GN/ Structure of wheat immune protein resolved
Genetics biweekly vol.38, 21st September — 6th October
TL;DR
- Scientists have unraveled how wheat protects itself from a deadly pathogen. Their findings could be harnessed to make important crop species more resistant to disease.
- Researchers have cloned mini-pigs with mutations in a gene that has recently been identified as a direct cause of Alzheimer’s disease. The results are particularly interesting for the pharmaceutical industry.
- Scientists have engineered mosquitoes that slow the growth of malaria-causing parasites in their gut, preventing transmission of the disease to humans.
- In a study using lab-grown cells, researchers specializing in aging report they have successfully delivered a common blood pressure drug directly to the inner membrane of mitochondria, the ‘power plants’ in the cells of humans, animals, plants and most other organisms.
- A new study has revealed that certain locations of DNA are copied faster than others, which could also have an effect on mutation rate.
- Tiny nets woven from DNA strands can ensnare the spike protein of the virus that causes COVID-19, lighting up the virus for a fast-yet-sensitive diagnostic test — and also impeding the virus from infecting cells, opening a new possible route to antiviral treatment, according to a new study.
- Researchers have modified a gene editing tool to serve as a highly sensitive diagnostic test for the presence of the SARS-CoV-2 virus.
- Bioscientists turn bacteria into self-assembling building blocks. The macroscale engineered living materials they form could be used to soak up targeted contaminants from the environment or as custom catalysts, among many possible applications.
- A new method allows scientists to determine all the molecules present in the lysosomes — the cell’s recycling centers — of mice. This could bring new understanding and treatment of neurodegenerative disorders.
- For an adequate immune response, it is essential that T lymphocytes recognize infected or degenerated cells. They do so by means of antigenic peptides, which these cells present with the help of specialized surface molecules (MHC I molecules). Using X-ray structure analysis, a research team has now been able to show how the MHC I molecules are loaded with peptides and how suitable peptides are selected for this purpose.
- 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
A wheat resistosome defines common principles of immune receptor channels
by Alexander Förderer, Ertong Li, Aaron W. Lawson, Ya-nan Deng, Yue Sun, Elke Logemann, Xiaoxiao Zhang, Jie Wen, Zhifu Han, Junbiao Chang, Yuhang Chen, Paul Schulze-Lefert, Jijie Chai in Nature
Scientists from the Max Planck Institute for Plant Breeding Research and the University of Cologne in Germany together with colleagues from China have unravelled how wheat protects itself from a deadly pathogen. Their findings could be harnessed to make important crop species more resistant to disease.
As a staple food for 40 % of the world’s population, it is hard to overstate the importance of wheat for food security. Crop resilience in a changing climate and resistance to infectious diseases will be the limiting factors for future food stability. In the case of wheat, one of the most economically significant pathogens is stem rust, a vicious fungus which can have devastating effects on yields.
Although stem rust has been infecting wheat since pre-Christian times, through the efforts of breeders and plant pathologists it had been possible to prevent any significant epidemics in the world’s major wheat growing areas in the last 50 years of the 20th century. Unfortunately, this rosy picture was shattered in 1998, with the emergence of a new, highly virulent variant of wheat stem rust in Uganda. Ug99, as it is known, can attack up to 80% of the world’s wheat varieties resulting, in some cases, in complete loss of yield from infected fields. In seeking to provide crops with resistance against new and emerging plant pathogens, plant scientists and breeders often scour wild varieties of some of our staple crops for genes that may provide effective immunity. The emergence of Ug99 lent particular impetus to such efforts and led to the identification of Sr35, a gene which protects against Ug99 when introduced into bread wheat.
Now, scientists led by Jijie Chai and Paul Schulze-Lefert from the University of Cologne and the Max Planck Institute for Plant Breeding Research in Cologne, Germany, and Yuhang Chen from the Chinese Academy of Sciences, China, have decoded the structure of the Sr35 wheat protein. This allowed them to explain how Sr35 protects Einkorn wheat against Ug99.
Sr35 is an example of a nucleotide-binding leucine-rich repeat (NLR) receptor inside plant cells that detects the presence of invading pathogens. NLR activation is triggered by the recognition of pathogen “effectors,” small proteins that are delivered into plant cells by invading microorganisms in order to weaken the plant. Each NLR typically binds to one type of effector. When Sr35 is activated, five receptors assemble together into a large protein complex, which the researchers term the “Sr35 resistosome.” Such resistosomes have the ability to act as channels in the plant cell membrane. This channel activity sets in motion powerful immune responses that culminate in the suicide of plant cells at the site of infection as a sort of self-sacrifice to protect the rest of the plant.
In this study, the researchers succeeded for the first time in resolving the structure and describing the immune function of a resistosome from a crop species. The scientists began by synthesizing both Sr35 and its corresponding Ug99 effector in insect cells, a strategy that allowed them to isolate and purify large amounts of Sr35 resistosomes, and used cryogenic electron microscopy, a technique in which samples are frozen to cryogenic temperatures allowing for the determination of biomolecular structures at atomic resolution.
