Genetics biweekly vol.40, 20th October — 2nd November
TL;DR
- New findings suggest an explanation for the century-old mystery of how chromosome recombination is regulated during sexual reproduction.
- Researchers have identified that finger-like cellular extensions called filopodia contribute to building a barrier surrounding breast tumors.
- Viral DNA in human genomes, embedded there from ancient infections, serve as antivirals that protect human cells against certain present-day viruses, according to new research.
- A new technology called RADARS allows scientists to detect and target specific cell types and states, opening up potential applications in diagnostics and therapeutics.
- Controlling gene activity is important for engineering plants for improved bioenergy crops and other applications. This research developed synthetic genes that use Boolean logic gates to achieve specific patterns of gene expression within a plant. The researchers used these gene circuits to redesign the root architecture by tuning the number of root branches.
- Preclinical studies in mice that model human COVID-19 suggest that an inexpensive, readily available amino acid might limit the effects of the disease and provide a new off-the-shelf therapeutic option for infections with SARS-CoV-2 variants and perhaps future novel coronaviruses.
- Researchers are working to reveal potential threats to the efficacy of CRISPR/Cas9 gene editing, even when it appears to be working as planned.
- When an actin filament bends during cell movement, older actin deforms differently than newer actin, allowing regulatory proteins to tell the two states apart.
- Inside cells, molecular droplets form defined compartments for chemical reactions. Not only sticky interactions between molecules, but also dynamic reactions can form such droplets, researchers have discovered. Their work has revealed a new regulatory mechanism by which life controls and organizes itself.
- The mTOR protein plays a central role in cell growth, proliferation and survival. Its activity varies according to the availability of nutrients and some growth factors, including hormones. This protein is implicated in several diseases, including cancer, where its activity frequently increases. To better understand its regulation, a team has identified the structure of the SEA complex — an interdependent set of proteins — responsible for controlling mTOR. The discovery of this structure allows a better understanding of how cells perceive nutrient levels to regulate their growth.
- 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
Joint control of meiotic crossover patterning by the synaptonemal complex and HEI10 dosage
by Stéphanie Durand, Qichao Lian, Juli Jing, Marcel Ernst, Mathilde Grelon, David Zwicker, Raphael Mercier in Nature Communications
In most higher organisms, including humans, every cell carries two versions of each gene, which are referred to as alleles. Each parent passes on one allele to each offspring. As they are linked together on chromosomes, adjacent genes are usually inherited together. However, this is not always the case. Why?
The answer is recombination, a process that shuffles the allele content between homologous chromosomes during cell division. Mechanistically, recombination is achieved by crossovers, where homologous chromosomes contact each other, resulting in the exchange of genetic material.
Crossovers have long fascinated scientists and especially plant breeders because manipulating the crossover process offers the potential of increasing genetic diversity and of assembling desired combinations of alleles that boost crop productivity. Crossovers are subject to a “Goldilocks principle”; at least one is required per chromosome pair for successful sexual reproduction; indeed, a lack of crossovers is a major cause of human trisomy such as in the case of Down’s Syndrome. Crossover numbers are also tightly regulated and generally do not exceed three. This limit on crossover number, and therefore, recombination, is achieved by crossover interference, a phenomenon through which crossovers inhibit additional crossovers in their vicinity. However, how this interference works has remained a mystery since it was first described some 120 years ago.
Now, a team led by Raphael Mercier at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, have found convincing evidence in support of a recently proposed model of crossover interference. Mercier and his team, together with collaborators, in work spearheaded by Stéphanie Durand, Qichao Lian, and Juli Jing, achieved these insights by manipulating the expression of proteins known to be involved in either promoting crossovers or in connecting chromosomes together in the model plant Arabidopsis thaliana, a species which Mercier and his colleagues use to gain fundamental insights into the mechanisms of heredity. Boosting expression of the pro-crossover protein HEI10 resulted in a significant increase in crossovers, as did disrupting the expression of the protein ZYP1, a constituent of the synaptonemal complex, a protein structure that forms between homologous chromosomes.
When the scientists combined the two interventions, they were surprised to observe a massive increase in crossovers, showing that HE10 dosage and ZYP1 jointly control CO patterning. Importantly, massively increasing crossovers in this way barely affected cell division.
The considerable increase in crossovers upon increasing HEI10 levels chimes well with an emerging model for how crossover number is regulated. This model, formulated by David Zwicker and his team at the Max Planck Institute for Dynamics and Self-Organization in Göttingen, Germany, is based on diffusion of the HEI10 protein along the synaptonemal complex and a coarsening process leading to well-spaced HEI10 foci that promote crossovers. In the model, HEI10 initially forms multiple small foci and is progressively consolidated into a small number of large foci that co-localize with sites of crossovers. In this simple model, increasing the levels of HEI10 will result in more foci and therefore more crossovers; thus, the formation of droplets along an axis appears to be the determinant of crossover sites.
