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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
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↗
DOE OSTI2022

The Complete Genome Sequence and Structure of the Oleaginous Rhodococcus opacus Strain PD630 Through Nanopore Technology

Rhodococcus bacterial strains are characterized by wide metabolic versatility and extraordinary resistance to environmental stresses. The high versatility and adaptability of Rhodococcus strains is partly related with large and complex genomes (up to 10.1 Mbp) including high genetic redundancy and the presence of several circular and linear (mega)plasmids, which harbour peculiar catabolic and biosynthetic genes. Within Rhodococcus genus, R. opacus strain PD630 is considered a model oleaginous strain for its ability to produce and accumulate lipids (mostly triacyglycerols, TAGs) using different carbon sources, including low-cost and renewable resources such as lignocellulose. Notably, under specific growth conditions, this strain is capable of accumulating up to 80% of its cellular dry weight in TAGs; that is a rare feature in the prokaryotic and eukaryotic kingdoms. Multi-omic approaches have been applied to obtain system-level information about metabolic and regulatory pathways involved in these biosynthetic processes. Novel molecular tools for genome editing (CRISPR/Cas9 and recombineering) have been recently developed highlighting the possible utilization of R. opacus PD630 as synthetic biology platform for lipids production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Systems Engineering of Rhodococcus opacus to Enable Production of Drop-in Fuels from Lignocellulose

Production of drop-in fuels from lignocellulose using Rhodococcus opacus PD630 (hereafter R. opacus) is a challenging goal. During the grant period we have pushed the field forward significantly in several areas of research. Towards the end goal of accelerating the adoption of R. opacus in biofuel production, during the grant period we have expanded the phenotypic characterization of R. opacus grown in single aromatic (model lignocellulosic) compounds or their mixtures, modeling the growth conditions in lignocellulosic biomass. Harnessing the power of adaptive evolution, we produced evolved R. opacus isolates with superior lignin valorization capabilities and identified differentially expressed genes and pathways after adaptation. We used next generation multi-omic techniques such as genomic, transcriptomic, and metabolomic analyses, to identify the catabolic pathways used by R. opacus to degrade aromatic compounds and funnel these degradation products into central metabolism, as well as the aromatic transport genes required for increased tolerance and utilization. Taking this information one step further, we identified endogenous transcription factors and regulatory mechanisms important for degradation of five model aromatic compounds. To accurately estimate R. opacus growth and consumption on model lignin compounds we pioneered the use of novel extraction procedures prior to GC-MS analysis. Alongside 13 C-metabolic flux analysis, we have elucidated the metabolic routes preferred by Rhodococcus opacus during aromatic compound degradation. Finally, we used in tandem lipidomics and high-resolution mass spectrometry to identify the modulation of mycolic acids and phospholipid membrane composition modification as a strategy for aromatic tolerance in R. opacus. Being a non-model organism, R. opacus lacks the breadth of tools and technical foundation which drive biofuel research in more well-understood microbes such as Escherichia coli. To reduce this burden for use, we designed and produced new tools for genomic manipulation and engineering in R. opacus. These engineering breakthroughs support efficient genomic editing, enabling gene overexpression, repression, and genetic alteration. Using these tools, we have generated synthetically engineered strains with increased lipogenesis and growth, both positive traits required for increased lignin valorization. Optimizing engineered strains for biofuel production from lignocellulose requires extremely sophisticated synthetic rewiring of metabolism. To facilitate systems-level reorganization of metabolism in R. opacus, we created a genome-scale model that accurately predicts metabolic flux and growth rates on the aromatic compound phenol. Lignin requires extensive pre-treatment before biological degradation by R. opacus. Towards an eventual goal of degrading real-world lignin, we developed new depolymerization processes to generate lignin breakdown products (LBP). We optimized LBP storage and composition analysis techniques, enabling accurate prediction of specific LBP compound integration into cell wall components. Overall, through the work funded by this grant we generated 20 manuscripts (17 published, 3 in review/preparation), methods for increased accuracy in metabolomics of aromatic compounds, multiple genetic tools for altering the R. opacus genome, genome scale models for predicting flux through metabolic pathways, as well as multi-omic data for community use. The work funded by this grant has increased the knowledge of aromatic degradation in bacteria and advanced our efforts to optimize R. opacus for lignin valorization.

09 BIOMASS FUELS↗
DOE OSTI2020

CRISPR-Cas “Non-Target” Sites Inhibit On-Target Cutting Rates

CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. Here, the number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

24 POWER TRANSMISSION AND DISTRIBUTION↗
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

CRISPR-COPIES: Web Tool

CRISPR/Cas system has emerged as a powerful genome-editing tool for metabolic engineering and human gene therapy. However, the conundrum of where to integrate heterologous genes on the chromosome using the CRISPR/Cas system remains an open question. Selecting a site for gene integration requires incorporation of complex criteria such as factors involved in CRISPR/Cas-mediated integration, genetic stability, and gene expression and therefore, usually requires strenuous characterization of sites on particular or different chromosomal locations. To address these issues, we developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. The tool applies ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. This submission contains the code we developed to create a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate CRISPR-COPIES will serve as a useful tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, rapid strain construction to produce valuable biochemicals, and human gene and cell therapy.