Alexander Förderer, who spearheaded the study says: “In the structure of Sr35 we could identify those parts of the protein that are important for Ug99 effector recognition. With this insight, I hope that we can generate new NLRs that can be applied in the field to protect elite wheat varieties against Ug99 and in this way contribute to global food security.”
Armed with their knowledge of the structure of the Sr35 resistosome, Alexander Förderer and his co-authors Ertong Li and Aaron W. Lawson set about determining whether they could now repurpose non-functional receptors of susceptible elite varieties of barley and wheat to recognize the Ug99 effector. They alighted on two proteins that, while similar to Sr35, do not recognize Ug99. When they swapped in the elements of Sr35 known to contact the Ug99 effector, the scientists could turn these proteins into receptors for the Ug99 effector.
According to Paul Schulze-Lefert, “This study also illustrates how nature has used a common design principle to build immune receptors. At the same time, these receptors have evolved in such a way that they have retained the flexibility to generate new receptor variants that can provide immunity to other microbial pathogens such as viruses, bacteria or nematodes.”
Jijie Chai points out that the insights gained in this study “open up the opportunity to improve crop resistance by engineering plant resistance proteins that recognise an array of different pathogen effectors.”
Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development
by Astrid Hoermann, Tibebu Habtewold, Prashanth Selvaraj, Giuseppe Del Corsano, Paolo Capriotti, Maria Grazia Inghilterra, Temesgen M. Kebede, George K. Christophides and Nikolai Windbichler in Science Advances
Scientists have engineered mosquitoes that slow the growth of malaria-causing parasites in their gut, preventing transmission of the disease to humans.
The genetic modification causes mosquitoes to produce compounds in their guts that stunt the growth of parasites, meaning they are unlikely to reach the mosquitoes’ salivary glands and be passed on in a bite before the insects die. So far, the technique has been shown to dramatically reduce the possibility of malaria spread in a lab setting, but if proven safe and effective in real-world settings it could offer a powerful new tool to help eliminate malaria.
The innovation, by researchers from the Transmission:Zero team at Imperial College London, is designed so it can be coupled with existing ‘gene drive’ technology to spread the modification and drastically cut malaria transmission. The team is looking towards field trials, but will thoroughly test the safety of the new modification before combining it with a gene drive for real-world tests. Collaborators from the Institute for Disease Modeling at the Bill and Melinda Gates Foundation also developed a model that, for the first time, can assess the impact of such modifications if used in a variety of African settings. They found that the modification developed by the Transmission:Zero team could be a powerful tool for bringing down cases of malaria even where transmission is high.
Malaria remains one of the world’s most devastating diseases, putting at risk about half of the world’s population. In 2021 alone, it infected 241 million and killed 627,000 people, mostly children aged below five years old in sub-Saharan Africa.
Co-first author of the study Dr Tibebu Habtewold, from the Department of Life Sciences at Imperial, said: “Since 2015, the progress in tackling malaria has stalled. Mosquitoes and the parasites they carry are becoming resistant to available interventions such as insecticides and treatments, and funding has plateaued. We need to develop innovative new tools.”
The disease is transmitted between people after a female mosquito bites someone infected with the malaria parasite. The parasite then develops into its next stage in the mosquito’s gut and travels to its salivary glands, ready to infect the next person the mosquito bites. However, only around 10% of mosquitoes live long enough for the parasite to develop far enough to be infectious. The team aimed to lengthen the odds even further, by extending the time it takes for the parasite to develop in the gut.
The Transmission:Zero team genetically modified the main malaria-carrying species of mosquito in sub-Saharan Africa: Anopheles gambiae. They were able to make it so that when mosquito takes a blood meal, it produces two molecules called antimicrobial peptides in its guts. These peptides, which were originally isolated from honeybees and African clawed frogs, impair the malaria parasite’s development. This caused a few days’ delay before the next parasite stage could reach the mosquito salivary glands, by which time most mosquitoes in nature are expected to die. The peptides work by interfering with the energy metabolism of the parasite, which also has some effect on the mosquito, causing them to have a shorter lifespan and further decreasing their ability to pass on the parasite.
Co-first author of the study Astrid Hoermann, from the Department of Life Sciences at Imperial, said: “For many years, we have been trying to no avail to make mosquitoes that cannot be infected by the parasite or ones that can clear all the parasites with their immune system. Delaying parasite’s development inside the mosquito is a conceptual shift that has opened many more opportunities to block malaria transmission from mosquitoes to humans.”
To use the genetic modification to prevent malaria spread in the real world, it needs to be spread from lab-bred mosquitoes to wild ones. Normal interbreeding would spread it to a certain degree, but because the modification has a ‘fitness cost’ in the form of reduced lifespan, it would likely be quickly eliminated thanks to natural selection. Gene drive is an additional genetic trick that can be added to mosquitoes that would cause the anti-parasite genetic modification to be preferentially inherited, making it spread more widely among any natural populations. Because this strategy is so new, it would require extremely careful planning to minimise risks before any field trials. The Transmission:Zero team is therefore creating two separate but compatible strains of modified mosquitoes — one with the anti-parasite modification and one with the gene drive. They can then test the anti-parasite modification on its own first, only adding in the gene drive once it has been shown to be effective.
