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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Data for Creating Yellow Seed Camelina sativa with Enhanced Oil Accumulation by CRISPR-Mediated Disruption of Transparent Testa 8

Camelina ( Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

Biofuels↗
DOE OSTI2025

Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing

Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infectEscherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targetedE. colicells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.

Science & Technology - Other Topics↗
DOE OSTI2024

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ~20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a–RNA–DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Populus VariantDB v3.2 facilitates CRISPR and functional genomics research

The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A miniature CRISPR–Cas10 enzyme confers immunity by inhibitory signalling

Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signalling that share mechanistic features with human antiviral systems1. In these pathways, including cyclic oligonucleotide-based antiphage signalling systems (CBASSs) and type III CRISPR systems in bacteria and cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS–STING) in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response2, 3–4. Here, in an unexpected inversion of this process, we show that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that represses a toxic effector. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing the spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TMβ. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TMβ-mediated cell death due to inner membrane collapse. These findings unveil a powerful defence strategy against virus-mediated immune suppression, expanding our understanding of the role of oligonucleotides in immunity.

Doherty, Erin E↗
DOE OSTI2025

Packaged delivery of CRISPR–Cas9 ribonucleoproteins accelerates genome editing

Effective genome editing requires a sufficient dose of CRISPR–Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy, we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least 2-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR–Cas9 RNPs required for experimental or clinical genome editing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

CRISPR interference↗
DOE OSTI2025

Characterizing and controlling CRISPR repair outcomes in nondividing human cells

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.

Ramadoss, Gokul N. [Gladstone Institutes, San Fran↗
DOE OSTI2025

Single-molecule live-cell RNA imaging with CRISPR–Csm

Abstract Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR–Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual nativeNOTCH2andMAP1Btranscripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation ofNOTCH2mRNA at the endoplasmic reticulum and directional transport ofMAP1BmRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Directed evolution expands CRISPR–Cas12a genome-editing capacity

CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

Ma, Enbo↗
DOE OSTI2026

Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing

CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST (VchCAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors and increased editing efficiency by 55.2-fold in E. coli, 5.6-fold in Pseudomonas putida, and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting VchCAST activity and enhances its efficiency as a bacterial genome editor.

Song, Leo C T↗
DOE OSTI2025

Leveraging CRISPR Cas9 RNPs and Cre- loxP in Picochlorum celeri for generation of field deployable strains and selection marker recycling

As new highly productive strains of algae are discovered and developed to meet the energy, chemical, and food requirements of the future, genetic engineering of those strains in a manner that yields deployable transformants is paramount. This study introduces the novel CRoxP ($\underline{\textrm{C}}$$\textrm{as9}$ $\underline{\textrm{R}}$$\textrm{NPs}$ coupled with an inducible $\underline{\textrm{CR}}$$\textrm{e}$-$\textrm{l}\underline{\textrm{oxP}}$) system for rapid generation of marker- and transgene-free strains of Picochlorum celeri. The CRoxP system allows reuse of selection markers without Cas9 expression in vivo, eliminating many of the bottlenecks associated with conventional CRISPR Cas9 use for precise genome editing. In P. celeri, transformants were generated with a turnaround time as short as 21 days between transformation and being ready for another round of transformation with the same selection marker by using the CRoxP system. As a use-case for CRoxP, depigmented strains of P. celeri were generated by multiplexed Cas9 disruption of major LHCII genes followed by either a second round of LHCII targeting, or knockout of an LHCI gene. One transformant tested in flask culture (R6) exhibited similar biomass production to the wild type with 46% less Chl a + b on a biomass basis. In photobioreactors and under diel light simulating a solar day, a transformant (LhcBM31) exhibited 34 g AFDW m –2 d –1 with 54% less Chl a + b on a biomass basis vs. wild type.

09 BIOMASS FUELS↗
DOE OSTI2025

CRISPR-CARB/nocap

Network Optimization and Causal Analysis of Perturb-seq (NOCAP) is a software package for causal inference of gene regulation networks using data from perturb-seq.

George, August↗