Mercier is excited by the team’s findings but is also already looking ahead: “These results are an exciting insight into a process that has baffled scientists for over a hundred years. Next, we want to better understand what controls the dynamics of the HEI10 droplets and how they promote crossovers. If we can get a better handle on how the process works, this may allow us selectively boost recombination during plant breeding, enabling the assembly of combinations of beneficial alleles that have remained out of reach.”
Cryo-EM structure of the SEA complex
by Lucas Tafur, Kerstin Hinterndorfer, Caroline Gabus, Chiara Lamanna, Ariane Bergmann, Yashar Sadian, Farzad Hamdi, Fotis L. Kyrilis, Panagiotis L. Kastritis, Robbie Loewith in Nature
The mTOR protein plays a central role in cell growth, proliferation and survival. Its activity varies according to the availability of nutrients and some growth factors, including hormones. This protein is implicated in several diseases, including cancer, where its activity frequently increases. To better understand its regulation, a team from the University of Geneva (UNIGE), in collaboration with researchers from the Martin Luther University (MLU) of Halle-Wittenberg in Germany, and the recently inaugurated Dubochet Center for Imaging (UNIGE-UNIL-EPFL), has identified the structure of the SEA complex — an interdependent set of proteins — responsible for controlling mTOR. The discovery of this structure allows a better understanding of how cells perceive nutrient levels to regulate their growth.
From yeast to humans, the mTOR protein (mammalian target of rapamycin) is the central controller of cell growth. This protein responds to various signals in the cell’s environment, such as nutrients and hormones, and regulates many fundamental cellular functions, such as protein and lipid synthesis, energy production by mitochondria and the organization of the cell’s structure. Disruptions in mTOR activity are the cause of several diseases, including diabetes, obesity, epilepsy and various types of cancer.
The laboratory of Robbie Loewith, Professor in the Department of Molecular and Cellular Biology at the UNIGE Faculty of Science and director of the National Center for Competence in Research in Chemical Biology, is interested in the regulation of mTOR, and in particular in the SEA complex, which is the direct sensor of nutrients and which controls the activity of mTOR. The SEA complex is composed of eight proteins. One part of the SEA complex (SEACIT) is involved in the inhibition of mTOR activity, while the other part (SEACAT) is involved in its activation.
In the absence of nutrients, the mTOR protein is blocked by the SEACIT subcomplex and cell growth is thus prevented. In contrast, in the presence of nutrients, the SEACAT subcomplex is thought to inhibit the SEACIT subcomplex, which can no longer block the mTOR protein. The central controller can then exert its activating role in cell growth by, for example, stimulating the production of proteins and lipids. How SEACAT regulates SEACIT is still not understood.
To determine the interactions between the proteins of the SEA complex, and thus better understand how they work, the researchers set out to determine the structure of this complex. After biochemically separating the SEA complex from all of the other components in the cell, the scientists used the technologies of the Dubochet Center for Imaging of UNIGE, UNIL and EPFL to obtain its molecular structure by cryo-electron microscopy (cryo-EM).
‘’By freezing the samples very quickly at -180°C, cryo-EM allows to obtain the structure of the proteins in their original state, i.e. in their functional three-dimensional form,’’ explains Lucas Tafur, a researcher in the Department of Molecular and Cellular Biology and first author of the study.
The biochemical activities of the different components of the complex were then tested in the laboratory. Despite the SEACAT subcomplex being in an active form (as when in the presence of nutrients), the researchers observed that the SEACIT subcomplex is still active and capable of blocking mTOR. ‘’This result is very unexpected since SEACAT has long been described as the direct inhibitor of SEACIT. We therefore expected SEACIT to be inactive in the presence of active SEACAT. Our results show that SEACAT acts more as a scaffold for the recruitment of other regulatory proteins and that its presence is therefore necessary but not sufficient for the inhibition of SEACIT,’’ explains Robbie Loewith, the last author of the study.
Obtaining the structure of the SEA complex has allowed to highlight missing links in the mTOR regulatory cascade. ‘’Of course, we now need to identify the as yet unknown partners that associate with this complex. These new factors could prove to be therapeutic targets for tumors where mTOR activity is exacerbated,’’ concludes Lucas Tafur.
