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At least 73 records · Page 4

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DOE OSTI2026

Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes

Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3 crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-null Nicotiana tabacum platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (k cat,air /K c,air ) under ambient conditions, driven by a significantly faster turnover rate (k cat,air ). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3 crops.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Ampk alpha2 T172 activation dictates exercise performance and energy transduction in skeletal muscle

Adenosine 5′-monophosphate–activated protein kinase (AMPK) is an energetic sensor for metabolic regulation and integration. Here, we used CRISPR-Cas9 to generate nonactivatable Ampkα knock-in (KI) mice with mutation of threonine-172 phosphorylation site to alanine (T172A), circumventing the limitations of previous genetic interventions that disrupt the protein stoichiometry. KI mice of Ampkα2, but not Ampkα1, demonstrated phenotypic changes with increased fat-to-lean mass, impaired endurance exercise capacity, and diminished mitochondrial maximal respiration and conductance in skeletal muscle. Integrated temporal multiomics analysis (proteomics/phosphoproteomics/metabolomics) in skeletal muscle at rest and during exercise establishes a pleiotropic yet imperative role of Ampkα2 T172 activation for glycolytic and oxidative metabolism, mitochondrial respiration, and contractile function. There is a substantial overlap of skeletal muscle proteomic changes in Ampkα2 T172A KI mice with that of patients with type 2 diabetes. Our findings suggest that Ampkα2 T172 activation is critical for exercise performance and energy transduction in skeletal muscle and may serve as a therapeutic target for type 2 diabetes.

Bioenergetics↗

DOE OSTI2026

Integrating CO2 Electrolysis with Gas Fermentation to Produce Valuable Fuels and Chemicals

Many industrial activities squander CO2, decreasing process yield. We envision a future where this waste carbon is instead captured, upgraded, and valorized directly at the point of emission. Within the CO2 Reduction and Upgrading Consortium (a collaboration of seven US national laboratories and industrial partners), we are pursuing this goal by developing and de-risking new technologies for low temperature CO2 electrolysis, coupled with biological upgrading of intermediates into more valuable compounds. One such process involves electrocatalytic reduction of CO2 to generate carbon monoxide (CO). As both a carbon and energy source, CO represents an attractive feedstock for microbial upgrading by certain syngas-fermenting species, such as the autotrophic bacterium Clostridium autoethanogenum. Our team has developed new genetic tools and optimized cultivation techniques to enhance C. autoethanogenum as a platform host for the biological conversion of syngas into value-added products. For example, we have created novel CRISPR-based genetic engineering techniques to build new, genome-reduced, platform strains of C. autoethanogenum with improved growth rates. Further, we ve introduced heterologous biochemical pathways into C. autoethanogenum to enable the production of high-value compounds from syngas, such as the isoprenoid precursor mevalonic acid. With the tools of electrochemistry and synthetic biology, there is virtually no limit to the spectrum of products that could be sustainably manufactured from CO2.

09 BIOMASS FUELS↗

DOE OSTI2024

Symbiotic bacteriophages exhibit multiple adaptive strategies in activated sludge flocs and contribute to floc stability

Despite the importance of phages for the dynamics and functions of microbial communities, it remains largely unexplored how symbiotic phages adapt in activated sludge systems and influence microbial aggregate stability, which is critical for clarification and reliable performance. Here, based on 12,127 phage contigs recovered from 12 activated sludge microbiomes, the symbiotic phages exhibited broad host ranges and infected dominant prokaryotes in activated sludge. Moreover, these phage communities exhibited high lysogenicity (49% to 66% lysogenic phages) and harbored diverse auxiliary metabolic genes (AMGs) that could enhance microbial aggregate stability, such as genes coding for lipopolysaccharide, peptidoglycan, and extracellular polysaccharide biosynthesis. Additionally, prokaryotic antiviral systems were widely distributed in dominant prokaryotes, particularly the Restriction-Modification (RM) and CRISPR-Cas systems, which could mitigate potential harmful phage infections. Overall, this study reveals the contemporary beneficial relationship between phages and prokaryotic hosts in activated sludge microbiomes and the potential benefits of symbiotic phages on floc stability.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Tailoring Microbial Fitness Through Computational Steering and CRISPRi-Driven Robustness Regulation

The widespread application of genetically modified microorganisms (GMMs) across diverse sectors underscores the pressing need for robust strategies to mitigate the risks associated with their potential uncontrolled escape. This study merges computational modeling with CRISPR interference (CRISPRi) to refine GMM metabolic robustness. Utilizing ensemble modeling, we achieved high-throughput in silico screening for enzymatic targets susceptible to expression alterations. Translating these insights, we developed functional CRISPRi, boosting fitness control via multiplexed gene knockdown. Our method, enhanced by an insulator-improved gRNA structure and an off-switch circuit controlling a compact Cas12m, resulted in rationally engineered strains with escape frequencies below National Institutes of Health standards. The effectiveness of this approach was confirmed under various conditions, showcasing its ability for secure GMM management. This research underscores the resilience of microbial metabolism, strategically modifying key nodes to halt growth without provoking significant resistance, thereby enabling more reliable and precise GMM control. A record of this paper's transparent peer review process is included in the supplemental information.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Zymomonas mobilis : bringing an ancient human tool into the genomic era

