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At least 883 records · Page 49

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DOE OSTI2020

Different tertiary interactions create the same important 3D features in a distinct flavivirus xrRNA

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific three-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana bat virus (TABV) and solved its structure by X-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A–C interactions, four distinct backbone kinks, several ordered Mg 2+ ions, and a C + –G–C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Data for "Viral-mediated Delivery of Morphogenic Regulators Enables Leaf Transformation in Sorghum bicolor (L.)"

Recent advancements in monocot transformation, using leaf tissue as explant material, have expanded the number of grass species capable of transgenesis. However, the complexity of vectors and reliance on inducible excision of essential morphogenic regulators have so far limited widespread application. Plant RNA viruses, such as Foxtail Mosaic Virus (FoMV), present a unique opportunity to express morphogenic regulator genes, such as Babyboom ( Bbm ), Wuschel2 ( Wus2 ), Wuschel-like homeobox protein 2a ( Wox2a ) and the GROWTH-REGULATING FACTOR 4 (GRF4) GRF-INTERACTING FACTOR 1 (GIF1) fusion protein transiently in leaf explant tissues. Furthermore, altruistic delivery of conventional and viral vectors could provide opportunities to simplify vectors used for leaf transformation—facilitating vector optimization and reducing reliance on morphogenic regulator gene integration. In this study, both viral and conventional T-DNA vectors were tested for their ability to promote the formation of embryonic calli, a critical step in leaf transformation protocols, using Sorghum bicolor leaf explants. Although conventional leaf transformation vectors yielded viable embryonic calli (43.2 ± 2.9%: GRF4-GIF1, 50.2 ± 3%: Bbm/Wus2), altruistic conventional vectors employing the GRF4-GIF1 morphogenic regulator resulted in improved efficiencies (61.3 ± 4.7%). Altruistic delivery was further enhanced with the use of viral vectors employing both GRF4-GIF1 and Bbm/Wus2 regulators, resulting in 75.1 ± 2.3% and 79.2 ± 2.5% embryonic calli formation, respectively. Embryonic calli generated from both conventional and viral vectors produced shoots expressing fluorescent reporters, which were confirmed using molecular analysis. This work provides an important proof-of-concept for the use of both altruistic vectors and viral-expressed morphogenic regulators for improving plant transformation.

gene editing↗

DOE OSTI2021

A quantitative model used to compare within-host SARS-CoV-2, MERS-CoV, and SARS-CoV dynamics provides insights into the pathogenesis and treatment of SARS-CoV-2

The scientific community is focused on developing antiviral therapies to mitigate the impacts of the ongoing novel coronavirus disease 2019 (COVID-19) outbreak. This will be facilitated by improved understanding of viral dynamics within infected hosts. Here, using a mathematical model in combination with published viral load data, we compare within-host viral dynamics of SARS-CoV-2 with analogous dynamics of MERS-CoV and SARS-CoV. Our quantitative analyses using a mathematical model revealed that the within-host reproduction number at symptom onset of SARS-CoV-2 was statistically significantly larger than that of MERS-CoV and similar to that of SARS-CoV. In addition, the time from symptom onset to the viral load peak for SARS-CoV-2 infection was shorter than those of MERS-CoV and SARS-CoV. These findings suggest the difficulty of controlling SARS-CoV-2 infection by antivirals. We further used the viral dynamics model to predict the efficacy of potential antiviral drugs that have different modes of action. The efficacy was measured by the reduction in the viral load area under the curve (AUC). Our results indicate that therapies that block de novo infection or virus production are likely to be effective if and only if initiated before the viral load peak (which appears 2–3 days after symptom onset), but therapies that promote cytotoxicity of infected cells are likely to have effects with less sensitivity to the timing of treatment initiation. Furthermore, combining a therapy that promotes cytotoxicity and one that blocks de novo infection or virus production synergistically reduces the AUC with early treatment. Our unique modeling approach provides insights into the pathogenesis of SARS-CoV-2 and may be useful for development of antiviral therapies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Mechanistic basis of post-treatment control of SIV after anti-α4β7 antibody therapy

