GN/ Molecular mechanisms of new gene-editing tool revealed
Genetics biweekly vol.58, 24th May — 7th June
TL;DR
- New research has determined the spatial structure of various processes of a novel gene-editing tool called ‘prime editor.’ Functional analysis based on these structures also revealed how a ‘prime editor’ could achieve reverse transcription, synthesizing DNA from RNA, without ‘cutting’ both strands of the double helix. Clarifying these molecular mechanisms contributes greatly to designing gene-editing tools accurate enough for gene therapy treatments.
- Under certain conditions, some algae are able to produce hydrogen — a much sought-after green energy source. Its production takes place in the unique catalytic center of the unicellular algae and is only possible if certain cofactors of the relevant proteins are present. Researchers have identified how such a cofactor, the so-called hydrogen cluster, is assembled. Specifically, they describe the previously unexplained role of the enzyme HydF, which is involved in the final steps of assembly.
- An international team has found a new RNA virus that they believe is hitching a ride with a common human parasite. The virus is associated with severe inflammation in humans infected with the parasite Toxoplasma gondii, leading the team to hypothesize that it exacerbates toxoplasmosis disease.
- Research work on symbiosis — a mutually beneficial relationship between living organisms — is pushing back against the newer theory of a ‘single-origin’ of root nodule symbiosis (RNS) — that all symbiosis between plant root nodules and nitrogen-fixing bacteria stems from one point — instead suggesting a ‘multiple-origin’ theory of sybiosis which opens a better understanding for genetically engineering crops.
- Scientists have discovered that the most widely-used class of antifungals in the world cause pathogens to self-destruct. The research could help improve ways to protect food security and human lives.
- A widely found gene in plants has been newly identified as a key transporter of a hormone that influences the size of corn. The discovery offers plant breeders a new tool to develop desirable dwarf varieties that could enhance the crop’s resilience and profitability.
- A new method of drug delivery using proline, an amino acid found in chicken feathers and skin tissue, could be used to limit the side effects of chemotherapy and repair important enzymes, new research suggests.
- Newly generated, complete genomes for the sex chromosomes of six primate species may inform conservation of these endangered species and shed light on sex-related genetic diseases in both humans and our closest living relatives.
- Researchers have demonstrated how B cells infected with the Epstein-Barr virus (EBV) can contribute to a pathogenic, inflammatory phenotype that contributes to multiple sclerosis.
- Scientists have designed a way to ‘cloak’ proteins in a generalized technique that could lead to repurposing things like antibodies for biological research and therapeutic applications.
- And more!
Overview
Genetic technology is defined as the term that 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
According to Global Genetic Engineering Market Research Report: The genetic engineering market is projected to grow from USD 1.36 Billion in 2023 to USD 7.73 Billion by 2032, exhibiting a compound annual growth rate (CAGR) of 24.20% during the forecast period (2023–2032).
Growing demand for synthetic genes and increased use of CRISPR genome editing technology across various biotechnology industries are the key market drivers enhancing the market growth. In addition, it’s projected that increased government financing, a rise in the output of genetically modified crops, and an increase in genomics studies will all contribute to the expansion.
Latest Research
Structural basis for pegRNA-guided reverse transcription by a prime editor
by Yutaro Shuto, Ryoya Nakagawa, Shiyou Zhu, Mizuki Hoki, Satoshi N. Omura, Hisato Hirano, Yuzuru Itoh, Feng Zhang, Osamu Nureki in Nature
Joint research led by Yutaro Shuto, Ryoya Nakagawa, and Osamu Nureki of the University of Tokyo determined the spatial structure of various processes of a novel gene-editing tool called “prime editor.” Functional analysis based on these structures also revealed how a “prime editor” could achieve reverse transcription, synthesizing DNA from RNA, without “cutting” both strands of the double helix. Clarifying these molecular mechanisms contributes greatly to designing gene-editing tools accurate enough for gene therapy treatments.
The 2020 Nobel Prize in Chemistry was awarded to Jennifer Doudna and Emmanuelle Charpentier for developing a groundbreaking yet simple way to edit DNA, the “blueprint” of living organisms. While their discovery opened new avenues for research, the accuracy of the method and safety concerns about “cutting” both strands of DNA limited its use for gene therapy treatments. As such, research has been underway to develop tools that do not have these drawbacks.