Evolution and antiviral activity of a human protein of retroviral origin
by John A. Frank, Manvendra Singh, Harrison B. Cullen, Raphael A. Kirou, Meriem Benkaddour-Boumzaouad, Jose L. Cortes, Jose Garcia Pérez, Carolyn B. Coyne, Cédric Feschotte in Science
Viral DNA in human genomes, embedded there from ancient infections, serve as antivirals that protect human cells against certain present-day viruses, according to new research.
Previous studies have shown that fragments of ancient viral DNA — called endogenous retroviruses — in the genomes of mice, chickens, cats and sheep provide immunity against modern viruses that originate outside the body by blocking them from entering host cells. Though this study was conducted with human cells in culture in the lab, it shows that the antiviral effect of endogenous retroviruses likely also exists for humans. The research is important because further inquiry could uncover a pool of natural antiviral proteins that lead to treatments without autoimmune side effects. The work reveals the possibility of a genome defense system that has not been characterized, but could be quite extensive.
“The results show that in the human genome, we have a reservoir of proteins that have the potential to block a broad range of viruses,” said Cedric Feschotte, professor of molecular biology and genetics in the College of Agriculture and Life Sciences. John Frank, Ph.D. ’20, a former graduate student in Feschotte’s lab and now a postdoctoral researcher at Yale University, is the study’s first author.
Endogenous retroviruses account for about 8% of the human genome — at least four times the amount of DNA that make up the genes that code for proteins. Retroviruses introduce their RNA into a host cell, which is converted to DNA and integrated into the host’s genome. The cell then follows the genetic instructions and makes more virus. In this way, the virus hijacks the cell’s transcriptional machinery to replicate itself. Typically, retroviruses infect cells that don’t pass from one generation to the next, but some infect germ cells, such as an egg or sperm, which opens the door for retroviral DNA to pass from parent to offspring and eventually become permanent fixtures in the host genome.
In order for retroviruses to enter a cell, a viral envelope protein binds to a receptor on the cell’s surface, much like a key into a lock. The envelope is also known as a spike protein for certain viruses, such as SARS-CoV-2. In the study, Frank, Feschotte and colleagues used computational genomics to scan the human genome and catalog all the potential retroviral envelope protein-coding sequences that may have retained receptor binding activity. Then they ran more tests to detect which of these genes were active — that is, expressing retroviral envelope gene products in specific human cell types.
“We found clear evidence of expression,” Feschotte said, “and many of them are expressed in the early embryo and in germ cells, and a subset are expressed in immune cells upon infection.”
Once the researchers had identified antiviral envelope proteins expressed in different contexts, they focused on one, Suppressyn, because it was known to bind a receptor called ASCT2, the cellular entry point for a diverse group of viruses called Type D retroviruses. Suppressyn showed a high level of expression in the placenta and in very early human embryonic development. They then ran experiments in human placental-like cells, as the placenta is a common target for viruses.
The cells were exposed to a type D retrovirus called RD114, which is known to naturally infect feline species, such as the domestic cat. While other human cell types not expressing Suppressyn could be readily infected, the placental and embryonic stem cells did not get infected. When the researchers experimentally depleted placental cells of Suppressyn, they became susceptible to RD114 infection; when Suppressyn was returned to the cells, they regained resistance. In addition, the researchers did reverse experiments, using an embryonic kidney cell line normally susceptible to RD114. The cells became resistant when the researchers experimentally introduced Suppressyn into these cells.
The study shows how one human protein of retroviral origin blocks a cell receptor that allows viral entry and infection by a broad range of retroviruses circulating in many non-human species. In this way, Feschotte said, ancient retroviruses integrated into the human genome provide a mechanism for protecting the developing embryo against infection by related viruses.
Future work will explore the antiviral activity of other envelope-derived proteins encoded in the human genome, he said.
Bending forces and nucleotide state jointly regulate F-actin structure
by Matthew J. Reynolds, Carla Hachicho, Ayala G. Carl, Rui Gong, Gregory M. Alushin in Nature
Inside the leading edge of a crawling cell, intricate networks of rod-like actin filaments extend toward the cell membrane at various angles, lengthening protein by protein. Upon impact, the crisscrossing rods glance off the membrane and bend as the collective force of myriad filaments pushes the cell forward.
How flexible these filaments are, and how effectively they recruit essential regulatory proteins to their cause, depends on the properties of the individual actin proteins composing them. Now, a new study provides high-resolution structures showing how two key biochemical states of actin work jointly with bending forces to determine how actin can interact with other proteins.
“When you add force to the mix, you see substantial changes,” says Rockefeller’s Gregory Alushin. “We provide clear evidence that these biochemical changes in actin are only readable through the mechanical properties of the filaments.”
