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Results for “Genome engineering”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

The Cas9 endonuclease is widely used for genome engineering applications by programming its single-guide RNA, and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer, we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Approaches to genetic tool development for rapid domestication of non-model microorganisms

Abstract Non-model microorganisms often possess complex phenotypes that could be important for the future of biofuel and chemical production. They have received significant interest the last several years, but advancement is still slow due to the lack of a robust genetic toolbox in most organisms. Typically, “domestication” of a new non-model microorganism has been done on an ad hoc basis, and historically, it can take years to develop transformation and basic genetic tools. Here, we review the barriers and solutions to rapid development of genetic transformation tools in new hosts, with a major focus on Restriction-Modification systems, which are a well-known and significant barrier to efficient transformation. We further explore the tools and approaches used for efficient gene deletion, DNA insertion, and heterologous gene expression. Finally, more advanced and high-throughput tools are now being developed in diverse non-model microbes, paving the way for rapid and multiplexed genome engineering for biotechnology.

09 BIOMASS FUELS↗
DOE OSTI2019

Data from Highly Efficient Single-Pot Scarless Golden Gate Assembly

Golden Gate assembly is one of the most widely used DNA assembly methods due to its robustness and modularity. However, despite its popularity, the need for BsaI-free parts, the introduction of scars between junctions, as well as the lack of a comprehensive study on the linkers hinders its more widespread use. Here, we first developed a novel sequencing scheme to test the efficiency and specificity of 96 linkers of 4-bp length and experimentally verified these linkers and their effects on Golden Gate assembly efficiency and specificity. We then used this sequencing data to generate 200 distinct linker sets that can be used by the community to perform efficient Golden Gate assemblies of different sizes and complexity. We also present a single-pot scarless Golden Gate assembly and BsaI removal scheme and its accompanying assembly design software to perform point mutations and Golden Gate assembly. This assembly scheme enables scarless assembly without compromising efficiency by choosing optimized linkers near assembly junctions.

Conversion↗
DOE OSTI2017

A Platform for Genome-Scale Design, Redesign, and Optimization of Bacterial Systems

This project was a collaboration between the Gill and Knight labs at University of Colorado Boulder, the Arkin lab at University of California Berkeley, and the Maness group at the National Renewable Energy Laboratory. There were two main objectives for this project: Objective 1: Integrated, genome-scale strategy for “chassis” strain design and construction. Objective 2: Genome “re-design” framework using multiplex synbio and genome-engineering technologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Bioproduction and Evaluation of Renewable Butyl Acetate as a Desirable Bioblendstock for Diesel Fuel

The objective of this project was to develop an integrated bioprocess for efficient butyl acetate (BA) production through strain development using systematic genome engineering and MicroNiche Engineering™ technology (MNE). BA's potential as a bioblendstock for diesel fuel was evaluated through droplet experiments and Mixing Controlled Compression Ignition (MCCI) engine testing. The project aimed to achieve 30 g/L BA production with a yield of 0.4 g/g and a productivity of 0.6 g/L/h. The suitable composition of the blended fuel (BA/biodiesel) would be determined for MCCI engine application.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2024

Design, Synthesis, and Validation: Genome Scale Optimization of Energy Flux through Compartmentalized Metabolic Networks in a Model Photosynthetic Eukaryotic Microbe (Final Report)

Photosynthetic organisms have recently gained considerable attention for a role in development of renewable energy sources. Genome-enabled systems biology methodology and modeling, coupled with high throughput genome engineering strategies, present opportunities to develop sustainable and economical applications such as fuel production within the next 10 to 15 years. However, optimization of light-driven metabolism for biomass or biofuel production will require significant advances in methodological throughput as well as improvements in detailed systems biology understanding of photosynthetic processes and cellular metabolism. The ability of diatoms to thrive in upwelling-induced, periodically nutrient-rich conditions makes them the base for the world’s shortest and most energy-efficient food webs. Diatom photosynthesis is estimated to account for between 25% and 40% of the 45-50 billion tons of organic carbon fixed annually in the sea.

60 APPLIED LIFE SCIENCES↗
NASA NTRSDate not supplied

Selection Factors for Space Crops

NASA is actively researching space crop production to determine its potential to contribute to food system security on long duration missions beyond Low Earth Orbit. Our near-term focus is on nutrient and variety supplementation of prepackaged food with fresh produce that requires little or no processing. The longer-term goal is caloric replacement to become less dependent on Earth, and this will require cultivation of staple crops, processing and cooking equipment, integration with spacecraft air, water, and power systems, and automation. There are numerous technology and knowledge gaps remaining for sustainable space crop production systems, but one high-impact area is in the development of crops specifically customized to meet the needs of controlled environment crop production, astronaut health and well-being, and space-unique environments. Modern crop breeding and genome engineering tools are allowing for rapid development of new genotypes with incredible specificity. Targeted aspects to optimize crops for space have been identified and characterized into five categories: plant growth and development, plant physiology, produce nutrition, produce organoleptic acceptability, and postharvest characteristics. Within each category there are several targets that further the development of crop production systems for spaceflight, such as crop size and harvest index, tolerance to specific environmental stresses, optimizing target nutrients that are low or degrade in the packaged diet, maintenance time requirements, and less indigestible structural material. NASA-funded PIs are already beginning to develop candidate crops, and spaceflight testing and validation of novel space crops is on the horizon. Crops developed for space also have the potential to benefit terrestrial controlled environment agriculture crop production systems. This research was supported by NASA’s Space Biology and Human Research Programs.