The prime editing system is one such tool, a molecule complex consisting of two components. One component is the prime editor, which combines a SpCas9 protein, used in the first CRISPR-Cas gene editing technology, and a reverse transcriptase, an enzyme that transcribes RNA into DNA. The second component is the prime editing guide RNA (pegRNA), a modified guide RNA that identifies the target sequence within the DNA and encodes the desired edit. In this complex, the prime editor works like a “word processor,” accurately replacing genomic information. The tool has already been successfully implemented in living cells of organisms such as plants, zebrafish, and mice. However, precisely how this molecule complex executes each step of the editing process has not been clear, mostly due to a lack of information on its spatial structure.
“We became curious about how the unnatural combination of proteins Cas9 and reverse transcriptase work together,” says Shuto, the first author of the paper.
The research team used cryogenic electron microscopy, an imaging technique that makes observations possible at a near-atomic scale. The method required samples to be in glassy ice to protect them from the potential damage by the electron beams, posing some additional challenges.
“We found the prime editor complex to be unstable under experimental conditions,” explains Shuto. “So, it was very challenging to optimize the conditions for the complex to stay stable. For a long time, we could only determine the structure of Cas9.”
Finally overcoming the challenges, the researchers succeeded in determining the three-dimensional structure of the prime editor complex in multiple states during reverse transcription on the target DNA. The structures revealed that the reverse transcriptase bound to the RNA-DNA complex that formed along the “part” of the Cas9 protein associated with DNA cleavage, the splitting of a single strand of the double helix. While performing the reverse transcription, the reverse transcriptase maintained its position relative to the Cas9 protein. The structural and biochemical analyses also indicated that the reverse transcriptase could lead to additional, undesired insertions. These findings have opened new avenues for both basic and applied research. So, Shuto lays out the next steps.
“Our structure determination strategy in this study can also be applied to prime editors composed of a different Cas9 protein and reverse transcriptase. We want to utilize the newly obtained structural information to lead to the development of improved prime editors.”
A Conserved Binding Pocket in HydF is Essential for Biological Assembly and Coordination of the Diiron Site of [FeFe]-Hydrogenases
by Rieke Haas, Oliver Lampret, Shanika Yadav, Ulf-Peter Apfel, Thomas Happe in Journal of the American Chemical Society
A research team from Bochum has identified a crucial detail that’s essential for the production of hydrogen using biocatalysts.
Under certain conditions, some algae are able to produce hydrogen — a much sought-after green energy source. Its production takes place in the unique catalytic center of the unicellular algae and is only possible if certain cofactors of the relevant proteins are present. Researchers at Ruhr University Bochum, Germany, have identified how such a cofactor, the so-called hydrogen cluster, is assembled. Specifically, they describe the previously unexplained role of the enzyme HydF, which is involved in the final steps of assembly.
“Iron-sulphur ([FeS]) clusters are essential, widely distributed protein cofactors that perform a wide variety of functions in the cell,” explains lead author Rieke Haas from the Photobiotechnology research group headed by Professor Thomas Happe at Ruhr University Bochum. They are involved, for example, in catalyzing chemical reactions, transferring of electrons, sensing changing environmental conditions and synthesizing other complex metal cofactors.
The hydrogen-producing [FeFe] hydrogenases of algae also have an [FeS] cluster — a unique catalytic center. The fact that it facilitates the production of the green energy carrier hydrogen under mild reaction conditions makes it a key research priority for future-oriented energy production.
“In addition to iron and sulphur atoms, its cofactor contains other ligands that make the conversion of hydrogen possible,” explains Rieke Haas. “This means that the biosynthesis of the cofactor requires a complex sequence of different synthesis steps in order to provide all the necessary components.”
To to this, the organism needs a biosynthetic apparatus tailored to this process, which includes three enzymes that are responsible for the main synthesis steps. In particular, the role of the enzyme HydF, which is involved in the final assembly steps, has remained largely unexplained.
The researchers used site-specific mutagenesis to gain new insights into how the cofactor precursor is integrated into the enzyme and how individual amino acids are involved in anchoring and synthesis. HydF plays a role during the synthesis of a ligand that is essential for the delivery of protons for hydrogen turnover. Using methods such as hydrogen production measurements and ATR-FTIR spectroscopy, the team gathered a more detailed understanding of how HydF works and, in particular, the role of specific amino acids. By providing insights into the previously unknown function of the maturation enzyme HydF, these new findings may help to shed light on the biosynthesis of the unique cofactor of [FeFe]-hydrogenases.
A parasite odyssey: An RNA virus concealed in Toxoplasma gondii
by Purav Gupta, Aiden Hiller, Jawad Chowdhury, Declan Lim, Dillon Yee Lim, Jeroen P J Saeij, Artem Babaian, Felipe Rodriguez, Luke Pereira, Alejandro Morales-Tapia in Virus Evolution
An international team led by researchers at the University of Toronto has found a new RNA virus that they believe is hitching a ride with a common human parasite.
