Search NASA⌕ Search

SEARCH · Search NASA

Results for “cas9”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane ( x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I ( MgCh ) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Editorial: Advanced in situ characterization of biological interfaces and materials

Rapid changes are occurring on this dynamic planet–in 2020 anthropogenic mass was reported to not only match but exceed the mass of natural origins (Elhacham et al., 2020). These indications do not bold well for humanity in the coming generations, as we face 2 degree overall temperature increases over the next 5 years, CO 2 is spewing into the atmosphere at the Gigatonne (Gt) rate per year without any signs of mitigation in the foreseeable future, ocean water levels are rising to incredible levels, drinking water availability is disappearing, humanity is heavily reliant on the “drill-and-fill” culture, and two wars are currently being fought in Gaza and Ukraine (Carr et al., 2024). And even with these pressures, humanity continues to eke out impressive scientific and technological achievements in the recent past including mRNA vaccines to counter a global pandemic (Hogan and Pardi, 2022) and the development of CRISPR-Cas9 drugs (Parums, 2024). All these developments are only possible from the incremental methodological improvements taking place presently.

36 MATERIALS SCIENCE↗
DOE OSTI2022

In vivo Functional Characterization of Hydrophilic X2 Modules in the Cellulosomal Scaffolding Protein

As part of free cellulases or scaffolding proteins in cellulosomes, the hydrophilic non-catalytic X2 module is widely distributed in cellulolytic Clostridia or other Firmicutes bacteria. Previous biochemical studies suggest that X2 modules might increase the solubility and substrate binding affinity of X2-bearing proteins. However, their in vivo biological functions remain elusive. Here we employed CRISPR-Cas9 editing to genetically modify X2 modules by deleting the conserved motif (NGNT) from the CipC scaffoldin. Both single and double X2 mutants (X2-N: near the N terminus of CipC; X2-C: near the C terminus of CipC) presented similar stoichiometric compositions in isolated cellulosomes as the wildtype strain (WT). These X2 mutants had an elongated adaptation stage during growth on cellulose compared to cellobiose. Compared to WT, the double mutant ΔX2-NC reduced cellulose degradation by 15% and the amount of released soluble sugars by 63%. Since single X2 mutants did not present such obvious physiological changes as ΔX2-NC, there seems to be a functional redundancy between X2 modules in CipC. The in vivo adhesion assay revealed that ΔX2-NC decreased cell attachment to cellulose by 70% but a weaker effect was also overserved in single X2 mutants. These results highlight the in vivo biological role of X2 in increasing cellulose degradation efficiency by enhancing the binding affinity between cells and cellulose, which provides new perspectives for microbial engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Advances in genetic tools for metabolic engineering of non-conventional yeasts

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Rapid Cis–Trans Coevolution Driven by a Novel Gene Retroposed from a Eukaryotic Conserved CCR4–NOT Component in Drosophila

Young, or newly evolved, genes arise ubiquitously across the tree of life, and they can rapidly acquire novel functions that influence a diverse array of biological processes. Previous work identified a young regulatory duplicate gene in Drosophila, Zeus that unexpectedly diverged rapidly from its parent, Caf40, an extremely conserved component in the CCR4–NOT machinery in post-transcriptional and post-translational regulation of eukaryotic cells, and took on roles in the male reproductive system. This neofunctionalization was accompanied by differential binding of the Zeus protein to loci throughout the Drosophila melanogaster genome. However, the way in which new DNA-binding proteins acquire and coevolve with their targets in the genome is not understood. Here, by comparing Zeus ChIP-Seq data from D. melanogaster and D. simulans to the ancestral Caf40 binding events from D. yakuba, a species that diverged before the duplication event, we found a dynamic pattern in which Zeus binding rapidly coevolved with a previously unknown DNA motif, which we term Caf40 and Zeus-Associated Motif (CAZAM), under the influence of positive selection. Interestingly, while both copies of Zeus acquired targets at male-biased and testis-specific genes, D. melanogaster and D. simulans proteins have specialized binding on different chromosomes, a pattern echoed in the evolution of the associated motif. Using CRISPR-Cas9-mediated gene knockout of Zeus and RNA-Seq, we found that Zeus regulated the expression of 661 differentially expressed genes (DEGs). Our results suggest that the evolution of young regulatory genes can be coupled to substantial rewiring of the transcriptional networks into which they integrate, even over short evolutionary timescales. Our results thus uncover dynamic genome-wide evolutionary processes associated with new genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Protoplast-Based Transient Expression and Gene Editing in Shrub Willow ( Salix purpurea L .)