Actin filaments are long polymers of actin proteins, linked end to end. Actin proteins within a filament can exist in one of two important biochemical states. Actin newly added to the polymer contains a phosphate molecule and aged actin does not; otherwise, the two states are more or less identical. But actin-binding proteins can tell them apart, and they will bind or ignore a filament based on the state of its actin.
How actin-binding proteins distinguish between these states is a long-standing mystery. Some have proposed that phosphate somehow changes the shape of actin, allowing actin-binding proteins to pick it out of the crowd in vivo. Indeed, many enzymes can switch between shapes when other molecules latch onto them, in a process known as allosteric regulation. It made some sense to assume that actin would be no different. But without knowing exactly what the two biochemical states of actin looked like, this was merely a guess. Alushin wondered whether there might be more to the story.
“How proteins are controlled is an old question,” he says. “It had been a while since new ideas had been explored.”
Matthew Reynolds, a graduate student in Alushin’s lab, began working on high-resolution structures of each state. Upon examining these structures, where bound phosphate and water molecules were clearly resolved, the team found that the two actin states were still effectively indistinguishable. Whether or not actin was bound to phosphate, the structures featured nearly identical filament lattices and protein backbones. Had standard allosteric regulation been involved, there would have been marked changes in actin when it was bound to phosphate — the sort of major differences that regulatory proteins could have used to distinguish one type of actin from another. But the differences observed seemed far too minor for actin-binding proteins to be able to tell them apart.
In search of an alternate explanation, the team developed a machine learning approach to find the relatively small number of bent filaments in their cryo-electron microscopy images in order to analyze their structures. They then determined structures of bent filaments in both biochemical states, where the scale of bending matched that found in cells when filaments glance off the membrane during locomotion.
“Developing a way to capture this subset of images was crucial,” Alushin says. “This was a case where a methodological advance was needed for the scientific advance.”
When bent, actin that contained phosphate looked very different from actin without phosphate, such that actin-binding proteins would be able to easily distinguish between the two states. “The change in the biochemical state of the filament biases the ways in which the filament can deform when force is applied,” Reynolds says.
A new model began to emerge: while an actin protein in a filament can flex in many ways when the polymer bends, that flexibility is limited when a phosphate cramps its style. Imagine a flexible tube containing little donuts, side by side. Some of the donuts have open holes, others have golf balls in their holes, but they are otherwise identical. When the tube bends, the donuts will all squish and change shape, but those with golf balls will deform differently than the others.
Similarly, the two states of actin are essentially indistinguishable before the filament bends but, once force is applied, those with phosphate squish differently than those without it.
“What will matter is the deformability of the protein,” Alushin says. “If there’s a hole in the middle, it can flex in one way. If you fill that hole with phosphate, it won’t be able to squish in the same way.”
The results explain how actin-binding proteins can distinguish between biochemical states of actin, and they reveal a model of protein regulation that involves biochemical states and force working in concert. In future studies, Alushin hopes to investigate whether other proteins are similarly co-regulated.
“Our study of actin is a first glimpse into this phenomenon, but one limitation right now is that we don’t have structures of other force-responsive proteins in action,” he says. “It would be worthwhile looking into these proteins as it becomes technically possible to do so.”
Catalysis-Induced Phase Separation and Autoregulation of Enzymatic Activity
by Matthew W. Cotton, Ramin Golestanian, Jaime Agudo-Canalejo in Physical Review Letters
Inside cells, molecular droplets form defined compartments for chemical reactions. Not only sticky interactions between molecules, but also dynamic reactions can form such droplets, as it was found by researchers from the Max Planck Institute for Dynamics and Self-Organization (MPI-DS) and the University of Oxford. They revealed a new regulatory mechanism by which life controls and organizes itself.
Traditionally, cellular organelles defined by a membrane have been considered the functional units of a cell. In recent years, it was shown that also molecular droplets formed inside the cell provide a micro-environment for important reactions. Such droplets are not enclosed by a membrane, and arise from phase separation. Hence, they form dynamically and can be regulated according to the needs of the cell.
In the department of Living Matter Physics, managing director Ramin Golestanian and coworkers aim to reveal the organizational principles of living matter.
“The formation of droplets in cells so far was ascribed to attractive, sticky interactions between molecules — similar to how droplets form in non-living, equilibrium systems, such as droplets of oil in a vinaigrette,” explains Jaime Agudo-Canalejo, group leader at the MPI-DS. “We now found that the nonequilibrium drive provided by enzymatic reactions can cause the formation of enzyme-rich droplets, even without any stickiness. Instead, the enzymes are pushed against each other by the chemical fluxes they create” he continues.