Treating macaques with an anti-α4β7 antibody under the umbrella of combination antiretroviral therapy (cART) during early SIV infection can lead to viral remission, with viral loads maintained at < 50 SIV RNA copies/ml after removal of all treatment in a subset of animals. Depletion of CD8+ lymphocytes in controllers resulted in transient recrudescence of viremia, suggesting that the combination of cART and anti-α4β7 antibody treatment led to a state where ongoing immune responses kept the virus undetectable in the absence of treatment. A previous mathematical model of HIV infection and cART incorporates immune effector cell responses and exhibits the property of two different viral load set-points. While the lower set-point could correspond to the attainment of long-term viral remission, attaining the higher set-point may be the result of viral rebound. Here we expand that model to include possible mechanisms of action of an anti-α4β7 antibody operating in these treated animals. We show that the model can fit the longitudinal viral load data from both IgG control and anti-α4β7 antibody treated macaques, suggesting explanations for the viral control associated with cART and an anti-α4β7 antibody treatment. This effective perturbation to the virus-host interaction can also explain observations in other nonhuman primate experiments in which cART and immunotherapy have led to post-treatment control or resetting of the viral load set-point. Interestingly, because the viral kinetics in the various treated animals differed—some animals exhibited large fluctuations in viral load after cART cessation—the model suggests that anti-α4β7 treatment could act by different primary mechanisms in different animals and still lead to post-treatment viral control. This outcome is nonetheless in accordance with a model with two stable viral load set-points, in which therapy can perturb the system from one set-point to a lower one through different biological mechanisms.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Full scale structural, mechanical and dynamical properties of HIV-1 liposomes

Enveloped viruses are enclosed by a lipid membrane inside of which are all of the components necessary for the virus life cycle; viral proteins, the viral genome and metabolites. Viral envelopes are lipid bilayers that adopt morphologies ranging from spheres to tubes. The envelope is derived from the host cell during viral replication. Thus, the composition of the bilayer depends on the complex constitution of lipids from the host-cell’s organelle(s) where assembly and/or budding of the viral particle occurs. Here, molecular dynamics (MD) simulations of authentic, asymmetric HIV-1 liposomes are used to derive a unique level of resolution of its full-scale structure, mechanics and dynamics. Analysis of the structural properties reveal the distribution of thicknesses of the bilayers over the entire liposome as well as its global fluctuations. Moreover, full-scale mechanical analyses are employed to derive the global bending rigidity of HIV-1 liposomes. Finally, dynamical properties of the lipid molecules reveal important relationships between their 3D diffusion, the location of lipid-rafts and the asymmetrical composition of the envelope. Overall, our simulations reveal complex relationships between the rich lipid composition of the HIV-1 liposome and its structural, mechanical and dynamical properties with critical consequences to different stages of HIV-1’s life cycle.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Identification of mosquito proteins that differentially interact with alphavirus nonstructural protein 3, a determinant of vector specificity

Chikungunya virus (CHIKV) and the closely related onyong-nyong virus (ONNV) are arthritogenic arboviruses that have caused significant, often debilitating, disease in millions of people. However, despite their kinship, they are vectored by different mosquito subfamilies that diverged 180 million years ago (anopheline versus culicine subfamilies). Previous work indicated that the nonstructural protein 3 (nsP3) of these alphaviruses was partially responsible for this vector specificity. To better understand the cellular components controlling alphavirus vector specificity, a cell culture model system of the anopheline restriction of CHIKV was developed along with a protein expression strategy. Mosquito proteins that differentially interacted with CHIKV nsP3 or ONNV nsP3 were identified. Six proteins were identified that specifically bound ONNV nsP3, ten that bound CHIKV nsP3 and eight that interacted with both. In addition to identifying novel factors that may play a role in virus/vector processing, these lists included host proteins that have been previously implicated as contributing to alphavirus replication.