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Status on Genetic Resistance to Rice Blast Disease in the Post-Genomic Era

Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production, necessitating the development of resistant cultivars through genetic improvement. Breakthroughs in rice genomics, including the complete genome sequencing of japonica and indica subspecies and the availability of various sequence-based molecular markers, have greatly advanced the genetic analysis of blast resistance. To date, approximately 122 blast-resistance genes have been identified, with 39 of these genes cloned and molecularly characterized. The application of these findings in marker-assisted selection (MAS) has significantly improved rice breeding, allowing for the efficient integration of multiple resistance genes into elite cultivars, enhancing both the durability and spectrum of resistance. Pangenomic studies, along with AI-driven tools like AlphaFold2, RoseTTAFold, and AlphaFold3, have further accelerated the identification and functional characterization of resistance genes, expediting the breeding process. Future rice blast disease management will depend on leveraging these advanced genomic and computational technologies. Emphasis should be placed on enhancing computational tools for the large-scale screening of resistance genes and utilizing gene editing technologies such as CRISPR-Cas9 for functional validation and targeted resistance enhancement and deployment. These approaches will be crucial for advancing rice blast resistance, ensuring food security, and promoting agricultural sustainability.

Pedrozo, Rodrigo↗
DOE OSTI2024

Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo

Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2015

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation.

molecular cloning↗
NASA NTRS2019

Mitigations to Reduce the Law of Unintended Consequences for Autonomy and Other Technological Advances

The United Nations states that Earth’s population is expected to reach just under 10 billion people (9.7) by the year 2050. To meet the demands of 10 billion people, governments, multinational corporations and global leaders are relying on autonomy and technological advances to augment and/or accommodate human efforts to meet the required needs of daily living. Genetically modified organisms (GMOs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-edited plants and cloning will be utilized to expand human food supply. Biomimetic implants are expected to improve life expectancy with 3D printed body parts. Human functioning will be extended with wearables and cybernetic implants continuing humanity’s path toward transhumanism. Families will be strengthened with 3 parent households. Disease will surely be eradicated using the CRISPR-CAS9 genetic engineering revolution to design out undesirable human traits and to design in new capabilities. With autonomous cars, trucks and buses on our roads and on-demand autonomous aircraft delivering pizzas, medical prescriptions and groceries in the air and multi-planet vehicles traversing space, utopia will finally arrive! Or will it? All of these powerful, man-made, technological systems will experience unintended consequences with certainty. Instead of over-reacting with hysteria and fear, we should be seeking answers to the following questions - “What skills are required to architect socially-healthy technological systems for 2050?” “What mindsets should we embody to ameliorate hubris syndrome and to build our future technological systems with deliberation, soberness and social responsibility?”

Morris, A. Terry↗
NASA NTRSDate not supplied

Conserved Function of RNA Binding Motif Protein48 (RBM48) and Armadillo Repeat Containing 7 (ARMC7) in Maize and Human U12 Splicing

Splicing of pre-mRNA is fundamental for genes containing introns. Emerging data points to a deeply conserved role of this process in eukaryotic cell differentiation and proliferation. The vast majority of introns termed U2-type introns are spliced by a major spliceosome; however, there also exist rare and more conserved U12-type introns that are spliced by a minor spliceosome. Mutations that disrupt U12 splicing inhibit cell differentiation in both maize endosperm and human blood cells. However, the mechanism underlying this process is not well understood. The maize RNA Binding Motif Protein 48 (RBM48) plays an essential role as a U12 splicing factor and is required for proper maize kernel development. Using human cell lines, CRISPR-Cas9 knockdown of RBM48 demonstrated a conserved function in human U12 intron splicing. RBM48 and Armadillo Repeat Containing 7 (ARMC7) protein interact in both maize and human as part of the activated minor spliceosome. Here we show RBM48 and ARMC7 co-localization in the nucleus of human cell lines. Vertebrate ARMC7 is normally localized in the cytosol, whereas RBM48 is found in the nucleus. Our data suggests that the interaction plays a role in regulating ARMC7 localization or the efficiency of U12 intron splicing. We also performed comprehensive transcriptome profiling and identified a common subset of conserved Minor Intron Containing Genes (MIGs) impacted in both human and maize RBM48 knockout mutants. Of importance, the vast majority of these MIGs are associated with developmental defects in both plants and animals. This suggests that aberrant splicing of these targets has a high likelihood of mediating abnormal cell phenotypes. These data support evolutionarily conserved U12 splicing mechanisms between maize and humans with both RBM48 and ARMC7 having roles in the activated spliceosome.