Zymomonas mobilis is an ethanologenic bacterium that has been used for over 1500 years to produce alcoholic beverages. Recently, this microbe has become a top candidate for biofuel production due to its efficient metabolism. Z. mobilis is being developed to utilize lignocellulosic biomass as a feedstock and synthesize a range of valuable chemicals and fuels. Genetic and metabolic engineering strategies are crucial to reach these goals. Recent advances include genome engineering, CRISPR editing, and CRISPRi knockdown of genes. Metabolic engineering has enabled redirection of carbon from the natural product ethanol to chemicals such as 2,3-butanediol and polyhydroxybutyrate. Finally, the approaches summarized here will streamline the development of Z. mobilis as an industrial chassis for sustainable liquid fuels and chemicals.

Boismier, Emma C. [Michigan State Univ., East Lans↗

DOE OSTI2025

Two deeply conserved non-coding sequences control PLETHORA1/2 expression and coordinate embryo and root development

Conserved non-coding sequences (CNSs) are integral elements of transcriptional regulation. Transcriptional tuning of PLETHORA (PLT) genes that encode master regulators of plant development is vital for embryogenesis and meristematic function. However, how the expression of PLT genes is modulated through CNSs remains unclear. Through motif-based mining of upstream sequences in 120 angiosperm genomes, we identified 21 conserved and lineage-specific CNSs, two of which are unusually long, similar, and colinear within eudicots. Using Arabidopsis thaliana, we demonstrate that these two deeply conserved elements, which we named BOX1 and BOX2, control PLT1 and PLT2 expression. CRISPR mutants within these elements specifically reduced PLT expression levels, and reporter lines revealed that deletion of either or both BOXes altered and/or abrogated the PLT2 expression pattern in the root tip, affecting the ability to rescue the plt1 plt2 double mutant. We further show that the influence of these elements on expression patterns is already exerted during embryogenesis and functional in the context of the early embryo. Finally, we reveal the existence of a BOX-mediated autoregulatory feedback loop that, in large part, explains CNS influence on expression patterns. We thus uncover a transcriptional mechanism by which genes encoding master regulators of embryo and root meristem development are regulated.

PLETHORA↗

DOE OSTI2025

Nickase fidelity drives EvolvR-mediated diversification in mammalian cells

Abstract In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, existing genomic diversification modalities for mammalian genomic loci exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to most missense mutations. Here, we engineer CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within genomic loci in mammalian cells. We demonstrate that EvolvR generates both transition and transversion mutations throughout a mutation window of at least 40 bp and implement EvolvR to evolve previously unreported drug-resistantMAP2K1variants via substitutions not achievable with deaminases. Moreover, we discover that the nickase’s mismatch tolerance limits EvolvR’s mutation window and substitution biases in a gRNA-specific fashion. To compensate for gRNA-to-gRNA variability in mutagenesis, we maximize the number of gRNA target sequences by incorporating a PAM-flexible nickase into EvolvR. Finally, we find a strong correlation between predicted free energy changes underlying R-loop formation and EvolvR’s performance using a given gRNA. The EvolvR system diversifies all four nucleotides to enable the evolution of mammalian cells, while nuclease and gRNA-specific properties underlying nickase fidelity can be engineered to further enhance EvolvR’s mutation rates.

Science & Technology - Other Topics↗

DOE OSTI2025

A global soil plasmidome resource unveils functional and ecological roles of plasmids in soil microbiomes

Plasmids play significant roles in microbial adaptation to ecosystems, yet their dynamics remain poorly understood due to identification challenges. We present the Global Soil Plasmidome Resource (GSPR), a comprehensive dataset of 98,728 plasmid sequences amassed from 6860 terrestrial microbial communities and isolates. We explore this resource through various computational approaches, including phylogenetic diversity analysis, host prediction, and extensive functional annotation, to understand the contribution of plasmids to the genetic and functional diversity in soil, correlating these findings with sample type, as well as the soil habitat they were retrieved from. Our analysis reveals insights into plasmid-encoded functions such as effector modules, quorum sensing, and stress resistance, which may contribute to their persistence and microbial adaptation in soil. Furthermore, CRISPR analysis suggests a prevalent role of these elements related to intra-plasmid competition. By contrasting plasmids from cultivated and uncultivated organisms, we identify important functions that expand existing knowledge of plasmid roles in these habitats. This study represents a notable step forward in elucidating plasmid diversity and function within soil microbiomes and establishes a foundational framework for exploring their roles in natural environments.