The virus, called Apocryptovirus odysseus, along with 18 others that are closely related to it, was discovered through a computational screen of human neuron data — an effort aimed at elucidating the connection between RNA viruses and neuroinflammatory disease. The virus is associated with severe inflammation in humans infected with the parasite Toxoplasma gondii, leading the team to hypothesize that it exacerbates toxoplasmosis disease.
“We discovered A. odysseus in human neurons using the open-science Serratus platform to search through more than 150,000 RNA viruses” said Purav Gupta, first author on the study, recent high school graduate and current undergraduate student at U of T’s Donnelly Centre for Cellular and Biomolecular Research. “Serratus identifies RNA viruses from public data by flagging an enzyme called RNA-dependent RNA polymerase, which facilitates replication of viral RNA. This enzyme allows the virus to reproduce itself and for the infection to spread.”
The parasite T. gondii is far-reaching, infecting an estimated one-third of the global population. It can live in any non-blood cell type, including neurons, forming cysts inside cells. The parasite is transmitted to nearby cells when the infected cell ruptures.
T. gondii infections often go unnoticed because they only lead to symptoms in rare cases. Regardless, toxoplasmosis merits investigation considering how widespread it is and the potential effects it may have on pregnant women and those who are immunocompromised, Gupta said.
“We believe the virus and parasite work hand-in-hand to cause disease in the human host, where the virus hides inside the parasite, like a soldier in a trojan horse, to gain entry to the human brain,” said Gupta. “Our research marks the first time that scientists have connected toxoplasmosis to a virus.”
The newly discovered A. odysseus is found in two hypervirulent strains of the T. gondii parasite, referred to as RUB and COUGAR.
RUB has been documented in French Guinea to cause severe fever and organ failure, while COUGAR has been shown in British Columbia to be connected to ocular toxoplasmosis — the leading cause of infectious blindness. Researchers found the strains in different geographical locations at different times, demonstrating their potentially wide-ranging impacts.
Symptoms of toxoplasmosis can be aggravated by a hyperactivated human immune response. The virus-carrying parasite triggers this type of response when the immune system senses the foreign RNA of the virus.
“The group of 19 RNA viruses we found are strong biomarkers for parasitic infection,” said Artem Babaian, principal investigator on the study and assistant professor of molecular genetics at the Donnelly Centre and the Temerty Faculty of Medicine. “It’s obvious now that the A. odysseus virus could be a valuable marker of disease-causing infections, like severe toxoplasmosis, in humans or other animals. The next step is to test if this raises the possibility that treating a parasite’s viruses could be an effective means of treating symptoms that arise from parasitic infections.”
Zoonotic viruses that infect other living things in our environment in order to reach us are expected to cause the majority of emerging infectious diseases in humans, Babaian noted. “This study underscores the importance of looking beyond the viruses that infect humans directly into the extended virome,” he said.
Shifts in evolutionary lability underlie independent gains and losses of root-nodule symbiosis in a single clade of plants
by Heather R. Kates, Brian C. O’Meara, Raphael LaFrance, Gregory W. Stull, Euan K. James, Shui-Yin Liu, Qin Tian, Ting-Shuang Yi, Daniel Conde, Matias Kirst, Jean-Michel Ané, Douglas E. Soltis, Robert P. Guralnick, Pamela S. Soltis, Ryan A. Folk in Nature Communications
A Mississippi State faculty member’s work on symbiosis — a mutually beneficial relationship between living organisms — is pushing back against the newer theory of a “single-origin” of root nodule symbiosis (RNS) — that all symbiosis between plant root nodules and nitrogen-fixing bacteria stems from one point — instead suggesting a “multiple-origin” theory of symbiosis which opens a better understanding for genetically engineering crops.
Ryan A. Folk, an assistant professor in the MSU Department of Biological Sciences and herbarium curator, is an author on a paper examining (RNS), which allows plants to access atmospheric nitrogen converted into usable forms through a mutualistic relationship with soil bacteria. He joins investigators at the University of Florida and an international team.
“A story of a single origin has become very popular in recent years, particularly among those hoping to genetically engineer symbiosis in crop plants, but using genomic data from 13,000 species and sophisticated statistical models, we confidently identified a scenario involving multiple origins. Symbiosis is a complex trait and our work identifies ideal experimental systems for better understanding the molecular mechanisms that led to the gain of symbiosis,” Folk said. “Our work is the first major push-back against the idea of a single origin as advocated by those working on genome comparisons.”