The researchers explored this novel mechanism by formulating a model in which the effect of a multicomponent enzymatic reaction on the micro-environment is described. They also considered the underlying feedback mechanism due to which the induced phase separation can in turn affect the initial enzymatic reaction. “When the enzymatic activity gets too intense, phase separation occurs and acts to reduce it, providing a new form of autoregulation,” says Matthew Cotton, first author of the study. This complex interplay of molecular interactions can provide a dynamic environment for cellular processes. Hence, the model adds another piece to the complex puzzle of how life is able to organize itself.
MYO10-filopodia support basement membranes at pre-invasive tumor boundaries
by Emilia Peuhu, Guillaume Jacquemet, et al in Developmental Cell
Researchers at Turku University and Åbo Akademi University, Finland, have identified that finger-like cellular extensions called filopodia contribute to building a barrier surrounding breast tumours.
At the early stage of breast cancer malignant cells are imprisoned by a tissue barrier called a basement membrane that stops them from disseminating into other parts of the body. This early disease stage is typically not life-threatening, as surgery can remove the tumour. However, breast cancer can become lethal if it spreads and forms metastases. To escape and spread, tumour cells first need to break through their most proximal barrier, the basement membrane. Researchers at University of Turku and Åbo Akademi University have discovered that cellular structures called filopodia help preserve the basement membrane surrounding the tumour, blocking their escape.
“These results are very surprising as we previously thought that these cancer cells’ sticky fingers were only used to invade nearby tissues. Now we find that these structures can also help contain the tumour,” says InFLAMES group leader Professor Johanna Ivaska, University of Turku.
These sticky fingers are generated by a protein called Myosin-10. The research teams led by Professor Ivaska, Docent at the Institute of Biomedicine at the University of Turku Dr. Emilia Peuhu, and InFLAMES group leader, Associate Professor of Cell Biology Dr. Guillaume Jacquemet from Åbo Akademi University, found that cancer cells lacking Myosin-10 cannot build and maintain their surrounding barrier, the basement membrane. This makes it easier for cancer cells to escape.
“Remove Myosin-10, and the tumours are clearly more aggressive. Their basement membrane is almost completely gone and they spread more freely to the surrounding tissue,” says Dr. Peuhu.
For several years, the Ivaska and Jacquemet teams have focused their efforts on understanding how cancer cells use filopodia to move and invade surrounding tissue. Their previous findings highlight that filopodia are used by cancer cells to disseminate once they escape the primary tumour. Now the teams found that filopodia have an opposite role at the early stage of the disease.
“We’ve been looking into developing anti-filopodia strategies to treat cancers, but our new results clearly emphasize that targeting filopodia or Myosin-10 too early could actually make things worse,” says Dr. Jacquemet.
Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR
by Kaiyi Jiang, Jeremy Koob, Xi Dawn Chen, Rohan N. Krajeski, Yifan Zhang, Verena Volf, Wenyuan Zhou, Samantha R. Sgrizzi, Lukas Villiger, Jonathan S. Gootenberg, Fei Chen, Omar O. Abudayyeh in Nature Biotechnology
Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have developed a system that can detect a particular RNA sequence in live cells and produce a protein of interest in response. Using the technology, the team showed how they could identify specific cell types, detect and measure changes in the expression of individual genes, track transcriptional states, and control the production of proteins encoded by synthetic mRNA.
The platform, called Reprogrammable ADAR Sensors, or RADARS, even allowed the team to target and kill a specific cell type. The team said RADARS could one day help researchers detect and selectively kill tumor cells, or edit the genome in specific cells. The study was led by co-first authors Kaiyi Jiang (MIT), Jeremy Koob (Broad), Xi Chen (Broad), Rohan Krajeski (MIT), and Yifan Zhang (Broad).
“One of the revolutions in genomics has been the ability to sequence the transcriptomes of cells,” said Fei Chen, a core institute member at the Broad, Merkin Fellow, assistant professor at Harvard University, and co-corresponding author on the study. “That has really allowed us to learn about cell types and states. But, often, we haven’t been able to manipulate those cells specifically. RADARS is a big step in that direction.”
“Right now, the tools that we have to leverage cell markers are hard to develop and engineer,” added Omar Abudayyeh, a McGovern Institute Fellow and co-corresponding author on the study. “We really wanted to make a programmable way of sensing and responding to a cell state.”
Jonathan Gootenberg, who is also a McGovern Institute Fellow and co-corresponding author, says that their team was eager to build a tool to take advantage of all the data provided by single-cell RNA sequencing, which has revealed a vast array of cell types and cell states in the body.
“We wanted to ask how we could manipulate cellular identities in a way that was as easy as editing the genome with CRISPR,” he said. “And we’re excited to see what the field does with it.”
