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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Maize tissue culture, transformation, and genome editing

The importance of maize (Zea mays L.) to global agriculture, world economy, and food security is widely known and increasing. Current maize breeding programs are deeply integrated with recent and rapid technological advances in genome sequencing, computational biology, and new genotyping and phenotyping technologies. Transformation and genome editing capabilities are a central hub to an array of advanced molecular and breeding approaches to crop improvement. Tissue culture and somatic embryogenesis play essential and central roles in maize transformation biology. Synergistic applications of maize transformation, advanced genomics, and genome editing provide a potent interdependent triad for functional genomics research and advanced molecular breeding. Implementation of advanced capabilities to transform maize and conduct genome editing will profoundly influence the dynamics of global agriculture ushering in a new era of varietal development and molecular breeding. With over 60.9 Mha planted in 2019 alone, biotech maize accounts for 31% of the world’s maize production. Up to 10% higher yields are achieved using new varieties generated using genetic modification technologies compared to similar conventional varieties. By extension, the impact new varietal releases developed through genome editing will likely be more significant. Further, advances in transformation and genome editing technologies will facilitate an even wider applicability for the development of new varieties with increasingly complex traits. The introduction of biochemical pathways and the use of synthetic biology have become increasingly more attainable. The future is of genotype independent maize transformation and genome editing, as the working platform will impact world agriculture, global food security, and plant science well into the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Building an expanded bio-based economy through synthetic biology

The field of synthetic biology is essential to the continued development of a bio-based economy, creating mechanisms to supply carbon needed in the economy by both converting existing end-of-life wastes as well as by creating novel, purpose-grown and sustainable feedstocks. Here, we first discuss the near- and long-term resources available for use as feedstocks for bioconversion as well as the output molecules needed for building the foundation of an expanded bio-based economy. We then outline the organisms and phenotypic traits that are needed for the performance-advantaged chassis organisms of the future. Furthermore, we detail the advances, challenges, and opportunities in both microbial and plant synthetic biology relevant to expanding the bio-based economy. Finally, we explore technologies that have and will further enable advances in synthetic biology and the greater bio-based economy.

09 BIOMASS FUELS↗
DOE OSTI2024

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Predicting synthetic mRNA stability using massively parallel kinetic measurements, biophysical modeling, and machine learning

Abstract mRNA degradation is a central process that affects all gene expression levels, though it remains challenging to predict the stability of a mRNA from its sequence, due to the many coupled interactions that control degradation rate. Here, we carried out massively parallel kinetic decay measurements on over 50,000 bacterial mRNAs, using a learn-by-design approach to develop and validate a predictive sequence-to-function model of mRNA stability. mRNAs were designed to systematically vary translation rates, secondary structures, sequence compositions, G-quadruplexes, i-motifs, and RppH activity, resulting in mRNA half-lives from about 20 seconds to 20 minutes. We combined biophysical models and machine learning to develop steady-state and kinetic decay models of mRNA stability with high accuracy and generalizability, utilizing transcription rate models to identify mRNA isoforms and translation rate models to calculate ribosome protection. Overall, the developed model quantifies the key interactions that collectively control mRNA stability in bacterial operons and predicts how changing mRNA sequence alters mRNA stability, which is important when studying and engineering bacterial genetic systems.

Cetnar, Daniel P.↗
DOE OSTI2021

Species- and site-specific genome editing in complex bacterial communities

Knowledge of microbial gene functions comes from manipulating the DNA of individual strains in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here, we describe a generalizable strategy for editing the genomes of specific organisms within microbial communities. We identified genetically tractable bacteria within a community using Environmental Transformation Sequencing (ET-Seq), an approach in which non-targeted transposon integrations are mapped and quantified following community delivery. We next developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. To illustrate the utility of our approach, we selectively edited closely related strains, measured gene fitness, and enriched targeted members within soil and infant gut microbiota. These results establish a new paradigm for targeted community editing relevant to research and applications on medical, agricultural, and industrial microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing

Abstract CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

High-throughput functional variant screens via in vivo production of single-stranded DNA

Significance We report a methodology for the pooled construction of mutants bearing precise genomic sequence variations and multiplex phenotypic characterization of these mutants using next-generation sequencing (NGS). Unlike existing techniques depending on CRISPR-Cas–directed genomic breaks for genome editing, this strategy instead uses single-stranded DNA produced by a retron element for recombineering. This enables libraries of millions of elements to be constructed and offers relaxed design constraints which permit natural DNA or random variation to be used as inputs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2

CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Harnessing Machine Learning for Agnostic Biodetection

The United States’ current list-based approach to biodefense is limited because it considers only known biological agents. Alternatively, developing and adopting a system based on agent-agnostic signatures would enable detection and characterization of both known and novel agents, thereby engendering greater adaptability in the face of an evolving threat landscape. Machine learning (ML) could aid in such a transition, as it can recognize and encode highly complex patterns from multiple input data modalities and has already demonstrated success in many healthcare and defense applications. Functionalizing ML for environmental biodetection requires understanding current technical capabilities. In this article, we provide a systematic review of existing ML platforms and discuss anticipated development efforts needed to achieve effective ML-enabled, agnostic biodetection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Crystal structures of ‘ALternative Isoinformational ENgineered’ DNA in B-form

The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host–guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5'-CTTBPPBBSSZZSAAG, 5'-CTTSSPBZPSZBBAAG and 5'-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, and B:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters of P:Z and B:S pairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further, Z can participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Digital Droplet PCR and Mesocosm-Based Methods to Evaluate Biocontainment Strategies in a Native Soil Ecosystem

Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect the complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees within a complex soil microbiome and differentiation between closely related strains. To this end, we have developed an approach that utilizes soil mesocosms and integrated digital droplet PCR (ddPCR) system to evaluate the efficacy of novel biocontainment strategies. We demonstrate the utility of this approach by modeling contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, strains of Synechocystis sp. PCC 6803 contained via gene knockout or toxin anti-toxin system, and strains of Escherichia coli that are contained via genomic recoding. We also show that ddPCR can be used to detect gene copies from E. coli equal to those counted by traditional spot plating assays. The resultant data demonstrates that this system has broad utility across diverse microbial chassis and biocontainment strategies and enables researchers to track the fate of our contaminating microbe with high sensitivity in the soil. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae ( Xoo ), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Further, through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21 . These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

BETO 2021 Peer Review: Algae Biotechnology Partnership - Technology Area Session: Advanced Algae Systems

Development of advanced genetic and genomic tools for targeted algal metabolic engineering pursuits will be integral to achieving target biomass productivity and, ultimately, the BETO goal of cost-competitive biofuels derived from algal biomass by 2022. However, at present, broad-host-range tool development is currently hindered by strain-specific negative regulatory mechanisms. Indeed, the biological processes controlling algal transcription and translation are subject to complex host regulation, which often presents hurdles for targeted genetic engineering strategies. Advanced genetic approaches offer a means to rewire these regulatory systems and/or introduce novel functionality into algal biocatalysts. Synthetic systems biology approaches also present a means to construct novel genetic regulatory networks and rewire natural biological systems to establish an “orthogonal central dogma,” wherein non-native control elements are introduced into or evolved in host microbes for bypass of host control. To this end, the Algae Biotechnology Partnership aims to develop advanced genetic editing tools and synthetic and orthogonal genetic regulatory systems to enable universal metabolic engineering strategies in top-candidate deployment algal strains. Successful development will ultimately open the door for targeted strain-engineering strategies, aimed at maximizing algal outdoor biomass production, composition, and strain robustness.

biocatalysis↗