Co-lead author Dr Nikolai Windbichler, from the Department of Life Sciences at Imperial, said: “We are now aiming to test whether this modification can block malaria transmission not just using parasites we have reared in the lab but also from parasites that have infected humans. If this proves to be true, then we will be ready to take this to field trials within the next two to three years.”
With partners in Tanzania, the team have set up a facility to generate and handle genetically modified mosquitoes and conduct some first tests. These include collecting parasites from locally infected schoolchildren, to ensure the modification works against the parasites circulating in relevant communities. They are also fully risk assessing any potential releases of modified mosquitoes, taking into account any potential hazards and making sure they have buy-in from the local community. But they are hopeful that their intervention can ultimately help to eradicate malaria.
Co-lead author Professor George Christophides, from the Department of Life Sciences at Imperial, said: “History has taught us that there is no silver bullet when it comes to malaria control, thus we will have to use all the weapons we have at our disposal and generate even more. Gene drive is one such very powerful weapon that in combination with drugs, vaccines and mosquito control can help stop the spread of malaria and save human lives.”
A genetically modified minipig model for Alzheimer’s disease with SORL1 haploinsufficiency
by Olav M. Andersen, Nikolaj Bøgh, Anne M. Landau, Gro G. Pløen, Anne Mette G. Jensen, et al in Cell Reports Medicine
For decades, researchers from all over the world have been working hard to understand Alzheimer’s disease. Now, a collaboration between the Department of Biomedicine and the Department of Clinical Medicine at Aarhus University has resulted in a flock of minipigs that could lead to a major step forward in the research and treatment of Alzheimer’s.
The cloned pigs were born with a mutation in the gene SORL1, which is interesting because the mutations are found in up to 2–3% of all early onset Alzheimer’s cases in human beings. Due to the gene mutation, the pigs develop signs of Alzheimer’s at a young age. This gives the researchers an opportunity to follow the early signs of the disease, as the pigs show changes in the same biomarkers that are used to make the diagnosis in humans.
“By following the changes over time in the pigs, we can better understand the earliest changes in the cells. Later, these changes lead to the irreversible alterations in the brain that are the cause of dementia. But now we can follow the pigs before they lose their memory, change their behaviour, etc., which will make it possible to test new drugs that can be used at an early stage to prevent SORL1-associated Alzheimer’s disease,” says Associate Professor Olav Michael Andersen, who is the first author of the study.
“Pigs resemble human beings in many ways, which is why this increases the possibilities of producing drugs that will work to counteract Alzheimer’s. It is important to have a workable animal model to bridge the gap between research and drug development,” he explains.
Since the 1990s, researchers have known of three genes which — if they mutate — can directly cause Alzheimer’s disease. Through intense research over the past 20 years, it has now definitively been established that a mutation in a fourth gene, namely SORL1, can also directly cause the widespread dementia disorder. If this gene is defective, the person carrying the genetic defect will develop Alzheimer’s.
“We have created an animal model for Alzheimer’s in minipigs by changing one of just four genes that are currently known to be directly responsible for the disease. The pigs can be used in the pharmaceutical industry to develop new drugs — and at the same time, this can provide researchers with better possibilities to understand the early changes in the brains of people who will later develop Alzheimer’s,” says Olav Michael Andersen.
Researchers have also previously developed pig models for Alzheimer’s and other diseases by means of cloning. This is done by removing the hereditary material from an unfertilised egg cell taken from a porker, after which the cell is fused with a skin cell from another pig. In this study, the researchers had previously used CRISPR-Cas9-based gene editing to destroy the SORL1 gene in a skin cell taken from a minipig of the Göttingen breed. The result is a reconstructed embryo, i.e. a cloned egg, which develops into a new individual with the same genetic characteristics as the gene-edited skin cell. This means that the cloned minipigs are born with a damaged SORL1 gene.
“The pigs resemble Alzheimer’s patients who have the SORL1 gene defects — in contrast to previous pig models for Alzheimer’s, which have had one or more mutated human genes inserted in the hope of accelerating the disease,” says Associate Professor Charlotte Brandt Sørensen, who has been responsible for the development of the genetically modified, cloned pigs.
As the mutation is inherited, researchers can now breed pigs that show the first signs of Alzheimer’s before they reach the age of three.
The study has major perspectives, says Associate Professor Olav Michael Andersen.
“We know from human genetics that when the SORL1 gene is destroyed, we develop Alzheimer’s. We have shown that if we destroy this gene in pigs, precisely the early changes occur in the animals’ brain cells that we had dared to hope for. This makes it possible to find biomarkers that reflect the initial, pre-clinical phase of the disease,” he says.
Net-Shaped DNA Nanostructures Designed for Rapid/Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus
by Neha Chauhan, Yanyu Xiong, Shaokang Ren, Abhisek Dwivedy, Nicholas Magazine, Lifeng Zhou, Xiaohe Jin, Tianyi Zhang, Brian T. Cunningham, Sherwood Yao, Weishan Huang, Xing Wang in Journal of the American Chemical Society
Tiny nets woven from DNA strands can ensnare the spike protein of the virus that causes COVID-19, lighting up the virus for a fast-yet-sensitive diagnostic test — and also impeding the virus from infecting cells, opening a new possible route to antiviral treatment, according to a new study.