The RADARS platform generates a desired protein when it detects a specific RNA by taking advantage of RNA editing that occurs naturally in cells.
The system consists of an RNA containing two components: a guide region, which binds to the target RNA sequence that scientists want to sense in cells, and a payload region, which encodes the protein of interest, such as a fluorescent signal or a cell-killing enzyme. When the guide RNA binds to the target RNA, this generates a short double-stranded RNA sequence containing a mismatch between two bases in the sequence — adenosine (A) and cytosine (c). This mismatch attracts a naturally occurring family of RNA-editing proteins called adenosine deaminases acting on RNA (ADARs).
In RADARS, the A-C mismatch appears within a “stop signal” in the guide RNA, which prevents the production of the desired payload protein. The ADARs edit and inactivate the stop signal, allowing for the translation of that protein. The order of these molecular events is key to RADARS’s function as a sensor; the protein of interest is produced only after the guide RNA binds to the target RNA and the ADARs disable the stop signal.
The team tested RADARS in different cell types and with different target sequences and protein products. They found that RADARS distinguished between kidney, uterine, and liver cells, and could produce different fluorescent signals as well as a caspase, an enzyme that kills cells. RADARS also measured gene expression over a large dynamic range, demonstrating their utility as sensors.
Most systems successfully detected target sequences using the cell’s native ADAR proteins, but the team found that supplementing the cells with additional ADAR proteins increased the strength of the signal. Abudayyeh says both of these cases are potentially useful; taking advantage of the cell’s native editing proteins would minimize the chance of off-target editing in therapeutic applications, but supplementing them could help produce stronger effects when RADARS are used as a research tool in the lab.
Abudayyeh, Chen, and Gootenberg say that because both the guide RNA and payload RNA are modifiable, others can easily redesign RADARS to target different cell types and produce different signals or payloads. They also engineered more complex RADARS, in which cells produced a protein if they sensed two RNA sequences and another if they sensed either one RNA or another. The team adds that similar RADARS could help scientists detect more than one cell type at the same time, as well as complex cell states that can’t be defined by a single RNA transcript.
Ultimately, the researchers hope to develop a set of design rules so that others can more easily develop RADARS for their own experiments. They suggest other scientists could use RADARS to manipulate immune cell states, track neuronal activity in response to stimuli, or deliver therapeutic mRNA to specific tissues.
“We think this is a really interesting paradigm for controlling gene expression,” said Chen. “We can’t even anticipate what the best applications will be. That really comes from the combination of people with interesting biology and the tools you develop.”
Synthetic genetic circuits as a means of reprogramming plant roots
by Jennifer A. N. Brophy, Katie J. Magallon, Lina Duan, Vivian Zhong, Prashanth Ramachandran, Kiril Kniazev, José R. Dinneny in Science
Controlling the activity of genes is an important step in engineering plants for improved bioenergy crops. This research developed synthetic genes that can be combined to achieve specific patterns of gene expression within the plant. The expression of the synthetic genes is programmed in the form of Boolean (“AND,” “OR,” and “NOT”) logic gates that work in a similar way to computer circuit boards. Using the synthetic gene circuits, the researchers successfully created predictable, novel expression patterns of fluorescent proteins. Finally, they used similar gene circuits to redesign root architecture by tuning the number of root branches.
To understand biological functions and design new biotechnology applications, scientists need to precisely manipulate gene expression. This is the process that converts instructions in DNA into proteins and other products that allow cells to do their jobs in an organism. Controlling specific patterns of gene expression in plants is challenging. One potential solution is synthetic genetic circuits. However, tuning circuit activity across different plant cell types has proven difficult. This research developed new genetic circuits that allow precise control of the root architecture. As roots are important for the uptake of water and nutrients, this approach will allow the design of tailored root architectures. This will in turn help researchers to engineer bioenergy crops with improved characteristics for growth in marginal lands.
To establish synthetic gene circuits capable of predictably regulating gene expression in plants, scientists adapted a large collection of bacterial gene regulators for use as synthetic activators or repressors of gene expression in plants, also known as transcription factors. Using a transient expression system, the researchers demonstrated that the synthetic transcription factors and their target DNA sequences (promoters) are able to direct specific and tunable control of gene expression. They designed synthetic promoters that responded to one synthetic transcription factor to work as simple logic gates that responded to one input, while more complex gates required synthetic promoters that responded to multiple inputs. The research found these logic gates to control expression in predictable ways according to the specific Boolean rules encoded in the engineered genes.