Folk said the single origin idea would suggest that the genetic engineering of crops, such as rice and maize, to work with nitrogen-fixing bacteria is a “lower hurdle” to cross.
“Our results, which point to multiple origins, complicates the picture because it suggests a lesser role for shared genetic machinery,” Folk said. “This would make it harder to transform crop plants that are not legumes to engage in a similar nitrogen-fixing symbiosis, but multiple origins also means diverse machinery, or as we argue, an enhanced ‘evolutionary palette’ to guide such experiments,” he said.
Azoles activate type I and type II programmed cell death pathways in crop pathogenic fungi
by Martin Schuster, Sreedhar Kilaru, Gero Steinberg in Nature Communications
Scientists have discovered that the most widely-used class of antifungals in the world cause pathogens to self-destruct. The University of Exeter-led research could help improve ways to protect food security and human lives.
Fungal diseases account for the loss of up to a quarter of the world’s crops. They also pose a risk to humans and can be fatal for those with weakened immune systems.
Our strongest “weapon” against fungal plant diseases are azole fungicides. These chemical products account for to a quarter of the world agricultural fungicide market, worth more than £3 billion per year. Antifungal azoles are also widely used as a treatment against pathogenic fungi which can be fatal to humans, which adds to their importance in our attempt to control fungal disease.
Azoles target enzymes in the pathogen cell that produce cholesterol-like molecules, named ergosterol. Ergosterol is an important component of cellular bio-membranes. Azoles deplete ergosterol, which results in killing of the pathogen cell. However, despite the importance of azoles, scientists know little about the actual cause of pathogen death.
Funded by the BBSRC, the team of researchers, led by Professor Gero Steinberg, combined live-cell imaging approaches and molecular genetics to understand why the inhibition of ergosterol synthesis results in cell death in the crop pathogenic fungus Zymoseptoria tritic (Z. tritici). This fungus causes septoria leaf blotch in wheat, a serious disease in temperate climates, estimated to cause more than £250 million per year in costs in the UK alone due to harvest loss and fungicide spraying.
The Exeter team observed living Z. tritici cells, treated them with agricultural azoles and analysed the cellular response. They showed that the previously accepted idea that azoles kill the pathogen cell by causing perforation of the outer cell membrane does not apply. Instead, they found that azole-induced reduction of ergosterol increase the activity of cellular mitochondria, the “powerhouse” of the cell, required to produce the cellular “fuel” that drives all metabolic processes in the pathogen cell. While producing more “fuel” is not harmful in itself, the process leads to the formation of more toxic by-products. These by-products initiate a “suicide” programme in the pathogen cell, named apoptosis. In addition, reduced ergosterol levels also trigger a second “self-destruct” pathway, which causes the cell to “self-eat” its own nuclei and other vital organelles — a process known as macroautophagy. The authors show that both cell death pathways underpin the lethal activity of azoles. They conclude that azoles drive the fungal pathogen into “suicide” by initiating self-destruction.
The authors found the same mechanism of how azoles kill pathogen cells in rice-blast fungus Magnaporthe oryzae. The disease caused by this fungus kills up to 30 per cent of rice, an essential food crop for more than 3.5 billion people across the world. The team also tested other clinically relevant anti-fungal drugs that target the ergosterol biosynthesis, including Terbinafine, Tolfonate and Fluconazole. All initiated the same responses in the pathogen cell, suggesting that cell suicide is a general consequence of ergosterol biosynthesis inhibitors.
Lead author Professor Gero Steinberg, who holds a Chair in Cell Biology and is Director of the Bioimaging Centre at the University of Exeter, said: “Our findings rewrite common understanding of how azoles kill fungal pathogens. We show that azoles trigger cellular “suicide” programmes, which result in the pathogen self-destructing. This cellular reaction occurs after two days of treatment, suggesting that cells reach a “point of no return” after some time of exposure to azoles. Unfortunately, this gives the pathogen time to develop resistance against azoles, which explains why azole resistance is advancing in fungal pathogens, meaning they are more likely to fail to kill the disease in crops and humans.
“Our work sheds light on the activity of our most widely used chemical control agents in crop and human pathogens across the world. We hope that our results prove to be useful to optimise control strategies that could save lives and secure food security for the future.”
ZmPILS6 is an auxin efflux carrier required for maize root morphogenesis
by Craig L. Cowling, Arielle L. Homayouni, Jodi B. Callwood, Maxwell R. McReynolds, Jasper Khor, Haiyan Ke, Melissa A. Draves, Katayoon Dehesh, Justin W. Walley, Lucia C. Strader, Dior R. Kelley in Proceedings of the National Academy of Sciences
A widely found gene in plants has been newly identified as a key transporter of a hormone that influences the size of corn. The discovery offers plant breeders a new tool to develop desirable dwarf varieties that could enhance the crop’s resilience and profitability.
