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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants

CRISPR/Cas9 has been widely applied to various plant species accelerating the pace of plant genome editing and precision breeding in crops. Unintended effects beyond off-target nucleotide mutations are still somewhat unexplored. We investigated the degree and patterns of epigenetic changes after gene editing. We examined changes in DNA methylation in genome-edited promoters of naturally hypermethylated genes (AT1G72350 and AT1G09970) and hypomethylated genes (AT3G17320 and AT5G28770) from Arabidopsis. Transgenic plants were developed via Agrobacterium-mediated floral dip transformation. Homozygous edited lines were selected from segregated T2 plants by an in vitro digestion assay using ribonucleoprotein complex. Bisulfite sequencing comparisons were made between paired groups of edited and non-edited plants to identify changes in DNA methylation of the targeted loci. We found that directed mutagenesis via CRISPR/Cas9 resulted in no unintended morphological or epigenetic alterations. Phenotypes of wild-type, transgenic empty vector, and transgenic edited plants were similar. Epigenetic profiles revealed that methylation patterns of promoter regions flanking target sequences were identical among wild-type, transgenic empty vector, and transgenic edited plants. There was no effect of mutation type on epigenetic status. We also evaluated off-target mutagenesis effects in the edited plants. Potential off-target sites containing up to 4-bp mismatch of each target were sequenced. No off-target mutations were detected in candidate sites. Our results showed that CRISPR/Cas9 did not leave an epigenetic footprint on either the immediate gene-edited DNA and flanking DNA or introduce off-target mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Chapter 7 - CRISPR-Based Tools for Microbial Cell Factories

The development of microbial chassis for the production of a variety of biochemicals and biofuels is a growing area of research. How to efficiently manipulate genetic information to achieve optimal production of these compounds is a key area of focus in the field. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become a popular strategy for gene editing and regulation in many organisms due to its versatility and efficacy. Here, we describe methods developed utilizing CRISPR-Cas systems for engineering microbial cell factories (e.g., bacteria and yeast) at the single gene to genome scale for mutagenesis and transcriptional regulation of target genes. Finally, we provide a perspective on the challenges and opportunities for the applications of advanced CRISPR-Cas-based tools for engineering microbial cell factories.

BIOMASS FUELS↗
DOE OSTI2020

Using online tools at the Bovine Genome Database to manually annotate genes in the new reference genome

With the availability of a new highly contiguous Bos taurus reference genome assembly (ARS-UCD1.2), it is the opportune time to upgrade the bovine gene set by seeking input from researchers. Furthermore, advances in graphical genome annotation tools now make it possible for researchers to leverage sequence data generated with the latest technologies to collaboratively curate genes. For many years the Bovine Genome Database (BGD) has provided tools such as the APOLLO genome annotation editor to support manual bovine gene curation. The goal of this paper is to explain the reasoning behind the decisions made in the manual gene curation process while providing examples using the existing BGD tools. We will describe the sources of gene annotation evidence provided at the BGD, including RNAseq and Iso-Seq data. We will also explain how to interpret various data visualizations when curating gene models, and will demonstrate the value of manual gene annotation. The process described here can be applied to manual gene curation for other species with similar tools. With a better understanding of manual gene annotation, researchers will be encouraged to edit gene models and contribute to the enhancement of livestock gene sets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Data for "Viral Delivery of Recombinases Activates Heritable Genetic Switches in Plants"

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ∼0.6 to ∼1.5 kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. An excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

gene editing↗
DOE OSTI2023

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom

Abstract Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

RNAi-Mediated Manipulation of Cuticle Coloration Genes in Lygus hesperus Knight (Hemiptera: Miridae)

Cuticle coloration in insects is a consequence of the accumulation of pigments in a species-specific pattern. Numerous genes are involved in regulating the underlying processes of melanization and sclerotization, and their manipulation can be used to create externally visible markers of successful gene editing. To clarify the roles for many of these genes and examine their suitability as phenotypic markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for six genes (aaNAT, black, ebony, pale, tan, and yellow) were identified, with two variants for black. Sequence and phylogenetic analyses supported preliminary annotations as cuticle pigmentation genes. In accord with observable difference in color patterning, expression varied for each gene by developmental stage, adult age, body part, and sex. Knockdown by injection of dsRNA for each gene produced varied effects in adults, ranging from the non-detectable (black 1, yellow), to moderate decreases (pale, tan) and increases (black 2, ebony) in darkness, to extreme melanization (aaNAT). Based solely on its expression profile and highly visible phenotype, aaNAT appears to be the best marker for tracking transgenic L. hesperus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

CasCollect: targeted assembly of CRISPR-associated operons from high-throughput sequencing data