To implement synthetic gene circuits in a multicellular context, the researchers used Arabidopsis roots as a model system where endogenous promoters drove tissue-specific expression of the synthetic transcription factors. The gene circuits generated novel expression patterns that were the result of successfully performing logical operations. The researchers further used one of the logic gates to quantitatively control the expression of a hormone signaling regulator to tune the amount of root branching in the root system of Arabidopsis. These results demonstrate that it is now possible to program gene expression across plant cell types using genetic circuits, providing a roadmap to engineer more resilient bioenergy crops.
A GABA-receptor agonist reduces pneumonitis severity, viral load, and death rate in SARS-CoV-2-infected mice
by Jide Tian, Barbara J. Dillion, Jill Henley, Lucio Comai, Daniel L. Kaufman in Frontiers in Immunology
Preclinical studies in mice that model human COVID-19 suggest that an inexpensive, readily available amino acid might limit the effects of the disease and provide a new off-the-shelf therapeutic option for infections with SARS-CoV-2 variants and perhaps future novel coronaviruses.
A team led by researchers at the David Geffen School of Medicine at UCLA report that an amino acid called GABA, which is available over-the-counter in many countries, reduced disease severity, viral load in the lungs, and death rates in SARS-CoV-2-infected mice. This follows up on their previous finding that GABA consumption also protected mice from another lethal mouse coronavirus called MHV-1. In both cases, GABA treatment was effective when given just after infection or several days later near the peak of virus production. The protective effects of GABA against two different types of coronaviruses suggest that GABA may provide a generalizable therapy to help treat diseases induced by new SARS-CoV-2 variants and novel beta-coronaviruses.
“SARS-CoV-2 variants and novel coronaviruses will continue to arise, and they may not be efficiently controlled by available vaccines and antiviral medications. Furthermore, the generation of new vaccines is likely to be much slower than the spread of new variants,” said senior author Daniel L. Kaufman, a researcher and professor in Molecular and Medical Pharmacology at the David Geffen School of Medicine at UCLA. Accordingly, new therapeutic options are needed to limit the severity of these infections. Their previous studies showed that GABA administration protected mice from developing severe disease after infection with a mouse coronavirus called MHV-1. To more stringently test the potential of GABA as a therapy for COVID-19, they studied transgenic mice that when infected with SARS-CoV-2 develop severe pneumonia with a high mortality rate. “If our observations of the protective effects of GABA therapy in SARS-CoV-2-infected mice are confirmed in clinical trials, GABA could provide an off-the-shelf treatment to help ameliorate infections with SARS-CoV-2 variants. GABA is inexpensive and stable at room temperature, which could make it widely and easily accessible, and especially beneficial in developing countries.”
The researchers said that GABA and GABA receptors are most often thought of as a major neurotransmitter system in the brain. Years ago, they, as well as other researchers, found that cells of the immune system also possessed GABA receptors and that the activation of these receptors inhibited the inflammatory actions of immune cells. Taking advantage of this property, the authors reported in a series of studies that GABA administration inhibited autoimmune diseases such as type 1 diabetes, multiple sclerosis, and rheumatoid arthritis in mouse models of these ailments.
Other scientists who study gas anesthetics have found that lung epithelial cells also possess GABA receptors and that drugs that activate these receptors could limit lung injuries and inflammation in the lung. The dual actions of GABA in inflammatory immune cells and lung epithelial cells, along with its safety for clinical use, made GABA a theoretically appealing candidate for limiting the overreactive immune responses and lung damage due to coronavirus infection.
Working with colleagues at the University of Southern California, the UCLA research team in this study administered GABA to the mice just after infection with SARS-CoV-2, or two days later when the virus levels are near their peak in the mouse lungs. While the vast majority of untreated mice did not survive this infection, those given GABA just after infection, or two days later, had less illness severity and a lower mortality rate over the course of the study. Treated mice also displayed reduced levels of virus in their lungs and changes in circulating immune signaling molecules, known as cytokines and chemokines, toward patterns that were associated with better outcomes in COVID-19 patients. Thus, GABA receptor activation had multiple beneficial effects in this mouse model that are also desirable for the treatment of COVID-19.
The authors hope that their new findings will provide a springboard for testing the efficacy of GABA treatment in clinical trials with COVID-19 patients. Since GABA has an excellent safety record, is inexpensive and available worldwide, clinical trials of GABA treatment for COVID-19 can be initiated rapidly.
The authors also suspect that the anti-inflammatory properties of GABA-receptor activating drugs may also be useful for limiting inflammation in the central nervous system that is associated with long-COVID. Indeed, this approach was very successful in their previous studies of therapeutics for multiple sclerosis in mice, a disease which is caused by an inflammatory autoimmune response in the brain. The authors speculate that such drugs may reduce both the deleterious effects of coronavirus infection in the periphery and limit inflammation in the central nervous system.