A team of scientists led by Iowa State University spent years working to pinpoint the functions of the gene ZmPILS6. Now, they have been able to characterize it as an important driver of plant size and architecture, a carrier for an auxin hormone that helps govern growth in roots below ground and shoots, or stalks, above ground.
“A hallmark of the current age of science is that we have all this high-quality genome data, whether for corn or humans or other organisms, and now we have the task of figuring out what the genes actually do,” said Dior Kelley, assistant professor of genetics, development and cell biology at Iowa State, who led the research team.
The group used “reverse genetic screening” (from the gene to traits expressed in the plant), combined with other techniques, as they tracked their gene’s role in corn development. Reverse screens require multiple growing seasons and don’t always work, according to Kelley. It took seven years for her group to thoroughly characterize ZmPILS6 and verify it regulates plant growth.
When “knocked out” of modified, mutant plants, its absence suppressed root lateral formation and plant height. The research has led to a provisional patent for its potential to be used in breeding programs to create short stature corn that is still highly productive.
“I think of this as ‘pixie’ corn,” Kelley said. “There’s a lot of interest in it for all kinds of reasons, including reduced use of water and nutrients and its ability to withstand high winds.”
As they studied ZmPILS6 in corn, the researchers made another curious finding: The gene seemed to have opposite effects on plant growth than a comparable gene in Arabidopsis, a plant often used as a model for research.
“This was very unexpected,” Kelley said. “It illustrates that plant proteins, which have evolved in different contexts, can behave differently. It emphasizes the need to study genes directly within key crops of interest, rather than thinking we understand them based on how they work in other plants.”
Kelley gives a lot of the credit for the project’s success to a “great team of collaborators,” especially Craig Cowling, a doctoral student in Kelley’s lab who is the first author on the paper. “Craig was the one to really dig in, to confirm that this gene carries the plant hormone auxin, and it absolutely controls size in corn.”
“This project and being acknowledged as first author on a paper in this important journal has been a little unbelievable,” Cowling said. “It’s been a long journey for me. I never thought I would go to college when I was in high school in Des Moines, so I went into ROTC and then the Marines, where I worked around the world as a technology specialist. When I got out, I wanted to do something different. Thanks to some good mentors, I’ve figured out that I love working with and understanding plants.”
Metal-peptidic cages — Helical oligoprolines generate highly anisotropic nanospaces with emergent isomer control
by Ben E. Barber, Ellen M.G. Jamieson, Leah E.M. White, Charlie T. McTernan in Chem
A new method of drug delivery using proline, an amino acid found in chicken feathers and skin tissue, could be used to limit the side effects of chemotherapy and repair important enzymes, new research suggests.
Researchers have designed a cage (a box made of single molecules) from biologically compatible peptides, short amino acids that form the basis of proteins. These cages can house drugs of different sizes and transport them in the body with high levels of precision.
The negative side effects associated with chemotherapy, such as hair loss and nerve damage, are a result of ‘off-site toxicity’, where the treatment kills healthy cells surrounding tumours as well as the tumour itself. By creating a nano-sized cage to house the drug and carry it into the tumour before releasing it, this effect can be channelled more directly to the tumour, shielding healthy cells.
The cage can be tuned to different sizes, enabling different payloads of drugs. This flexible structure allows for chemotherapy drugs, antibiotics, and antivirals to potentially be delivered. Previously, cages of this kind could only be made using hydrocarbon molecules found in tar, which can often be toxic to humans. This structure, researchers believe, also opens the door for faulty enzymes to be replaced within the body, which has previously not been possible. Historically, enzymes, which are composed of proteins and perform important functions in the body, could only have their activities blocked by drugs. The blocking of this functionality would then have an impact in the body, like reducing inflammation. Now, the cages could replace this function which may lay the groundwork for a new form of treatment.
Principal author Dr Charlie McTernan, Lecturer in Chemistry at King’s College London and Group Leader at the Francis Crick Institute, said “What we’ve created is essentially a biologically compatible molecular teabag. We can fill this teabag, or cage made from widely available proline and collagen, with several different medicines and deliver them in a much more targeted way than we could before.”
“In time, we hope that this could mean that we can limit the hair loss, nausea, and other unpleasant side effects of chemotherapy. We might even be able to repair malfunctioning enzymes that have an influence on the development of cancer. The best part is we can do this sustainably and at scale.”