Researchers at the University of Illinois Urbana-Champaign and collaborators demonstrated the DNA nets’ ability to detect and impede COVID-19 in human cell cultures in a paper published in the Journal of the American Chemical Society.
“This platform combines the sensitivity of PCR and the speed and low cost of antigen tests,” said study leader Xing Wang, a professor of bioengineering and of chemistry at Illinois. “We need tests like this for a couple of reasons. One is to prepare for the next pandemic. The other reason is to track ongoing viral epidemics — not only coronaviruses, but also other deadly and economically impactful viruses like HIV or influenza.”
DNA is best known for its genetic properties, but it also can be folded into custom nanoscale structures that can perform functions or specifically bind to other structures much like proteins do. The DNA nets the Illinois group developed were designed to bind to the coronavirus spike protein — the structure that sticks out from the surface of the virus and binds to receptors on human cells to infect them. Once bound, the nets give off a fluorescent signal that can be read by an inexpensive handheld device in about 10 minutes.
The researchers demonstrated that their DNA nets effectively targeted the spike protein and were able to detect the virus at very low levels, equivalent to the sensitivity of gold-standard PCR tests that can take a day or more to return results from a clinical lab. The technique holds several advantages, Wang said. It does not need any special preparation or equipment, and can be performed at room temperature, so all a user would do is mix the sample with the solution and read it. The researchers estimated in their study that the method would cost $1.26 per test.
“Another advantage of this measure is that we can detect the entire virus, which is still infectious, and distinguish it from fragments that may not be infectious anymore,” Wang said. This not only gives patients and physicians better understanding of whether they are infectious, but it could greatly improve community-level modeling and tracking of active outbreaks, such as through wastewater.
In addition, the DNA nets inhibited the virus’s spread in live cell cultures, with the antiviral activity increasing with the size of the DNA net scaffold. This points to DNA structures’ potential as therapeutic agents, Wang said.
“I had this idea at the very beginning of the pandemic to build a platform for testing, but also for inhibition at the same time,” Wang said. “Lots of other groups working on inhibitors are trying to wrap up the entire virus, or the parts of the virus that provide access to antibodies. This is not good, because you want the body to form antibodies. With the hollow DNA net structures, antibodies can still access the virus.”
The DNA net platform can be adapted to other viruses, Wang said, and even multiplexed so that a single test could detect multiple viruses.
“We’re trying to develop a unified technology that can be used as a plug-and-play platform. We want to take advantage of DNA sensors’ high binding affinity, low limit of detection, low cost and rapid preparation,” Wang said.
A de novo matrix for macroscopic living materials from bacteria
by Sara Molinari, Robert F. Tesoriero, Dong Li, Swetha Sridhar, Rong Cai, Jayashree Soman, Kathleen R. Ryan, Paul D. Ashby, Caroline M. Ajo-Franklin in Nature Communications
Engineered living materials promise to aid efforts in human health, energy and environmental remediation. Now they can be built big and customized with less effort.
Bioscientists and synthetic biologists at Rice University have introduced centimeter-scale, slime-like colonies of engineered bacteria that self-assemble from the bottom up. It can be programmed to soak up contaminants from the environment or to catalyze biological reactions, among many possible applications. The creation of autonomous engineered living materials — or ELMs — has been a goal of bioscientist Caroline Ajo-Franklin since long before she joined Rice in 2019 with a grant from the Cancer Prevention and Research Institute of Texas (CPRIT).
“We’re making material from bacteria that acts like putty,” Ajo-Franklin said. “One of the beautiful things about it is how easy it is to make, merely needing a little motion, a few nutrients and bacteria.”
A study details the lab’s creation of flexible, adaptable ELMs using Caulobacter crescentus as a biological building block. While the bacteria themselves can easily be genetically modified for various processes, designing them to self-assemble has been a long and complicated process. It involved engineering the bacteria to display and secrete the biopolymer matrix that gives the material its form. C. crescentus already expresses a protein that covers its outer membrane like scales on a snake. The researchers modified the bacteria to express a version of that protein, which they call BUD (for bottom-up de novo, as in from scratch), with characteristics not only favorable to forming ELMs (dubbed BUD-ELMs) but also providing tags for future functionalization.
“We wanted to prove that it’s possible to grow materials from cells, like a tree grows from a seed,” said study lead author Sara Molinari, a postdoctoral researcher in Ajo-Franklin’s lab who earned her doctorate in Rice’s Systems, Synthetic and Physical Biology Ph.D. program. “The transformative aspect of ELMs is that they contain living cells that allow the material to self-assemble and self-repair in case of damage. Moreover, they can be further engineered to perform non-native functions, such as dynamically processing external stimuli.”
Molinari, who earned her Ph.D. in the lab of Rice bioscientist Matthew Bennett, said BUD-ELM is the most customizable example of an autonomously formed, macroscopic ELM. “It shows a unique combination of high performance and sustainability,” she said. “Thanks to its modular nature, it could serve as a platform to generate many different materials.” ELMs grow in a flask in about 24 hours, according to the researchers. First, a thin skin forms at the air-water interface, seeding the material. Constant shaking of the flask encourages the ELM to grow. Once it expands to a sufficient size, the material sinks to the bottom and grows no further.