maize↗
NASA NTRSDate not supplied

Engineered Yeast to Test Risks for Human Exploration of the Lunar Surface

Jessica W. Chau, Natalie N. Ball, Aditya Hindupur, Sandra T. Vu, Jennifer Gil Acevedo, Lauren C. Liddell, Chinmayee Govinda Raj, Gentry, Sergio R. Santa Maria, A. Mark Settles Crewed exploration of the Moon carries risks of long duration exposure to reduced gravity and to deep space radiation. The Lunar Explorer Instrument for space biology Applications (LEIA) investigates the effects of increased radiation and reduced gravity on yeast viability and growth in a Commercial Lunar Payload Services (CLPS) surface mission to the south polar region. LEIA conducts yeast genetics experiments to quantify growth, metabolism, and synthetic biology-enabled production of human nutrients, while taking real time measurements of biologically relevant radiation exposure on the lunar surface. We have engineered beta-carotene producing yeast strains to test the importance of selected DNA damage repair and reactive oxygen species (ROS) defense pathways in mitigating cellular damage from lunar surface radiation. Carotenoids are important dietary antioxidants, and beta-carotene is pro-vitamin A, which is needed for vision and immune function. Carotenoids are sensitive to ROS produced by ionizing radiation and NASA is testing on-demand production of carotenoids from yeast in the BioNutrients space flight experiments. In LEIA, we test the effects of deep space on carotenoid yield in engineered yeast strains. The LEIA team uses CRISPR-Cas9 to engineer yeast to express carotenoids as well as to generate loss-of-function mutations. We are generating mutations in the RAD51 DNA damage repair locus and three genes that function to reduce oxidative damage to the cell: SOD1, SOD2, and TSA1. These strains are tested for carotenoid production using microfluidics and LED spectroscopy to allow remote sensing of cellular growth and carotenoid levels. Keywords: synthetic biology, oxidative stress tolerance, biosensors, space radiation, beyond low Earth orbit, lunar surface, CRISPR/Cas9, gene editing, desiccation, carotenoids.

synthetic biology↗
NASA NTRSDate not supplied

Precision Fermentation: Relieving the Crabtree Effect in S. cerevisiae through Genetic Engineering

Nutrient degradation in the current NASA pre-packaged food system poses significant challenges to crew health in missions beyond low earth orbit as certain vitamins and nutrients have been shown to degrade during extended storage, and resupply and fresh food items cannot be readily provided. BioNutrients(BN) uses synthetic biology in an effort to supplement the NASA food system by delivering in situ production of nutrients and therapeutics produced by genetically engineered microbes. One yeast S. cerevisiae strain utilized in BN is genetically modified to produce two types of antioxidants: zeaxanthin and β-carotene. Zeaxanthin is a non-provitamin A carotenoid and has been implicated in eye health and vision performance, and β-carotene is a provitamin A carotenoid. Microbial production of high-value nutrients can aid our understanding and address the issue of nutrient degradation and loss in the NASA food system. Although S. cerevisiae is an excellent candidate for a long shelf life, on-demand nutrient production system, its space applications are limited by the Crabtree effect where respiration is shut down in favor of fermentative metabolism when glucose is present in high concentrations, causing the release of unwanted ethanol. Current and likely future life support systems are negatively impacted by ethanol and ethanol release is strictly limited.To improve feasibility of using S. cerevisiae, we explored ways to reduce the Crabtree effect, thereby limiting its ethanol production. Specifically, we used CRISPR-cas9 to genetically modify various genes in this pathway

E Zaroff↗
DOE OSTI2022

Multiplex RNA-guided genome engineering

Methods of multiplex genome engineering in cells using Cas9 is provided which includes a cycle of steps of introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are members of a co-localization complex for the target DNA, and introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, and wherein the cycle is repeated a desired number of times to multiplex DNA engineering in cells.

Church, George M.↗
DOE OSTI2021

CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The major trimeric antenna complexes serve as a site for qH-energy dissipation in plants

Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as nonphotochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants’ survival under abiotic stress; however, qH location in the photosynthetic membrane is unresolved. Here, we tested whether we could isolate subcomplexes from plants in which qH was induced that would remain in an energy-dissipative state. Interestingly, we found that chlorophyll (Chl) fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, indicating that they serve as a site for qH-energy dissipation and providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein, and lipid contents of antenna with active or inactive qH but did not detect any evident differences. Finally, we investigated whether specific subunits of the major antenna complexes were required for qH but found that qH was insensitive to trimer composition. Because we previously observed that qH can occur in the absence of specific xanthophylls, and no evident changes in pigments, proteins, or lipids were detected, we tentatively propose that the energy-dissipative state reported here may stem from Chl–Chl excitonic interaction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing

CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. Here, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems.

59 BASIC BIOLOGICAL SCIENCES↗