Unfortunately, there has been no pharmaceutical interest pursuing GABA therapy for COVID-19, presumably because it is not patentable and widely available as a dietary supplement. The authors hope for federal funding to continue this line of study.
The researchers emphasize that unless clinical trials are conducted and GABA is approved for treating COVID-19 by relevant governing bodies, it should not be consumed for the treatment of COVID-19 since it could pose health risks, such as dampening beneficial immune or physiological responses.
Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing
by So Hyun Park, Mingming Cao, Yidan Pan, Timothy H. Davis, Lavanya Saxena, Harshavardhan Deshmukh, Yilei Fu, Todd Treangen, Vivien A. Sheehan, Gang Bao in Science Advances
A Rice University lab is leading the effort to reveal potential threats to the efficacy and safety of therapies based on CRISPR-Cas9, the Nobel Prize-winning gene editing technique, even when it appears to be working as planned.
Bioengineer Gang Bao of Rice’s George R. Brown School of Engineering and his team point out in a paper published in Science Advances that while off-target edits to DNA have long been a cause for concern, unseen changes that accompany on-target edits also need to be recognized — and quantified. Bao noted a 2018 Nature Biotechnology paper indicated the presence of large deletions. “That’s when we started looking into what we can do to quantify them, due to CRISPR-Cas9 systems designed for treating sickle cell disease,” he said.
Bao has been a strong proponent of CRISPR-Cas9 as a tool to treat sickle cell disease, a quest that has brought him and his colleagues ever closer to a cure. Now the researchers fear that large deletions or other undetected changes due to gene editing could persist in stem cells as they divide and differentiate, thus have long-term implications for health.
“We do not have a good understanding of why a few thousand bases of DNA at the Cas9 cut site can go missing and the DNA double-strand breaks can still be rejoined efficiently,” Bao said. “That’s the first question, and we have some hypotheses. The second is, what are the biological consequences? Large deletions (LDs) can reach to nearby genes and disrupt the expression of both the target gene and the nearby genes. It is unclear if LDs could result in the expression of truncated proteins.
“You could also have proteins that misfold, or proteins with an extra domain because of large insertions,” he said. “All kinds of things could happen, and the cells could die or have abnormal functions.”
His lab developed a procedure that uses single-molecule, real-time (SMRT) sequencing with dual unique molecular identifiers (UMI) to find and quantify unintended LDs along with large insertions and local chromosomal rearrangements that accompany small insertions/deletions (INDELs) at a Cas9 on-target cut site.
“To quantify large gene modifications, we need to perform long-range PCR, but that could induce artifacts during DNA amplification,” Bao said. “So we used UMIs of 18 bases as a kind of barcode.”
“We add them to the DNA molecules we want to amplify to identify specific DNA molecules as a way to reduce or eliminate artifacts due to long-range PCR,” he said. “We also developed a bioinformatics pipeline to analyze SMRT sequencing data and quantified the LDs and large insertions.”
The Bao lab’s tool, called LongAmp-seq (for long-amplicon sequencing), accurately quantifies both small INDELs and large LDs. Unlike SMRT-seq, which requires the use of a long-read sequencer often only available at a core facility, LongAmp-seq can be performed using a short-read sequencer. To test the strategy, the lab team led by Rice alumna Julie Park, now an assistant research professor of bioengineering, used Streptococcus pyogenes Cas9 to edit beta-globin (HBB), gamma-globin (HBG) and B-cell lymphoma/leukemia 11A (BCL11A) enhancers in hematopoietic stem and progenitor cells (HSPC) from patients with sickle cell disease, and the PD-1 gene in primary T-cells. They found large deletions of up to several thousand bases occurred at high frequency in HSPCs: up to 35.4% in HBB, 14.3% in HBG and 15.2% in BCL11A genes, as well as on the PD-1 (15.2%) gene in T-cells.
Since two of the specific CRISPR guide RNAs tested by the Bao lab are being used in clinical trials to treat sickle cell disease, he said it’s important to determine the biological consequences of large gene modifications due to Cas9-induced double-strand breaks.
Bao said the Rice team is currently looking downstream to analyze the consequences of long deletions on messenger RNA, the mediator that carries code for ribosomes to make proteins.
“Then we’ll move on to the protein level,” Bao said. “We want to know if these large deletions and insertions persist after the gene-edited HSPCs are transplantation into mice and patients”
MISC
Subscribe to Paradigm!
Medium. Twitter. Telegram. Telegram Chat. Reddit. LinkedIn.
Main Sources
Research articles