“We found the shaking process influences how big of a material we get,” said co-author Robert Tesoriero Jr., a Ph.D. student in systems, synthetic and physical biology. “Partially, we’re looking for the optimal range of material we can get in a flask of about 250 millimeters. Currently it is about the size of a fingernail.”
“Getting to centimeter scale with a cell that is less than a micron in size means they collectively organize over four orders of magnitude, about 10,000 times bigger than a single cell,” Molinari added.
She said their functional materials are robust enough to survive in a jar on the shelf for three weeks at room temperature, meaning they can be transported with no refrigeration. The lab proved that the BUD-ELM could successfully remove cadmium from a solution and was able to perform biological catalysis, enzymatically reducing an electron carrier to oxidize glucose. Because BUD-ELMs carry tags for attachment, Ajo-Franklin said it should be relatively easy to modify them for optical, electrical, mechanical, thermal, transport and catalytic applications.
“There’s a lot of room to play around, which I think is the fun part,” Tesoriero said.
“The other big question is that while we love Caulobacter crescentus, it’s not the most popular kid on the block,” Ajo-Franklin said. “Most people have never heard of it. So we’re really interested in knowing if these rules we’ve discovered in Caulobacter can be applied to other bacteria.”
She said ELMs could be especially useful for environmental remediation in low-resource settings. C. crescentusis ideal for this as it requires less nutrients to grow than many bacteria.
“One of my dreams is to use the material to remove heavy metals from water, and then when it reaches the end of its lifetime, pull off a little part and grow it on the spot into fresh material,” Ajo-Franklin said. “That we could do it with minimal resources is really a compelling idea to me.”
Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection
by Jie Yang, Yang Song, Xiangyu Deng, Jeffrey A. Vanegas, Zheng You, Yuxuan Zhang, Zhengyan Weng, Lori Avery, Kevin D. Dieckhaus, Advaith Peddi, Yang Gao, Yi Zhang, Xue Gao in Nature Chemical Biology
An engineered CRISPR-based method that finds RNA from SARS-CoV-2, the virus that causes COVID-19, promises to make testing for that and other diseases fast and easy.
Collaborators at Rice University and the University of Connecticut further engineered the RNA-editing CRISPR-Cas13 system to boost their power for detecting minute amounts of the SARS-CoV-2 virus in biological samples without the time-consuming RNA extraction and amplification step necessary in gold-standard PCR testing. The new platform was highly successful compared to PCR, finding 10 out of 11 positives and no false positives for the virus in tests on clinical samples directly from nasal swabs. The researchers showed their technique finds signs of SARS-CoV-2 in attomolar (10–18) concentrations. The study led by chemical and biomolecular engineer Xue Sherry Gao at Rice’s George R. Brown School of Engineering and postdoctoral researchers Jie Yang of Rice and Yang Song of Connecticut appears in Nature Chemical Biology.
Cas13, like its better-known cousin Cas9, is part of the system by which bacteria naturally defend themselves against invading phages. Since its discovery, CRISPR-Cas9 has been adapted by scientists to edit living DNA genomes and shows great promise to treat and even cure diseases. And it can be used in other ways. Cas13 on its own can be enhanced with guide RNA to find and snip target RNA sequences, but also to find “collateral,” in this case the presence of viruses like SARS-CoV-2.
“The engineered Cas13 protein in this work can be readily adapted to other previously established platforms,” Gao said. “The stability and robustness of engineered Cas13 variants make them more suitable for point-of-care diagnostics in low-resource setting areas when expensive PCR machines are not available.”
Yang said wild-type Cas13, drawn from a bacterium, Leptotrichia wadei, cannot detect attomolar level of viral RNA within a time frame of 30 to 60 minutes, but the enhanced version created at Rice does the job in about half an hour and detects SARS-CoV-2 in much lower concentrations than the previous tests. She said the key is a well-hidden, flexible hairpin loop near Cas13’s active site.
“It’s in the middle of the protein near the catalytic site that determines Cas13’s activity,” Yang said. “Since Cas13 is large and dynamic, it was challenging to find a site to insert another functional domain.”
The researchers fused seven different RNA binding domains to the loop, and two of the complexes were clearly superior. When they found their targets, the proteins would fluoresce, revealing the presence of the virus.
“We could see the increased activity was five- or six-fold over wild-type Cas13,” Yang said. “This number seems small, but it’s quite astonishing with a single step of protein engineering.
“But that was still not enough for detection, so we moved the whole assay from a fluorescence plate reader, which is quite large and not available in low-resource settings, to an electrochemical sensor, which has higher sensitivity and can be used for point-of-care diagnostics,” she said.
With the off-the-shelf sensor, Yang said the engineered protein was five orders of magnitude more sensitive in detecting the virus compared to the wild-type protein. The lab wants to adapt its technology to paper strips like those in home COVID-19 antibody tests, but with much higher sensitivity and accuracy. “We hope that will make testing more convenient and with lower cost for many targets,” Gao said. The researchers are also investigating improved detection of the Zika, dengue and Ebola viruses and predictive biomarkers for cardiovascular disease. Their work could lead to rapid diagnosis of the severity of COVID-19.
