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At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Development, optimization, and application of an episomal plasmid system for Rhodotorula toruloides

Rhodotorula toruloides is an emerging oleaginous yeast with strong potential as a microbial cell factory for the production of acetyl-CoA-derived bioproducts. However, engineering of this organism has been limited by the absence of a functional episomal plasmid system, a foundational genetic tool for rapid gene expression, pathway testing, and CRISPR-based genome engineering. Here, we report the first episomal plasmid system for R. toruloides . Through systematic screening of candidate autonomously replicating sequences (ARSs) from diverse sources, we identified multiple functional ARS elements and selected C63F4, a fragment derived from Contig 63 of R. toruloides CBS14, because of its stable performance. The resulting pC63F4 plasmid was maintained episomally, supported GFP reporter expression, exhibited a copy number of 2.39 ± 0.13, and showed good stability during long term cultivation. To overcome poor transformation efficiency, we developed a Cre- loxP -mediated in vivo re-circularization strategy that enabled reliable delivery of the episomal plasmid. Using this improved system, we demonstrated functional episomal expression of metabolic engineering genes and multi-gene pathways for the production of triacetic acid lactone, fatty alcohols, and limonene. Finally, we leveraged this platform to establish a redesigned CRISPR system that enables seamless genome editing in R. toruloides for the first time, while also simplifying marker recycling. Together, this work establishes a long-needed episomal plasmid platform and associated CRISPR toolkit that will accelerate metabolic engineering, synthetic biology, and fundamental studies in R. toruloides .

CRISPR-Cas9↗
DOE OSTI2023

Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes

The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. Furthermore, this work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii, Stutzerimonas stutzeri (Pseudomonas stutzeri), and a new isolate of Klebsiella variicola. All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A genome resource for green millet Setaria viridis enables discovery of agronomically valuable loci

Abstract Wild and weedy relatives of domesticated crops harbor genetic variants that can advance agricultural biotechnology. Here we provide a genome resource for the wild plant green millet ( Setaria viridis ), a model species for studies of C 4 grasses, and use the resource to probe domestication genes in the close crop relative foxtail millet ( Setaria italica ). We produced a platinum-quality genome assembly of S. viridis and de novo assemblies for 598 wild accessions and exploited these assemblies to identify loci underlying three traits: response to climate, a ‘loss of shattering’ trait that permits mechanical harvest and leaf angle, a predictor of yield in many grass crops. With CRISPR–Cas9 genome editing, we validated Less Shattering1 ( SvLes1 ) as a gene whose product controls seed shattering. In S. italica , this gene was rendered nonfunctional by a retrotransposon insertion in the domesticated loss-of-shattering allele SiLes1-TE (transposable element). This resource will enhance the utility of S. viridis for dissection of complex traits and biotechnological improvement of panicoid crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Apomixis in Farmers’ Fields: Overview, Case Studies from Forage Grasses and Considerations for Future Apomictic Crops

Apomixis occurs naturally in several commercially important species from diverse plant families. While in some of these species apomixis is yet to be exploited in breeding schemes aimed at fixing heterosis, genetic progress and cultivar development, in other species apomixis has been integrated at different stages of breeding. Some of the most relevant examples come from the subfamily Panicoideae, the second largest subfamily of the Poaceae, and are the main focus of this review. The subfamily encompasses many tropical and sub-tropical grasses and grains of worldwide economic importance. Apomictic tropical forages are prime examples of how apomixis can be used and exploited in the development of marketable cultivars, which are essential to the meat and milk production industries globally. The main commercial forages used as grass pastures covering millions of hectares in tropical and sub-tropical regions are polyploids exhibiting gametophytic apomixis that belong to the genus Urochloa spp. (brachiariagrasses) and to the species Megathyrsus maximus (guineagrass). Buffel grass (Cenchrus ciliaris) and Paspalum spp. are other important apomictic forages bred and used in these regions. Breeding involves large germplasm collections from the centers of origin of the species, and for most of them, sexually reproducing diploid plants have been found. Chromosomically duplicated plants that maintain sexual reproduction are used in crosses with apomictic genotypes for the development and selection of cultivars to be marketed or used as progenitors in subsequent breeding cycles. The peculiarities of each genus/species breeding programs, the cultivars obtained from these programs, and the impact of use of marker assisted selection in cultivar development are presented. In addition, the test or implementation of new technologies such as high throughput phenotyping, and the use of machine learning methods for trait prediction and genomic selection are positively impacting the selection and speed of development of new polyploid apomictic cultivars. Furthermore, genetic transformation techniques, including genome editing, provide an additional layer for design of tailor-made, customer-oriented cultivars.

