist in Coon’s lab, to identify the functions of as many mitochondrial proteins as possible.
The researchers used CRISPR-Cas9 technology to remove individual genes from a human cell line. The procedure created a set of related cell lines, each derived from the same original cell line but with a single gene deleted. The missing genes coded for 50 mitochondrial proteins of unknown function and 66 mitochondrial proteins with known functions.
Then, they examined each cell line for clues to the role each missing gene normally plays in keeping the mitochondria running properly. The researchers monitored the cells’ growth rates and quantified the levels of 8,433 proteins, 3,563 lipids and 218 metabolites for each cell line. They used the data to build the MITOMICS (mitochondrial orphan protein multi-omics CRISPR screen) app, equipping it with tools to analyze and identify the biological processes that faltered when a specific protein went missing.
After validating the approach with mitochondrial proteins of known function, the researchers proposed possible biological roles for many mitochondrial proteins of unknown function. With further investigation, they were able to tie three proteins to three separate mitochondrial conditions.
“It is very exciting to see how our mass spectrometry technology platform can generate data on this scale but more importantly, data that can directly help us to understand human disease,” Coon said.
One condition is a multisystemic disorder caused by defects in the main energy-producing pathway. Co-author Robert Taylor, PhD, DSc, a professor of mitochondrial pathology at Newcastle University in Newcastle-upon-Tyne, U.K., identified a patient with clear signs of the disorder but no mutations in the usual suspect genes. The researchers identified a new gene in the pathway and showed that the patient carried a mutation in it.
Separately, Pagliarini and colleagues noticed that disrupting one gene, RAB5IF, eliminated a protein encoded by a different gene, TMCO1, that has been linked to cerebrofaciothoracic dysplasia. The condition is characterized by distinctive facial features and severe intellectual disability. In collaboration with co-author Nurten Akarsu, PhD, a professor of human genetics at Hacettepe University in Ankara, Turkey, the researchers showed that a mutation in RAB5IF was responsible for one case of cerebrofaciothoracic dysplasia and two cases of cleft lip in one Turkish family.
A third gene, when disrupted, led to problems with sugar storage, contributing to a fatal autoinflammatory syndrome. Data regarding that syndrome were published last year in a paper led by Bruno Reversade, PhD, of A*STAR, Singapore’s Agency for Science, Technology and Research.
“We focused primarily on the three conditions, but we found data connecting about 20 other proteins to biological pathways or processes,” said Pagliarini, a professor of cell biology & physiology, of biochemistry & molecular biophysics and of genetics. “We can’t chase down 20 stories in one paper, but we made hypotheses and put them out there for us and others to test.”
To aid scientific discovery, Pagliarini, Coon and colleagues have made the MITOMICS app available to the public. They built in several user-friendly analysis tools so anyone can look for patterns and create plots just by clicking around. All of the data can be downloaded for more advanced analysis.
“The hope is that this large dataset becomes one of a number in the field that collectively help us to devise better biomarkers and diagnostics for mitochondrial diseases,” Pagliarini said. “Every time we discover a function of a new protein, it gives us a new opportunity to target a pathway therapeutically. Our long-term goal is to understand mitochondria at sufficient depth to be able to intervene therapeutically, which we can’t do yet.”
Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells
by Kazuaki Monde, Yorifumi Satou, Mizuki Goto, Yoshikazu Uchiyama, Jumpei Ito, et al in Journal of Virology
Using a next generation sequencing analysis to examine human endogenous retrovirus (HERV) integration sites, researchers from Kumamoto University, the National Institute of Genetics (Japan), and the University of Michigan (USA) have discovered that these ancient retroviruses can undergo retrotransposition (DNA sequence insertion with RNA mediation) into iPS cells. The team believes that their discovery places a spotlight on a possible risk that HERVs pose when using iPS cells in regenerative medicine.
The study of ancient retroviruses embedded in our genome requires knowledge about our coexistence with viral threats throughout history. We know that HERVs occupy approximately 8% of the human genome and obtain mutations and deletions over long periods. HERVs are also expressed in early embryos and play several physiological roles in human development. For example, HERV-W and HERV-FRD Env proteins are important for placental formation, and HERV-K is thought to protect host cells from exogenous retrovirus infection. However, uncontrollable HERV-K expression is also thought to be associated with various diseases, including various cancers and neurological diseases, but the details of this association is not well known in humans.