“Different viruses have different sequences,” Yang said. “We can design guide RNA to target a specific sequence that we can then detect, which is the power of the CRISPR-Cas13 system.”
But because the project began just as the pandemic took hold, SARS-CoV-2 was a natural focus. “The technology is quite amenable to all the targets,” she said. “This makes it a very good option to detect all kinds of mutations or different coronaviruses.”
“We are very excited about this work as a combinational effort of structure biology, protein engineering and biomedical device development,” Gao added. “I greatly appreciate all the efforts from my lab members and collaborators.”
Nature-inspired delivery of mitochondria-targeted angiotensin receptor blocker
by Jude M Phillip, Ran Lin, Andrew Cheetham, David Stern, Yukang Li, Yuzhu Wang, Han Wang, David Rini, Honggang Cui, Jeremy D Walston, Peter M Abadir in PNAS Nexus
In a study using lab-grown cells, Johns Hopkins Medicine researchers specializing in aging report they have successfully delivered a common blood pressure drug directly to the inner membrane of mitochondria, the “power plants” in the cells of humans, animals, plants and most other organisms.
Developing ways to directly target these energy-producing parts of the cell for delivery of drugs has long been a goal for researchers because mitochondria drive, control or play a role in almost every biological process, including natural cell death and aging. Alterations or declines in mitochondrial activity and pathways are closely aligned with decreased organ function and frailty. But because of the mitochondria’s double-membrane structure, scientists have found it challenging to get drug molecules to penetrate the inner membrane and gain access to core functions of the organelles.
The new study, reports on a method that essentially hijacks a system already used by mitochondria to transport oxygen and other chemicals to the inner membrane.
“Our study shows that we can use the body’s natural mitochondrial transport system to deliver drugs much more precisely,” says Peter Abadir, M.D., associate professor of geriatric medicine and gerontology at the Johns Hopkins University School of Medicine.
For the study, the researchers lab-synthesized three naturally occurring transport proteins that interact with mitochondria. They then fused a commonly prescribed blood pressure medication (losartan) to each of these three proteins to determine which had the highest success rate penetrating the inner membrane of the mitochondria. These fused proteins, dubbed mtLOS1, mtLOS2 and mtLOS3, when introduced to lab-grown cells in separate trials, were able to transport the drug directly to the mitochondria at a significantly higher concentration than was possible with free losartan not fused to the transport protein. This could be seen under a microscope using florescence.
In a proof-of-concept experiment, the researchers also tested a “scrambled” version of mtLOS, which was unable to penetrate the inner membrane. Abadir says further research is needed, but the goal is to use mtLOS or other natural transport pathways to deliver medicines that directly and efficiently target the biochemical imbalances and losses linked to chronic inflammation and weakened organ function characteristic of aging and many disorders.
“We know people age in part because of mitochondrial decline, and scientists have been trying to get therapies directly into the organelle to counteract this decline for decades,” says Abadir. “This is another attempt at delivering compounds using the body’s natural systems, which may greatly reduce negative side effects both short and long term.”
Structure of an MHC I–tapasin–ERp57 editing complex defines chaperone promiscuity
by Ines Katharina Müller, Christian Winter, Christoph Thomas, Robbert M. Spaapen, Simon Trowitzsch, Robert Tampé in Nature Communications
As task forces of the adaptive immune system, T lymphocytes are responsible for attacking and killing infected or cancerous cells. Such cells, like almost all cells in the human body, present on their surface fragments of all the proteins they produce inside. If these include peptides that a T lymphocyte recognises as foreign, the lymphocyte is activated and kills the cell in question. It is therefore important for a robust T-cell response that suitable protein fragments are presented to the T lymphocyte. The research team led by Simon Trowitzsch and Robert Tampé from the Institute of Biochemistry at Goethe University Frankfurt has now shed light on how the cell selects these protein fragments or peptides.
Peptide presentation takes place on so-called major histocompatibility complex class I molecules (MHC I). MHC I molecules are a group of very diverse surface proteins that can bind myriads of different peptides. They are anchored in the cell membrane and form a peptide-binding pocket with their outward-facing part. Like all surface proteins, MHC I molecules take the so-called secretory pathway: they are synthesised into the cell’s cavity system (endoplasmic reticulum (ER) and Golgi apparatus) and folded there. Small vesicles then bud off from the cavity system, migrate to the cell membrane and fuse with it.
The maturation process of the MHC I molecules is very strictly controlled: in the ER, proteins known as “chaperones” help them fold. The chaperone tapasin is essential for peptide loading in this process. “When an MHC I molecule has bound a peptide, tapasin checks how tight the binding is,” says Trowitzsch, explaining the chaperone’s task. “If the bond is unstable, the peptide is removed and replaced by a tightly binding one.” However, it has not yet been possible to clarify how exactly tapasin performs this task — especially because the loading process is extremely fast.