Bioinformatics↗
DOE OSTI2024

CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

Abstract The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

High-Throughput Functional Genomics for Energy Production

Functional genomics remains a foundational field for establishing genotype-phenotype relationships that enable strain engineering. High-throughput (HTP) methods accelerate the Design-Build-Test-Learn cycle that currently drives synthetic biology towards a forward engineering future. Trackable mutagenesis techniques including transposon insertion sequencing and CRISPR-Cas-mediated genome editing allow for rapid fitness profiling of a collection, or library, of mutants to discover beneficial mutations. Due to the relative speed of these experiments compared to adaptive evolution experiments, iterative rounds of mutagenesis can be implemented for next-generation metabolic engineering efforts to design complex production and tolerance phenotypes. Further, the expansion of these mutagenesis techniques to novel bacteria are opening up industrial microbes that show promise for establishing a bio-based economy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Editorial: Proceedings of ICPSBBB 2018 - 2nd International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology

Plant synthetic biology combines engineering principles with plant biology toward the design, construction, and testing of plants with new traits (Liu and Stewart, 2015). Synthetic biology applies engineering principles, using defined genetic parts with the aim of achieving predictable outcomes. The field aims at providing fuels, pharmaceuticals, bioactive metabolites and increased crop yield. Synthetic biology is increasingly forming a framework in genetic transformation of plants allowing us to improve traits and crop performance. The conference on Plant Synthetic Biology, Bioengineering, and Biotechnology covers broad aspects of plant synthetic biology, ranging from tools to the design of predictable functions and precision genome editing, to re-designed photosynthesis, plant bioengineering techniques, and rational design of novel plant traits. This Frontiers in Plant Science virtual issue on the “Research Topic Proceedings of ICPSBBB 2018 - 2nd International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology” consists of 8 publications, including four reviews and four original research articles, which fall into the following general topics: quantitative and genetic parts, evolution, expression of transgenes and epigenetic changes, engineering of stress tolerance and bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of sequence-specific cytosine deamination by double-stranded DNA deaminase toxin DddA

The interbacterial deaminase toxin DddA catalyzes cytosine-to-uracil conversion in double-stranded (ds) DNA and enables CRISPR-free mitochondrial base editing, but the molecular mechanisms underlying its unique substrate selectivity have remained elusive. Here, we report crystal structures of DddA bound to a dsDNA substrate containing the 5'-TC target motif. These structures show that DddA binds to the minor groove of a sharply bent dsDNA and engages the target cytosine extruded from the double helix. DddA Phe1375 intercalates in dsDNA and displaces the 5' (-1) thymine, which in turn replaces the target (0) cytosine and forms a noncanonical T–G base pair with the juxtaposed guanine. This tandem displacement mechanism allows DddA to locate a target cytosine without flipping it into the active site. Biochemical experiments demonstrate that DNA base mismatches enhance the DddA deaminase activity and relax its sequence selectivity. On the basis of the structural information, we further identified DddA mutants that exhibit attenuated activity or altered substrate preference. Our studies may help design new tools useful in genome editing or other applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Development of male-sterile lines of Setaria viridis to accelerate C 4 model plant genetics

Setaria viridis is a diploid C 4 grass in the Poaceae family, notable for its rapid life cycle of 6–8 weeks from sowing to seed—much shorter than the 4–5 months required by crops such as Zea mays and Sorghum bicolor . This fast growth makes S. viridis a valuable model for C 4 crop research. Genetic crosses are essential for studying gene function, but manual crossing is labor-intensive and time-consuming. Here, to address this, we developed a male-sterile line by targeting the S. viridis ortholog of Setaria italica NO POLLEN 1 ( SiNP1 ), which encodes a glucose–methanol–choline oxidoreductase required for pollen exine formation. Using Cas9 and TREX2 -mediated genome editing, we generated SiNP1 knockouts in both the S. viridis ME034V and A10.1 backgrounds that were fully male-sterile. Backcrossing T 0 male-sterile plants to ME034V wild-type followed by selfing yielded a stable BC 1 F 2 line homozygous for a 59 bp deletion in the S. viridis NO POLLEN 1 gene, easily genotyped by PCR and maintained by heterozygous siblings. Using this line, we developed a simple and efficient crossing protocol that eliminates the need for emasculation. This method enables a single person to perform up to 100 crosses per day—compared to 15 using traditional methods—and yields 20–32 F 1 hybrid seeds per panicle with 100% genetic purity. We also quantified pollen flow and outcrossing frequencies under greenhouse conditions to develop optimal bagging strategies and prevent unintended pollination. This resource accelerates genetic research in S. viridis , enhancing its utility as a premier C 4 model for mapping and functional genomics.

C4 research↗
DOE OSTI2021

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain ?cre-1, while glucose repression was still mostly functional in ?exo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in ?exo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in ?exo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Gabriel, Raphael↗
DOE OSTI2025

Improving the Transformation Efficiency of Synechococcus sp. PCC 7002 via Methylome-Guided Premethylation of DNA

Cyanobacteria are promising microbial platforms for a diverse set of biotechnology applications, from living materials to photosynthetic chemical production, but are less well characterized than commonly engineered microbes such as Escherichia coli. This study facilitates genetic engineering in Synechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain, by identifying ten native methylation motifs and designing shuttle strains that mimic the native methylation state by expressing a subset of heterologous methyltransferases. DNA methylation in E. coli with as few as two active methyltransferases increased transformation efficiency up to 30-fold across four distinct integration sites in PCC 7002. This work provides an experimental framework to bypass native restriction-modification systems for efficient genome editing and metabolic engineering in nonmodel bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for Comparison of Genotyping Assays for Detection of Targeted CRISPR/Cas Mutagenesis in Highly Polyploid Sugarcane

Sugarcane ( Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

Genomics↗