Byers, Nathaniel M. (ORCID:0000000157725940)↗

DOE OSTI2024

Using intrahost single nucleotide variant data to predict SARS-CoV-2 detection cycle threshold values

Over the last four years, each successive wave of the COVID-19 pandemic has been caused by variants with mutations that improve the transmissibility of the virus. Despite this, we still lack tools for predicting clinically important features of the virus. In this study, we show that it is possible to predict the PCR cycle threshold (Ct) values from clinical detection assays using sequence data. Ct values often correspond with patient viral load and the epidemiological trajectory of the pandemic. Using a collection of 36,335 high quality genomes, we built models from SARS-CoV-2 intrahost single nucleotide variant (iSNV) data, computing XGBoost models from the frequencies of A, T, G, C, insertions, and deletions at each position relative to the Wuhan-Hu-1 reference genome. Our best model had an R 2 of 0.604 [0.593–0.616, 95% confidence interval] and a Root Mean Square Error (RMSE) of 5.247 [5.156–5.337], demonstrating modest predictive power. Overall, we show that the results are stable relative to an external holdout set of genomes selected from SRA and are robust to patient status and the detection instruments that were used. This study highlights the importance of developing modeling strategies that can be applied to publicly available genome sequence data for use in disease prevention and control.

COVID19↗

DOE OSTI2020

Pressurized DNA state inside herpes capsids—A novel antiviral target

Drug resistance in viruses represents one of the major challenges of healthcare. As part of an effort to provide a treatment that avoids the possibility of drug resistance, we discovered a novel mechanism of action (MOA) and specific compounds to treat all nine human herpesviruses and animal herpesviruses. The novel MOA targets the pressurized genome state in a viral capsid, “turns off” capsid pressure, and blocks viral genome ejection into a cell nucleus, preventing viral replication. This work serves as a proof-of-concept to demonstrate the feasibility of a new antiviral target—suppressing pressure-driven viral genome ejection —that is likely impervious to developing drug resistance. This pivotal finding presents a platform for discovery of a new class of broad-spectrum treatments for herpesviruses and other viral infections with genome-pressure-dependent replication. A biophysical approach to antiviral treatment such as this is also a vital strategy to prevent the spread of emerging viruses where vaccine development is challenged by high mutation rates or other evasion mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Dynamic metabolic reprogramming in dendritic cells: An early response to influenza infection that is essential for effector function

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Complementary antibody lineages achieve neutralization breadth in an HIV-1 infected elite neutralizer

Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG + B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient’s remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

High throughput analysis of B cell dynamics and neutralizing antibody development during immunization with a novel clade C HIV-1 envelope

A protective HIV-1 vaccine has been hampered by a limited understanding of how B cells acquire neutralizing activity. Our previous vaccines expressing two different HIV-1 envelopes elicited robust antigen specific serum IgG titers in 20 rhesus macaques; yet serum from only two animals neutralized the autologous virus. Here, we used high throughput immunoglobulin receptor and single cell RNA sequencing to characterize the overall expansion, recall, and maturation of antigen specific B cells longitudinally over 90 weeks. Diversification and expansion of many B cell clonotypes occurred broadly in the absence of serum neutralization. However, in one animal that developed neutralization, two neutralizing B cell clonotypes arose from the same immunoglobulin germline and were tracked longitudinally. Early antibody variants with high identity to germline neutralized the autologous virus while later variants acquired somatic hypermutation and increased neutralization potency. The early engagement of precursors capable of neutralization with little to no SHM followed by prolonged affinity maturation allowed the two neutralizing lineages to successfully persist despite many other antigen specific B cells. The findings provide new insight into B cells responding to HIV-1 envelope during heterologous prime and boost immunization in rhesus macaques and the development of selected autologous neutralizing antibody lineages.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease – 3rd CIAO AOP Design Workshop

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project “Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework” aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and positioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowdsourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, integrating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

The D614G mutation in Spike: increased infectivity and neutralizing Ab sensitivity and the underlying mechanism [Slides]

Evidence is presented that there are now more SARS-CoV-2 viruses circulating in the human population globally that have the G614 form that was originally identified from the first human cases in Wuhan, China. Follow-up studies show that patients infected with G614 shed more viral nucleic avid compared with those with D614 and G614-bearing viruses show significantly higher infectious titers in vitro than their D614 counterparts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Lab Home Testing of Residential Isolation Space Control to Minimize Infectious Disease Transmission in Existing Single-Family Homes