Proline is very straight and rigid in shape, while also being soluble in water, which makes it uniquely suited for drug delivery, as water makes up roughly 60% of the human body. By binding the peptide to small amounts of metal such as palladium, the researchers could create a tuneable structure they could rapidly increase or decrease in size.
As proline and collagen are widely available and don’t rely on chains of hydrocarbons like previous methods, the team hope to sustainably scale up their current production in the lab.
The complete sequence and comparative analysis of ape sex chromosomes
by Kateryna D. Makova, Brandon D. Pickett, Robert S. Harris, et al in Nature
Newly generated, complete “end-to-end” reference genomes for the sex chromosomes of five great ape species and one lesser ape species — produced by an international collaborative team led by researchers at Penn State, the National Human Genome Research Institute and the University of Washington — highlight extremely rapid changes on the male-specific Y chromosome among ape species. These findings shed light on the evolution of sex chromosomes and inform understanding of diseases related to genes on these chromosomes in both apes and humans.
“The Y chromosome is important for human fertility, and the X chromosome harbors genes critical for reproduction, cognition and immunity,” said Kateryna Makova, Verne M. Willaman Chair of Life Sciences, professor of biology at Penn State and leader of the research team. “Our study opens doors for many future investigations of sex chromosomes, how they evolved, and diseases associated with them. The living non-human great ape species we studied are all endangered. The availability of their complete sex chromosome sequences will facilitate studies of their sex-specific dispersal in the wild and of their genes important for reproduction and fertility.”
Such reference genomes act as a representative example that are useful for future studies of these species. The team found that, compared to the X chromosome, the Y chromosome varies greatly across ape species and harbors many species-specific sequences. However, it is still subject to purifying natural selection — an evolutionary force that protects its genetic information by removing harmful mutations.
“Researchers sequenced the human genome in 2001, but it wasn’t actually complete,” Makova said. “The technology available at the time meant that certain gaps weren’t filled in until a renewed effort led by the Telomere-to-Telomere, or T2T, Consortium in 2022–23. We leveraged the experimental and computational methods developed by the Human T2T Consortium to determine the complete sequences for the sex chromosomes of our closest living relatives — great apes.”
The team produced complete sex chromosome sequences for five species of great apes — chimpanzee, bonobo, gorilla, Bornean orangutan and Sumatran orangutan, which comprise most great ape species living today — as well as a lesser ape, siamang. They generated sequences for one individual of each species. The resulting reference genomes act as a map of genes and other chromosomal regions, which can help researchers sequence and assemble the genomes of other individuals of that species. Previous sex chromosome sequences for these species were incomplete or — for the Bornean orangutan and siamang — did not exist.
“The Y chromosome has been challenging to sequence because it contains many repetitive regions, and, because traditional short-read sequencing technology decodes sequences in short bursts, it is difficult to put the resulting segments in the correct order,” said Karol Pál, postdoctoral researcher at Penn State and a co-first author of the study. “T2T methods use long-read sequencing technologies that overcome this challenge. Combined with advances in computational analysis, on which we collaborated with Adam Phillippy’s group at the NHGRI, this allowed us to completely resolve repetitive regions that were previously difficult to sequence and assemble. By comparing the X and Y chromosomes to each other and among species, including to the previously generated human T2T sequences of the X and the Y, we learned many new things about their evolution.”
“Sex chromosomes started like any other chromosome pair, but the Y has been unique in accumulating many deletions, other mutations and repetitive elements because it does not exchange genetic information with other chromosomes over most of its length,” said Makova, who is also the director of the Center for Medical Genomics at Penn State.
As a result, across the six ape species, the research team found that the Y chromosome was much more variable than the X over a variety of characteristics, including size. Among the studied apes, the X chromosome ranges in size from 154 million letters of the ACTG alphabet — representing the nucleotides that make up DNA — in chimpanzee and human to 178 million letters in gorilla. In contrast, the Y chromosome ranges from 30 million DNA letters in siamang to 68 million letters in Sumatran orangutan.
The amount of DNA sequence shared between species was also more variable on the Y. For example, about 98% of the X chromosome aligns between human and chimpanzee, but only about a third of the Y aligns between them. The researchers found that this is in part because the Y chromosome is more likely to be rearranged or have portions of its genetic material duplicated.
Additionally, the percentage of the chromosome occupied by sequences that are repeated is highly variable on the Y. Whereas, depending on the species, 62% to 66% of the X chromosomes are occupied by repetitive elements, 71% to 85% of the Y chromosomes are occupied by them. These percentages are higher on both the X and the Y than in other chromosomes in the human genome.