The biochemists and structural biologists from Goethe University Frankfurt have now succeeded for the first time in visualising the short-lived interaction between chaperone and MHC I molecule by means of X-ray structure analysis. To do this, they produced variants of the two interaction partners that were no longer embedded in the membrane, purified them and brought them together. A trick helped to capture the loading complex in action for crystallisation: first, the research team loaded the MHC I molecule with a high-affinity peptide so that a stable complex was created. A light signal triggered cleavage of the peptide, which greatly reduced its ability to bind the MHC I molecule. Immediately, tapasin entered the scene and remained bound to the MHC I molecule that lacks its peptide.
“The photo-induced cleavage of the peptide was pivotal to the success of our experiment,” says Tampé. “With the help of this optochemical biology, we can now systematically reproduce complex cellular processes one by one.”
X-ray structure analysis of the crystals revealed how tapasin widens the peptide-binding pocket of the MHC I molecule, thereby testing the strength of the peptide bond. For this purpose, the interaction partners form a large contact area; for stabilisation, a loop of tapasin sits on top of the widened binding pocket.
“This is the first time we have shown the process of loading at high resolution,” Tampé is pleased to report. The images also reveal how a single chaperone can interact with the enormous diversity of MHC I molecules, says the biochemist: “Tapasin binds precisely the non-variable regions of the MHC I molecules.”
However, the new structure not only improves our understanding of the complex processes involved in loading MHC I molecules. It should also help select suitable candidates for vaccine development.
Speed variations of bacterial replisomes
by Deepak Bhat, Samuel Hauf, Charles Plessy, Yohei Yokobayashi, Simone Pigolotti in eLife
Cell division is fundamental for life, allowing organisms to grow, repair tissues, and reproduce. For a cell to divide, all the DNA inside the cell (the genome) must first be copied, in a process called DNA replication. But the precise dynamics of replisomes — the protein machinery that copies DNA — has been difficult for scientists to determine.
Now, researchers at the Okinawa Institute of Science and Technology (OIST) in Japan have developed a new model that can determine variations in the speed at which replisomes copy bacterial genomes. The model, combined with experiments, shows that certain sections of DNA are copied faster than others and reveals an intriguing link between replication speed and error rate.
“The machines that copy DNA are amazing — they are very fast and very precise,” said Simone Pigolotti, an Associate Professor at OIST who heads the Biological Complexity Unit. “Understanding these machines can tell us what is important for cells — what mistakes are tolerable, what mistakes are not, how fast replication should be.”
The model relies on measuring how abundant different DNA locations are within a population of bacterial cells that are constantly dividing. In bacteria, to start DNA replication, two replisomes attach to the DNA at a set origin point and head in opposite directions along the loop of DNA, copying DNA until they meet on the other side. This means that the DNA closest to the origin point is copied first, while DNA closest to the termination point is copied last.
“If you let a population of bacteria freely grow, then at any given point in time, most cells will be in the process of cell division. Because DNA replication always starts from the same location, this means that if you then sequence all the DNA, there will be a higher abundance of DNA that is closest to the origin point, and a much lower amount of DNA that is closer to the end point,” explained Prof. Pigolotti.
In the study, researchers from the Nucleic Acid Chemistry and Engineering Unit at OIST cultured Escherichia coli (E. coli) bacteria at different temperatures. The Sequencing Section then sequenced the bacteria’s DNA. By analyzing features of the distribution curve, the researchers were able to determine the exact speed of the protein machinery. They found that as the temperature increased, the replication speed increased. Even more interestingly, the researchers discovered that the replisomes varied their speed at different points along the genome. One potential reason for their fluctuating speed, Prof. Pigolotti speculates, is that there may be limits on resources needed for replication, such as nucleotides — the building blocks of DNA.
In E. coli, when conditions are good, a single bacterial cell can divide every 25 minutes. But the process of replicating DNA takes longer — around 40 minutes. Therefore, in order to keep up at high growth rates, multiple copies of the genome are replicated at the same time, which increases the number of replisomes at work. Competition for nucleotides could then cause the replisomes to slow down. Additional evidence backs up this hypothesis. At low temperatures and in nutrient-poor cultures, when the growth rate of the bacteria is low and only one genome would be copied at a time, these fluctuations in replication speed disappear. Intriguingly, the researchers also found that the oscillations seen for replication speed also matched the oscillations in mutation rate documented in other studies. When they overlaid the two patterns, they found that areas of the genome that were copied faster also had a higher mutation rate.
“This seems intuitive — if we think of an action, like typing on a keyboard, the faster we type, the more likely that we will make a mistake,” said Prof. Pigolotti. “So we think that when the replisomes go faster, their error rate is higher.”
For Prof. Pigolotti, the next step is to determine how the speed of replication changes in mutant strains of E. coli, such as ones that are missing proteins that assist in replication. He is also curious to see if the pattern holds in other strains of bacteria.
“It’s a really exciting research direction,” said Prof. Pigolotti. “And all the work was done in collaboration with other units here. It’s the kind of interdisciplinary collaboration that can only happen at OIST.”