Existing evidence strongly suggests that viral infectious diseases can be transmitted via an airborne route across distances in indoor environments. Accordingly, the risk of airborne transmission within homes should be managed. The public health emergency associated with SARS-CoV-2 makes controlling airborne transmission of respired viruses in indoor environments critical, especially in poorly ventilated indoor environments. The effectiveness of engineering interventions requiring minor efforts that create a negative-pressure isolation zone (IZ) for a contagious person has yet to be tested for existing residential homes. To mitigate the risk of airborne virus transmission and maximize health protection for the population in existing single-family homes, the relative effectiveness of several control strategies are investigated in this report. While very high-efficiency MERV filtration, high ventilation rates, and other controls can help be effective, most occupants are not likely to have the time or means for advanced measures found in hospitals. This project focused on testing relatively simple efforts that utilize existing or easy to acquire materials and simple processes.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗

DOE OSTI2021

Genetics and Genomics of Pathogen Resistance in Switchgrass

This project investigates the genetic and genomic bases of resistance/tolerance to disease in switchgrass (Panicum virgatum) populations developed as bioenergy feedstock. The focus is placed populations adapted to the Central Great Plains derived from the hybridization of upland and lowland ecotypes, represented by cultivars Summer and Kanlow, respectively. Resistance was investigated against the rust disease caused by the fungus Puccinia novo-panici (syn. P emaculata) and mosaic disease caused by the virus complex of Panicum mosaic virus and its satellite. Objectives of the project were: 1) to elucidate the genetic and genomic bases of resistance/tolerance in locally adapted upland and lowland switchgrass breeding populations with contrasting disease symptoms under field conditions; 2) determine the molecular underpinnings of why cv Kanlow has broad resistance/tolerance to pathogens; and 3) discover the molecular differences that permit systemic viral infections in some switchgrass plants but not in other genetically-related plants.

09 BIOMASS FUELS↗

DOE OSTI2021

Science & Technology Review - July 2021 Issue

In the early weeks of the COVID-19 pandemic, Lawrence Livermore researchers mobilized to address emerging challenges and develop capabilities for detecting the SARS-CoV-2 virus and fighting COVID-19, the diseased caused by the virus. As described in the article beginning on p. 4, the Laboratory’s Innovation and Partnerships Office provided technology transfer expertise to deploy existing, in-development, and new technologies to help detect SARS-CoV-2 and treat COVID-19.

36 MATERIALS SCIENCE↗

DOE OSTI2020

Doing Pandemic R&D as if There’s No Mañana: Charting a course to zig and zag our way out of the pandemonium we’re in

Since the beginnings of Sandia National Laboratories, Sandians have made the unthinkable not only thinkable, but also plannable and doable. Over time, we’ve tempered ourselves, studying and devising ways to deter, defend against, and blunt threats of mass death, destruction, and disruption. Now we, and all within our social circles, are living one of the nightmares that only a small fraction of our fellow Sandians have pondered, tackled, and anticipated––a virus that bypasses militaries, zips through borders, hitches to domestic travelers and commuters, makes its hosts unknowing dangers to others, overwhelms hospitals and clinics, hobbles governments, and takes away the lives and livelihoods of compromised and marginalized people everywhere. Taken by surprise, Sandians have dispersed and re-congregated into three interconnected camps. Those who determinedly and defiantly carry on with the work they’ve been doing since before the pandemic hit. Those who are doggedly mustering and redirecting their abilities, experience, education, and energies to get us to the other side of this crisis (without ending up on the Other Side in the process). And those who are in fight-or-flight mode because this virus is pushing them to their personal limits, directly threatening them and/or the lives, jobs, and businesses that matter to them. From these three camps (which some of us migrate between on a daily, hourly, or even minute-by-minute basis), we make our individual and collective attempts to hold it together, do our work, and live another day. While there is much about this that is beyond our control, we are anything but helpless. Each of us has specialized powers and influence that can be brought to bear on the big and small parts of this gnarly problem. It all starts with a personal and collective understanding of the situation we’re in.

99 GENERAL AND MISCELLANEOUS↗

DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