Fiamenghi, Mateus B↗

DOE OSTI2025

Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida

Advances in genome engineering have improved our ability to perturb microbial metabolic networks, yet bioproduction campaigns often struggle with parsing complex metabolic datasets to efficiently enhance product titers. We address this challenge by coupling laboratory automation with machine learning to systematically optimize the production of isoprenol, a sustainable aviation fuel precursor, in Pseudomonas putida. The simultaneous downregulation through CRISPR interference of combinations of up to four gene targets, guided by machine learning, permitted us to increase isoprenol titer 5-fold in six consecutive design-build-test-learn cycles. Moreover, machine learning enabled us to swiftly explore a vast experimental design space of 800,000 possible combinations by strategically recommending approximately 400 priority constructs. High-throughput proteomics allowed us to validate CRISPRi downregulation and identify biological mechanisms driving production increases. Our work demonstrates that ML-driven automated design-build-test-learn cycles, when combined with rigorous data validation, can rapidly enhance titers without specific biological knowledge, suggesting that it can be applied to any host, product, or pathway.

Carruthers, David N↗

DOE OSTI2025

Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis

Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation1. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

Weiss, Trevor↗

DOE OSTI2025

Transcription factor binding divergence drives transcriptional and phenotypic variation in maize

Regulatory elements are essential components of plant genomes that have shaped the domestication and improvement of modern crops. However, their identity, function and diversity remain poorly characterized, limiting our ability to harness their full power for agricultural advances using induced or natural variation. Here, in this study, we mapped transcription factor (TF) binding for 200 TFs from 30 families in two distinct maize inbred lines historically used in maize breeding. TF binding comparison revealed widespread differences between inbreds, driven largely by structural variation, that correlated with gene expression changes and explained complex quantitative trait loci such as Vgt1, an important determinant of flowering time, and DICE, an herbivore resistance enhancer. CRISPR–Cas9 editing of TF binding regions validated the function and structure of regulatory regions at various loci controlling plant architecture and biotic resistance. Our maize TF binding catalogue identifies functional regulatory regions and enables collective and comparative analysis, highlighting its value for agricultural improvement.

Galli, Mary [Rutgers Univ., Piscataway, NJ (United↗

DOE OSTI2025

CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

An expanded registry of candidate cis -regulatory elements

Mammalian genomes contain millions of regulatory elements that control the complex patterns of gene expression. Previously, the ENCODE consortium mapped biochemical signals across hundreds of cell types and tissues and integrated these data to develop a registry containing 0.9 million human and 300,000 mouse candidate cis-regulatory elements (cCREs) annotated with potential functions. Here we have expanded the registry to include 2.37 million human and 967,000 mouse cCREs, leveraging new ENCODE datasets and enhanced computational methods. This expanded registry covers hundreds of unique cell and tissue types, providing a comprehensive understanding of gene regulation. Functional characterization data from assays such as STARR-seq, massively parallel reporter assay, CRISPR perturbation and transgenic mouse assays have profiled more than 90% of human cCREs, revealing complex regulatory functions. We identified thousands of novel silencer cCREs and demonstrated their dual enhancer and silencer roles in different cellular contexts. Integrating the registry with other ENCODE annotations facilitates genetic variation interpretation and trait-associated gene identification, exemplified by the identification of KLF1 as a novel causal gene for red blood cell traits. This expanded registry is a valuable resource for studying the regulatory genome and its impact on health and disease.

Moore, Jill E. [Univ. of Massachusetts, Worchester↗

DOE OSTI2025

High-density CRISPRi screens reveal diverse routes to improved acclimation in cyanobacteria

Cyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the acclimation of these microbes, it remains poorly understood and challenging to rewire. Here, this study uses a high-density, genome-wide CRISPR interference screen to examine the influence of gene-specific transcriptional variation on the growth of Synechococcus sp. PCC 7002 under environmental extremes. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Transcriptional repression of genes for core subunits of the NDH-1 complex, which are important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. Most genes with fitness-improving knockdowns were distinct to each light color, and dual-target transcriptional repression produced nonadditive effects. Findings reveal diverse routes to improved acclimation in cyanobacteria (e.g., attenuation of genes involved in CO 2 uptake, light harvesting, translation, and purine metabolism) and provide an approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Mechanism-guided engineering of a minimal biological particle for genome editing

The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

metagRoot: a comprehensive database of protein families associated with plant root microbiomes

The plant root microbiome is vital in plant health, nutrient uptake, and environmental resilience. To explore and harness this diversity, we present metagRoot, a specialized and enriched database focused on the protein families of the plant root microbiome. MetagRoot integrates metagenomic, metatranscriptomic, and reference genome-derived protein data to characterize 71 091 enriched protein families, each containing at least 100 sequences. These families are annotated with multiple sequence alignments, CRISPR elements, hidden Markov models, taxonomic and functional classifications, ecosystem and geolocation metadata, and predicted 3D structures using AlphaFold2. MetagRoot is a powerful tool for decoding the molecular landscape of root-associated microbial communities and advancing microbiome-informed agricultural practices by enriching protein family information with ecological and structural context. The database is available at https://pavlopoulos-lab.org/metagroot/ or https://www.metagroot.org.

Chasapi, Maria N↗

DOE OSTI2024

Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION1 knockout for biofuel production

Abstract The aviation industry’s growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays) and soybean (Glycine max) to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs) from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multiomics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to 3 d earlier at 10 °C. However, these alterations led to a tradeoff between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.

Rasoul, Amira (ORCID:0009000714290175)↗