Cenchrus↗
DOE OSTI2024

Development of modular expression across phylogenetically distinct diazotrophs

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here, we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes

Abstract RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes

Abstract CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

Kammerdiener, Elise K. [Oak Ridge National Laborat↗
DOE OSTI2021

Modeling temporal and hormonal regulation of plant transcriptional response to wounding

Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Distant-acting Enhancers in Development and Disease

Multiple converging lines of evidence from experimental and human genetic studies indicate that both common and rare sequence variants involving enhancers play major roles in Mendelian and complex human disease phenotypes. We use a combination of sequence-based molecular approaches including chromatin mapping and synthesis-enabled modification of enhancer sequences, large-scale transgenic mouse studies (http://enhancer.lbl.gov), and CRISPR genome editing in the mouse model to study the in vivofunction of enhancers in developmental, evolutionary, and disease-related processes. Using examples from our ongoing work, I will illustrate how these methods provide insight into the function and evolution of distant-acting regulatory sequences and offer a starting point for understanding their role in human disease. I will discuss how improved in vivo mouse assays can be used to study candidate pathogenic human mutations in enhancers at scale, revealing the extent to which human genetic studies alone can be insufficient to identify truly causal mutations in enhancers. I will also show how such assays can be used to explore the sensitivity of enhancers that are extremely conserved ("ultraconserved") in evolution to mutations, revealing a remarkable functional robustness to sequence changes that contrasts with apparent evolutionary constraints.

Visel, A↗
DOE OSTI2023

Haplotype‐resolved genome assembly of Populus tremula × P. alba reveals aspen‐specific megabase satellite DNA

SUMMARY Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717‐1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence‐sensitive analyses. Here we report a telomere‐to‐telomere genome for this hybrid aspen with two chromosome‐scale, haplotype‐resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array‐based and array‐less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus ) but PtaM147‐like sequences occur in LTR‐retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis‐driven research to probe into the function of the abundant and aspen‐specific PtaM147 satellite DNA.

Zhou, Ran↗
DOE OSTI2021

The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases

Tracing the origin of CRISPR-Cas CRISPR-Cas systems have transformed genome editing and other biotechnologies; however, the broader origins and diversity of RNA-guided nucleases have largely remained unexplored. Altae-Tran et al . show that three distinct transposon-encoded proteins, IscB, IsrB, and TnpB, are naturally occurring, reprogrammable RNA-guided DNA nucleases (see the Perspective by Rousset and Sorek). In addition to identifying diverse guide-encoding mechanisms, the authors elucidate the evolutionary relationship between IsrB, IscB, and CRISPR-Cas9. Overall, these newly characterized systems, called OMEGA (for obligate mobile element–guided activity) systems, are found in all domains of life and may be harnessed for biotechnology development. —DJ

Science & Technology - Other Topics↗
DOE OSTI2026

Expanding the genetic toolkit: adenine and cytosine base editors for gene disruption in Aspergillus niger

Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A‧T to G‧C, and cytosine base editors (CBEs), which convert C‧G to T‧A, offer a safer alternative by enabling predictable, target-specific single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger . For the first time, we constructed an ABE in A. niger , achieving up to 80% editing efficiency and inducing predictable A-to-G mutations at the intended intron sites, disrupting gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Leveraging this expanded scope, we established gene disruption approaches by targeting start codons via ABE-mediated A-to-G conversions (ATG-to-GTG and ATG-to-ACG) and CBE-mediated C-to-T conversion (ATG-to-ATA). Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Collectively, the scope-expanding strategies increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.

Aspergillus↗
DOE OSTI2021

CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Background: 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results: We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3 3 -factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4 K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7 G141S , results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions: The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Data for Development, Optimization, and Application of an Episomal Plasmid System for Rhodotorula toruloides

Rhodotorula toruloides is an emerging oleaginous yeast with strong potential as a microbial cell factory for the production of acetyl-CoA-derived bioproducts. However, engineering of this organism has been limited by the absence of a functional episomal plasmid system, a foundational genetic tool for rapid gene expression, pathway testing, and CRISPR-based genome engineering. Here, we report the first episomal plasmid system for R. toruloides . Through systematic screening of candidate autonomously replicating sequences (ARSs) from diverse sources, we identified multiple functional ARS elements and selected C63F4, a fragment derived from Contig 63 of R. toruloides CBS14, because of its stable performance. The resulting pC63F4 plasmid was maintained episomally, supported GFP reporter expression, exhibited a copy number of 2.39 ± 0.13, and showed good stability during long term cultivation. To overcome poor transformation efficiency, we developed a Cre-loxP-mediated in vivo re-circularization strategy that enabled reliable delivery of the episomal plasmid. Using this improved system, we demonstrated functional episomal expression of metabolic engineering genes and multi-gene pathways for the production of triacetic acid lactone, fatty alcohols, and limonene. Finally, we leveraged this platform to establish a redesigned CRISPR system that enables seamless genome editing in R. toruloides for the first time, while also simplifying marker recycling. Together, this work establishes a long-needed episomal plasmid platform and associated CRISPR toolkit that will accelerate metabolic engineering, synthetic biology, and fundamental studies in R. toruloides .

Gene Editing↗