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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Data for "Enhancing Lipid Production in Plant Cells through Automated High-Throughput Genome Engineering and Phenotyping"

Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

AI/ML↗
DOE OSTI2025

Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping

Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. Furthermore, these innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Unleashing the capacity of Rhodococcus for converting lignin into lipids

Bioconversion of bioresources/wastes (e.g., lignin, chemical pulping byproducts) represents a promising approach for developing a bioeconomy to help address growing energy and materials demands. Rhodococcus, a promising microbial strain, utilizes numerous carbon sources to produce lipids, which are precursors for synthesizing biodiesel and aviation fuels. However, compared to chemical conversion, bioconversion involves living cells, which is a more complex system that needs further understanding and upgrading. Various wastes amenable to bioconversion are reviewed herein to highlight the potential of Rhodococci for producing lipid-derived bioproducts. In light of the abundant availability of these substrates, Rhodococcus’ metabolic pathways converting them to lipids are analyzed from a “beginning-to-end” view. Based on an in-depth understanding of microbial metabolic routes, genetic modifications of Rhodococcus by employing emerging tools (e.g., multiplex genome editing, biosensors, and genome-scale metabolic models) are presented for promoting the bioconversion. Co-solvent enhanced lignocellulose fractionation (CELF) strategy facilitates the generation of a lignin-derived aromatic stream suitable for the Rhodococcus’ utilization. Novel alkali sterilization (AS) and elimination of thermal sterilization (ETS) approaches can significantly enhance the bioaccessibility of lignin and its derived aromatics in aqueous fermentation media, which promotes lipid titer significantly. In order to achieve value-added utilization of lignin, biodiesel and aviation fuel synthesis from lignin and lipids are further discussed. In conclusion, the possible directions for unleashing the capacity of Rhodococcus through synergistically modifying microbial strains, substrates, and fermentation processes are proposed toward a sustainable biological lignin valorization.

09 BIOMASS FUELS↗
DOE OSTI2025

Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis

Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation1. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

Weiss, Trevor↗
DOE OSTI2021

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production

Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies

Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Polysaccharide utilization loci in Bacteroides determine population fitness and community-level interactions

Polysaccharide utilization loci (PULs) are co-regulated bacterial genes that sense nutrients and enable glycan digestion. Human gut microbiome members, notably Bacteroides, contain numerous PULs that enable glycan utilization and shape ecological dynamics. To investigate the role of PULs on fitness and inter-species interactions, we develop a CRISPR-based genome editing tool to study 23 PULs in Bacteroides uniformis (BU). BU PULs show distinct glycan-degrading functions and transcriptional coordination that enables the population to adapt upon loss of other PULs. Exploiting a BU mutant barcoding strategy, we demonstrate that in vitro fitness and BU colonization in the murine gut are enhanced by deletion of specific PULs and modulated by glycan availability. PULs mediate glycan-dependent interactions with butyrate producers that depend on the degradation mechanism and glycan utilization ability of the butyrate producer. Thus, PULs determine community dynamics and butyrate production and provide a selective advantage or disadvantage depending on the nutritional landscape.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4 + T-cells

CRISPR–Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4 + T-cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira

ABSTRACT Leptospirosis, caused by pathogenic bacteria from the genusLeptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation ofLeptospirahas been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal toLeptospiracells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3′ end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenicLeptospirafrom multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulateLeptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis. IMPORTANCE Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenicLeptospiraspp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system toLeptospiraenhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique inLeptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in theLeptospira borgpeterseniibackground, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms ofLeptospiraand the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Microbiology↗
DOE OSTI2023

A landing pad system for multicopy gene integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. Furthermore, this study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A Landing Pad System for Multicopy Gene Integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

Fatma, Zia↗
DOE OSTI2023

Topologically associating domain boundaries are required for normal genome function

Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1,2,3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11–80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Topologically Associating Domain Boundaries are Commonly Required for Normal Genome Function

Topologically associating domain (TAD) boundaries are thought to partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotype, but the overall extent to which this occurs remains unknown. Here we show that TAD boundary deletions commonly disrupt normal genome function in vivo . We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80kb in size) from the genome in mice. All deletions examined resulted in at least one detectable molecular or organismal phenotype, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. For 5 of 8 (62%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and suggest that noncoding deletions affecting TAD boundaries should be carefully considered for potential pathogenicity in clinical genetics screening.

Rajderkar, Sudha↗
DOE OSTI2024

Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2021

A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9 s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Gene editing to enhance biotic stress tolerance in sugarcane

Sugarcane (Saccharum spp.) contributes approximately 80% of global sugar production and 40% of biofuel while serving as a promising feedstock for bioproducts (Brant et al., 2025). However, productivity faces mounting challenges from biotic threats including fungal, viral and bacterial diseases (Rott, 2018). Traditional breeding approaches are severely constrained by sugarcane’s complex polyploid genome containing 10–12 copies of "hom(oe)ologous genes within an approximately 10 Gb genome (Healey et al., 2024), extending breeding cycles to 12–15 years. Genome editing has revolutionized crop improvement by enabling targeted modifications without necessarily introducing foreign DNA, potentially circumventing regulatory hurdles while accelerating variety development (Li et al., 2022a). For sugarcane, these technologies offer unprecedented opportunities to enhance biotic stress resilience while improving yield and quality. This article examines current progress and prospects for developing biotic stress-tolerant sugarcane through gene editing, emphasizing technical advances, promising gene targets, and strategic approaches for trait stacking.

CRISPR-Cas systems↗