Since no one has yet discovered replication competent HERVs in our genome, it is thought that they are from an extinct (fossil) virus. In their current work, the research team from Japan and the US discovered that HERV-K is expressed in SOX2-expressing cells, such as those in early embryos, cancer stem cells and iPS cells. They also found that some HERV-K are newly integrated into the host genome in the absence of Env, the viral envelope glycoprotein. This integration was dependent on reverse transcriptase, integrase and protease, thus the researchers hypothesized that the HERV-K embedded in our genome is actually not from a fossil virus, but moves on the genome through the synthesis of proviral DNA reverse transcription. Interestingly, when the researchers compared the HERV-K integration sites between iPS and fibroblast cells from the same donor, they found new HERV-K integration sites in iPS cells. However, the new integration sites were rarely preserved and disappeared during long-term culturing. HERV-K is likely to be randomly integrated into genome, thus the possibility remains that HERV-K retrotransposed-cells predominantly survive depending on their integration site.
The movement of HERV-K on the genome might cause cancer and neurological diseases by altering the gene expression profile. The researchers believe that the risk of HERV-K transposition is low in iPS cells but suggest that monitoring HERV-K integration sites should be seriously considered to improve the safety of regenerative medicine using iPS cells.
Touch signaling and thigmomorphogenesis are regulated by complementary CAMTA3- and JA-dependent pathways
by Essam Darwish, Ritesh Ghosh, Abraham Ontiveros-Cisneros, Huy Cuong Tran, Marcus Petersson, Liesbeth De Milde, Martyna Broda, Alain Goossens, Alex Van Moerkercke, Kasim Khan, Olivier Van Aken in Science Advances
A 30-year-old genetic mystery has been solved. It has previously been established that touch can trigger stress reactions in plants. However, the molecular models for explaining this process have been quite spartan so far. Now researchers at Lund University in Sweden have found genetic keys that explain how plants respond so strongly to mechanical stimuli. Cracking this code could help lead to higher yields and improved stress resistance in crops in the future.
When you water your garden plants, they react directly at a biochemical level. When a knife edge cuts a rhubarb stalk, thousands of genes are activated, and stress hormones are released.
Unlike humans, plants can not feel pain, but they still react strongly to mechanical stimuli from human touch, hungry animals, wind and rain, for example. These external factors lead to the plant’s molecular defense system being activated quickly, which in turn can contribute to plants becoming more resistant and flowering later. Although the phenomenon has been known since Darwin, there are still many question marks. A new study has examined the complex regulating networks that affect how the plant’s defenses are strengthened by external influences.
“We exposed the plant thale cress to soft brushing, after which thousands of genes were activated and stress hormones were released. We then used genetic screening to find the genes that were responsible for this process,” explains Olivier Van Aken, biology researcher at Lund University.
Previous studies have shown that the plant hormone jasmonic acid is an important mediator in touch signaling. It has also been known that jasmonic acid is only part of the plant’s complex network of touch-sensitive responses, and that there are several unidentified pathways that have not yet been unveiled. After extensive laboratory work, the researchers were able to identify three new proteins that play a key role in the plants’ response to touch.
“Our results solve a scientific mystery that has eluded the world’s molecular biologists for 30 years. We have identified a completely new signaling pathway that controls a plant’s response to physical contact and touch. Now the search for more paths continues,” says Essam Darwish, biology researcher at Lund University.
What possible applications will the new results have? Olivier Van Aken is also studying a centuries-old Japanese agricultural technology that involves trampling grain during the growth phase, to obtain more abundant harvests. The researchers believe that there is a lot of hidden knowledge about how mechanical stimuli can lead to higher yields and improved stress resistance in crops. Knowledge that in the long run can change modern agriculture at its core.
“Given the extreme weather conditions and pathogen infections that climate change leads to, it is of utmost importance to find new ecologically responsible ways to improve crop productivity and resistance,” concludes Olivier Van Aken.
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