“We found the ape Y to be shrinking, accumulating many mutations and repeats, and losing genes,” Makova said. “So why hasn’t the Y chromosome disappeared, as some previous hypotheses suggested? In collaboration with Sergei Kosakovsky Pond from Temple University and others, we found that the Y chromosome still has a number of genes evolving under purifying selection — a type of natural selection that keeps gene sequences intact. Many of these genes are important for spermatogenesis. This means that the Y chromosome is unlikely to disappear any time soon.”
The researchers found that many genes on the Y chromosome seem to use two strategies to survive. The first takes advantage of genetic redundancy — the presence of multiple copies of the same gene on a chromosome — so that intact copies of the gene can compensate for copies that might acquire mutations. The team quantified this genetic redundancy by completing the landscape of multi-copy gene families on ape sex chromosomes for the first time.
The second survival strategy takes advantage of palindromes, where the sequence of letters in the DNA alphabet is followed by the same, but inverted sequence, for example, ACTG-GTCA. When located within a palindrome, genes benefit from the palindrome’s ability to correct mutations.
“We found that the Y chromosome can exchange genetic information with itself between the repeated sequences of the two palindrome arms, which fold so that the inverted sequences align,” Pál said. “When two copies of the same gene are located within palindromes, and one copy is hit by a mutation, the mutation can be rescued by the genetic exchange with another copy. This can compensate for the Y’s lack of genetic information exchange with the other chromosomes.”
The research team obtained the complete sequences of palindromes on ape sex chromosomes also for the first time, as they were previously difficult to sequence and study. They found that palindromes are particularly abundant and long on the ape Y chromosome, yet they are usually only shared among closely related species. In collaboration with Michael Schatz and his team at Johns Hopkins University, the researchers also studied the sex chromosomes of 129 individual gorillas and chimpanzees to better understand the genetic variation within each species and search for evidence of natural selection and other evolutionary forces acting on them.
“We obtained substantial new information from previously studied gorilla and chimpanzee individuals by aligning their sex chromosome sequencing reads to our new reference sequences,” said Zachary Szpiech, assistant professor of biology at Penn State and an author of the paper. “While increasing the sample size in the future will be very helpful to improve our ability to detect signatures of different evolutionary forces, this can be ethically and logistically challenging when working with endangered species, so it is critical that we can get the most out of the data we do have.”
The researchers explored a variety of factors that could explain variation on the Y chromosome within gorillas and within chimpanzees, and this analysis revealed additional signatures of purifying selection on the Y. This confirms the role of this type of natural selection on the Y, as was discovered in their previous analyses of genes.
“The powerful combination of bioinformatic techniques and evolutionary analyses that we used allows us to better explain the evolutionary processes acting on sex chromosomes in our closest living relatives, great apes,” said Christian Huber, assistant professor of biology at Penn State and an author of the paper. “Additionally, the reference genomes we produced will be instrumental for future studies of primate evolution and human diseases.”
Multiple sclerosis patient-derived spontaneous B cells have distinct EBV and host gene expression profiles in active disease
by Samantha S. Soldan, Chenhe Su, Maria Chiara Monaco,et al in Nature Microbiology
The Wistar Institute’s Paul M. Lieberman, Ph.D., and lab team led by senior staff scientist and first author, Samantha Soldan, Ph.D., have demonstrated how B cells infected with the Epstein-Barr virus (EBV) can contribute to a pathogenic, inflammatory phenotype that contributes to multiple sclerosis (MS); the group has also shown how these problematic B cells can be selectively targeted in a way that reduces the damaging autoimmune response of multiple sclerosis.
EBV — a usually inactive, or latent, herpesvirus — affects most of the human population; more than 90% of people carry the virus as a passive, typically symptomless infection. However, EBV infection has been linked to several diseases, including MS: an incurable, chronic autoimmune disease that causes the body’s immune system to attack the myelin sheath of neurons in the brain and nervous system. Because myelin sheathing facilitates fast nervous system signaling (the fatty insulation of myelin along a neuron’s axon allows electrical impulses to travel through neuronal networks faster), its degradation can cause a wide variety of symptoms in both type and severity that may include motor control disruption, sensory issues, and speech difficulties.
Though researchers know that EBV can contribute to the development of MS, the exact mechanisms by which it does so aren’t completely understood. The Lieberman lab, in seeking to understand how EBV contributes to the development of MS, collaborated with Steven Jacobson, Ph.D., of the Neuroimmunology Branch at the National Institute of Neurological Disorders and Stroke, who contributed cell line samples from patients. The research team analyzed spontaneous lymphoblastoid cell line (SLCL) cell samples from a healthy control group; a group of patients with active MS (as opposed to so-called stable MS; the disease is characterized by unpredictable periods of flare-ups and eased symptoms); and a group of patients with stable MS.