Space Crop Production↗
NASA NTRSDate not supplied

Selection Factors for Space Crops

NASA is actively researching space crop production to determine its potential to contribute to food system security on long duration missions beyond Low Earth Orbit. Our near-term focus is on nutrient and variety supplementation of prepackaged food with fresh produce that requires little or no processing. The longer-term goal is caloric replacement to become less dependent on Earth, and this will require cultivation of staple crops, processing and cooking equipment, integration with spacecraft air, water, and power systems, and automation. There are numerous technology and knowledge gaps remaining for sustainable space crop production systems, but one high-impact area is in the development of crops specifically customized to meet the needs of controlled environment crop production, astronaut health and well-being, and space-unique environments. Modern crop breeding and genome engineering tools are allowing for rapid development of new genotypes with incredible specificity. Targeted aspects to optimize crops for space have been identified and characterized into five categories: plant growth and development, plant physiology, produce nutrition, produce organoleptic acceptability, and postharvest characteristics. Within each category there are several targets that further the development of crop production systems for spaceflight, such as crop size and harvest index, tolerance to specific environmental stresses, optimizing target nutrients that are low or degrade in the packaged diet, maintenance time requirements, and less indigestible structural material. NASA-funded PIs are already beginning to develop candidate crops, and spaceflight testing and validation of novel space crops is on the horizon. Crops developed for space also have the potential to benefit terrestrial controlled environment agriculture crop production systems. This research was supported by NASA’s Space Biology and Human Research Programs.

Space Crop Production↗
DOE OSTI2024

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Constructs and methods for genome editing and genetic engineering of fungi and protists

Provided herein are constructs for genome editing or genetic engineering in fungi or protists, methods of using the constructs and media for use in selecting cells. The construct include a polynucleotide encoding a thymidine kinase operably connected to a promoter, suitably a constitutive promoter; a polynucleotide encoding an endonuclease operably connected to an inducible promoter; and a recognition site for the endonuclease. The constructs may also include selectable markers for use in selecting recombinations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Constructs and methods for genome editing and genetic engineering of fungi and protists

Provided herein are constructs for genome editing or genetic engineering its fungi or protists, methods of using the constructs and media for use in selecting cells. The construct include a polynucleotide encoding a thymidine kinase operably connected to a promoter, suitably a constitutive promoter; a polynucleotide encoding an endonuclease operably connected to an inducible promoter; and a recognition site for the endonuclease. The constructs may also include selectable markers for use in selecting recombinations.

Hittinger, Christopher Todd↗
DOE OSTI2022

Improved genome editing by an engineered CRISPR-Cas12a

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis - and trans -DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genome-scale Design and Engineering of Non-model Yeast Organisms for Production of Biofuels and Bioproducts

The overall goal of this project was to develop genome-scale design and engineering tools for two non-model yeast organisms including Rhodotorula toruloides and Issatchenkia orientalis to produce high-levels of fatty acids-derived products and organic acids, respectively. The project was performed between 9/15/2017 and 9/14/2024 (the last two-years were no-cost extensions). The team consisted of Huimin Zhao (Lead PI) and Christopher Rao (Co-PI) from the University of Illinois at Urbana-Champaign (UIUC), Costas Maranas (Co-PI) from the Pennsylvania State University, Joshua Rabinowitz (Co-PI) and Martin Wuhr (Co-PI) from Princeton University, and Yasuo Yoshikuni (Co-PI) from the DOE Joint Genome Institute. The team has made great progress in both tool development and fundamental understanding of these two non-model yeasts. In total, there were 40 research publications (one of them is still under review) and one patent application as well as numerous oral presentations.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

09 BIOMASS FUELS↗
DOE OSTI2016

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off‐target pathways through metabolite signalling

Summary The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE ( FPS ) and SQUALENE SYNTHASE ( SQS ) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both ( CN ) genomes to promote squalene biosynthesis. SQS levels were ~4300‐fold higher in C and CN lines than in N, but all accumulated ~150‐fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65–120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32‐fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000‐fold in CN ; 7784 transcripts by up to 1400‐fold in N; and 5224 transcripts by as much as 2200‐fold in C. Transporter‐related transcripts were induced, and cell cycle‐associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT ‐ PCR . The mechanism underlying these large changes likely involves metabolite‐mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