CLN3 is required for the clearance of glycerophosphodiesters from lysosomes
by Nouf N. Laqtom, Wentao Dong, Uche N. Medoh, Andrew L. Cangelosi, et al in Nature
Small but mighty, lysosomes play a surprisingly important role in cells despite their diminutive size. Making up only 1–3% of the cell by volume, these small sacs are the cell’s recycling centers, home to enzymes that break down unneeded molecules into small pieces that can then be reassembled to form new ones. Lysosomal dysfunction can lead to a variety of neurodegenerative or other diseases, but without ways to better study the inner contents of lysosomes, the exact molecules involved in diseases — and therefore new drugs to target them — remain elusive.
A new method allows scientists to determine all the molecules present in the lysosomes of any cell in mice. Studying the contents of these molecular recycling centers could help researchers learn how the improper degradation of cellular materials leads to certain diseases. Led by Stanford University’s Monther Abu-Remaileh, institute scholar at Sarafan ChEM-H, the study’s team also learned more about the cause for a currently untreatable neurodegenerative disease known as Batten disease, information that could lead to new therapies.
“Lysosomes are fascinating both fundamentally and clinically: they supply the rest of the cell with nutrients, but we don’t always know how and when they supply them, and they are the places where many diseases, especially those that affect the brain, start,” said Abu-Remaileh, who is an assistant professor of chemical engineering and of genetics.
Some proteins that are usually located in lysosomes are linked to a number of diseases. Mutations in the genetic instructions for making those proteins lead to these “lysosomal storage disorders,” as they are collectively called, but the functions of some of these proteins have long puzzled scientists. Information about how these proteins work could help scientists develop better ways to diagnose, monitor, or treat these diseases.
If scientists want to study the role a particular protein plays in the cell, they might either block or stimulate its function and see if certain molecules appear or disappear in response. But studying the contents of lysosomes is a problem of scale. “If something happens and a molecule grows in abundance 200-fold in the lysosome, you would see only a two-fold increase if you look at the whole cell,” said Nouf Laqtom, first author on the study. The revealing results get buried in the noise.
To quiet the noise, the researchers would have to separate lysosomes from everything else in the cell. They had previously developed a method to do just that in cells grown in labs, but they wanted to develop a way to do the same thing in mice.
The first step in their quest to isolate lysosomes was making a small change in the genes of the mice to install a little molecular tag on the surface of every lysosome in the entire animal. At any point when they want to stop and check on the molecules in the mouse lysosomes, like after fasting or feeding them a specific food, they turn on the tag in the cells they want to examine, and then remove the tissue and carefully grind it up to break open the cells without disrupting the lysosomes inside.
To fish lysosomes from the cellular sludge, the team relies on magnets. To their slurry they add tiny magnetic beads that are each decorated with molecular clamps that grab ahold of the lysosomal tag they had previously installed. They can selectively collect all the lysosomes using a second magnet, and then break apart the lysosomes to reveal the molecules that had been safely tucked inside. Mass spectrometry, a set of tools that determines the weights of different molecules in a mixture, then helps the researchers identify the individuals in their lysosomal molecular potpourri. Those that grow or decrease would point scientists to certain pathways or functions. Except for the little extra tag on each lysosome, these “LysoTag” mice are otherwise normal laboratory mice. Now, almost any researcher can use these mice to study the role of lysosomes in different diseases.
“These mice are freely available for anyone in the research community to use, and people are already starting to use them,” said Abu-Remaileh. “We hope that this will become the gold standard.”
The team was eager to apply their method to study the lysosomes found in brain cells to better understand the neurodegenerative lysosomal storage diseases, starting with CLN3 disease or juvenile Batten disease. “We really see this as one of the most urgent problems we can help solve,” said Abu-Remaileh. Caused by a mutation in the gene that codes for a protein called CLN3, juvenile Batten disease is fatal and leads to vision loss, seizures, and progressive motor and mental deterioration in children and young adults. The CLN3 protein is found on the membrane of the lysosome, but no one has ever determined its function in the cell or how its dysfunction leads to the observed symptoms. Using their LysoTag mice, the researchers collaborated with experts in both the Sarafan ChEM-H Metabolomics Knowledge Center and the Whitehead Institute Metabolomics Core Facility and found a dramatic increase in the amount of a kind of molecule called a glycerophosphodiester, or GPD for short, in mice with the CLN3 disease mutation. These GPDs are temporarily formed during the degradation of the fatty molecules that make up the membranes of every cell in our body.
In healthy cells, the GPDs do not accumulate in the lysosome; they get exported to a different part of the cell, where they are then degraded into smaller pieces. The researchers now believe that the CLN3 protein plays an important role in that export, either by directly shuttling out the molecules or by helping another protein do that job. They found GPD molecules in the cerebral spinal fluid of patients with CLN3 disease, which suggests that clinicians could potentially monitor GPD levels to measure the success of future treatments. The team is now determining which of the GPD molecules might be toxic and how the proteins involved in making and exporting GPDs could be targeted with new drugs. They also are applying their method to look at other diseases that involve mutations in lysosomal genes, like Parkinson’s disease.
“You can’t develop new ways to diagnose or treat diseases if you don’t know what is changing in the lysosomes,” said Laqtom, a former postdoctoral scholar in the Abu-Remaileh lab. “This method helps you make sure you’re looking in the right direction. It points you down the right path and keeps you from getting lost.”
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