B cells are crucial cells of the immune system that help regulate the body’s immune responses; they have also been implicated in autoimmune conditions due to their role as mediators of which biological signals warrant immune response. And B cells, when infected with EBV, become immortalized — that is, the cells are no longer constrained by senescence, so they can continue to divide an indefinite number of times — as “lymphoblastoid cell lines,” or LCLs. This immortalized B cell state can occur spontaneously within the body as a result of EBV infection, which is how the Lieberman lab was able to extract immortalized SLCL samples for study from the different patient groups.
Having obtained the matched samples, Dr. Lieberman and his team conducted genetic analyses of the SLCLs and confirmed that the MS-positive sample groups showed greater expression of genes associated with lytic EBV (“lytic” describes when latent viruses like EBV become active); they also saw increased inflammatory signaling and expression of the FOXP1 protein, the latter of which was shown to promote lytic EBV gene expression. As a whole, the group’s findings suggested a mechanism of lytic EBV in MS that promoted inflammation and disease.
Diving further, Lieberman’s group tested several antiviral compounds on all SLCL groups and found that one, TAF, reduced lytic EBV gene expression without killing the cells. TAF also significantly reduced the expression of inflammatory cytokines like IL-6 in the SLCLs from the patients with active MS. Finally, when cultured SLCLs from active MS, stable MS, and controls were administered TAF in the presence of antiviral T cells, the T cell response (a major factor in the autoimmune dysfunction of MS) was reduced in SLCLs from patients with MS but not reduced in the control SLCLs — an indication that TAF treatment has potential as a selectively cytotoxic anti-lytic treatment for MS.
“Our work with these SLCLs shows that the problematic inflammation signaling from lytic EBV can be selectively targeted in a way that demonstrably reduces damaging immune responses,” said Dr. Lieberman. “We’re excited about expanding this concept further; we have the potential to see whether TAF or other inhibitors of EBV might be a viable treatment for multiple sclerosis that can stop the autoimmune damage without causing wide-ranging and dangerous cell death.”
Bioreversible Anionic Cloaking Enables Intracellular Protein Delivery with Ionizable Lipid Nanoparticles
by Azmain Alamgir, Souvik Ghosal, Matthew P. DeLisa, Christopher A. Alabi in ACS Central Science
Cornell University scientists have designed a way to “cloak” proteins in a generalized technique that could lead to repurposing things like antibodies for biological research and therapeutic applications.
The “cloaked” proteins can be captured by lipid nanoparticles, which are akin to tiny bubbles of fat. These bubbles are small enough to sneak their hidden cargo into living cells, where the proteins uncloak and exert their therapeutic effect.
The lead author is doctoral student Azmain Alamgir, who works in the labs of the paper’s co-senior authors, Chris Alabi, associate professor of chemical and biomolecular engineering, and Matt DeLisa, professor of engineering.
For some drugs to impact a cell’s biology, and ultimately treat disease, they need to get inside the cell and reach a specific space. Protein-based therapeutics have many virtues — they can have more specific effects, with lower toxicity and diminished immune response — but ease of delivery is not one of them. Proteins are large and cumbersome and don’t freely diffuse into cells as easily as small molecules do.
“We had been looking for a clever way to efficiently get our engineered proteins inside of cells, especially in a translational context that would not only work in lab-cultured cells, but that would also be effective and safe in animal models and eventually in humans,” DeLisa said.
The researchers had the broad idea of using a bioconjugation approach that would allow the proteins to be loaded into lipid nanoparticles, which form around nucleic acids. A major advantage of this approach was that lipid nanoparticles were a key component in the successful COVID-19 vaccines developed by Pfizer-BioNTech and Moderna.
Those vaccines worked by delivering a payload in the form of messenger RNA, which are nucleic acids. The researchers now would use the same lipid nanoparticle delivery concept — the same materials even — but with a protein payload. The trick would be to make proteins look more like nucleic acids.
The researchers found they could accomplish this by “cloaking” the proteins with a negatively charged ion, so they would join with the positively charged lipids electrostatically.
“The crux of our strategy is conceptually very simple,” Alamgir said. “We’re taking proteins and specifically remodeling their surfaces with negative charges, so they look like nucleic acids and can similarly assemble into nanoparticles when formulated with the characteristic lipids.”
The team successfully demonstrated the cloaking method with lysine-reactive sulfonated compounds, killing cancer cells with ribonuclease A and inhibiting tumor signaling with monoclonal immunoglobulin G (IgG) antibodies.
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