Abstract CRISPR arrays and CRISPR-associated (Cas) proteins comprise a widespread adaptive immune system in bacteria and archaea. These systems function as a defense against exogenous parasitic mobile genetic elements that include bacteriophages, plasmids and foreign nucleic acids. With the continuous spread of antibiotic resistance, knowledge of pathogen susceptibility to bacteriophage therapy is becoming more critical. Additionally, gene-editing applications would benefit from the discovery of new cas genes with favorable properties. While next-generation sequencing has produced staggering quantities of data, transitioning from raw sequencing reads to the identification of CRISPR/Cas systems has remained challenging. This is especially true for metagenomic data, which has the highest potential for identifying novel cas genes. We report a comprehensive computational pipeline, CasCollect, for the targeted assembly and annotation of cas genes and CRISPR arrays—even isolated arrays—from raw sequencing reads. Benchmarking our targeted assembly pipeline demonstrates significantly improved timing by almost two orders of magnitude compared with conventional assembly and annotation, while retaining the ability to detect CRISPR arrays and cas genes. CasCollect is a highly versatile pipeline and can be used for targeted assembly of any specialty gene set, reconfigurable for user provided Hidden Markov Models and/or reference nucleotide sequences.

Podlevsky, Joshua D.↗
DOE OSTI2023

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

Church, George M.↗
DOE OSTI2024

Genome-wide identification and diversity of FAD2, FAD3 and FAE1 genes in terms of biotechnological importance in Camelina species

False flax, or gold-of-pleasure (Camelina sativa) is an oilseed that has received renewed research interest as a promising vegetable oil feedstock for liquid biofuel production and other non-food uses. This species has also emerged as a model for oilseed biotechnology research that aims to enhance seed oil content and fatty acid quality. To date, a number of genetic engineering and gene editing studies on C. sativa have been reported. Among the most common targets for this research are genes, encoding fatty acid desaturases, elongases, and diacylglycerol acyltransferases. However, the majority of these genes in C. sativa are present in multiple copies due to the allohexaploid nature of the species. Therefore, genetic manipulations require a comprehensive understanding of the diversity of such gene targets.

09 BIOMASS FUELS↗
DOE OSTI2025

Combining genome-wide association studies and expression quantitative trait nucleotide mapping with molecular and genetic validations to identify transcriptional networks regulating drought tolerance in Populus

Objectives: (i). To deploy a large-scale experimental drought trial for up to 1000 unique genotypes of Populus equipping the sites with controlled irrigation and drought treatments that are fully automated and monitored. FULLY COMPLETED (ii) To test the hypothesis that a suite of traits identified for drought tolerance in P. nigra can be measured in drought and control treatments in the wide germplasm collection of P. trichocarpa. FULLY COMPLETED (iii) To use established and novel GWAS model approaches to identify gene loci linked to drought tolerance traits on interest in P. trichocarpa. FULLY COMPLETED (iv) To undertake comparative analysis of GWAS results for drought tolerance traits in P. nigra and P. trichocarpa. PARTIALLY COMPLETED – remains active (v) Using RNAseq in P. trichocarpa, in droughted and control treatments to identify cis- and trans-regulated eQTN. FULLY COMPLETED (vi) Validate up to 50 cis-QTNs, from network hubs using transient protoplast assays. FULLY COMPLETED (vii) To establish Agrobacterium-based gene editing protocols in Populus. FULLY COMPLETED (viii) To utilize early leads from previous research to investigate at least 6 candidate genes for drought tolerance in Populus. FULLY COMPLETED (ix) To validate up to 20 candidate genes for drought tolerance in P. trichocarpa refined from the long-list tested in the transient assays for cis-acting hub gene targets. PARTIALLY COMPLETED- remains active.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva

CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species Trichoderma reesei and developed the first protocol to efficiently transform the non-model species, Sphaerulina musiva. This fungal pathogen constitutes a real threat to the genus Populus, a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineering tools in S. musiva can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Chapter 20: Advances and Application of CRISPR-Cas Systems

A new gene-editing technique called clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have revolutionized genome engineering because of its high efficiency, relatively low cost, and ease of use compared with other techniques such as zinc-finger nucleases and transcription activator-like effector nucleases. CRISPR-Cas systems have transformed biological research, quickly becoming the preferred method for engineering specific genome sequences in industrially relevant microbes, important food crops, and human cells. This powerful new tool has the potential for limitless applications ranging from the treatment of human diseases to improving food security to the generation of renewable and sustainable bioproducts and biofuels. In this chapter, we will discuss advances and applications for these CRISPR-Cas systems in biological engineering.

bacterial↗
DOE OSTI2021

The SvFUL2 transcription factor is required for inflorescence determinacy and timely flowering in Setaria viridis

Inflorescence architecture in cereal crops directly impacts yield potential through regulation of seed number and harvesting ability. Extensive architectural diversity found in inflorescences of grass species is due to spatial and temporal activity and determinacy of meristems, which control the number and arrangement of branches and flowers, and underlie plasticity. Timing of the floral transition is also intimately associated with inflorescence development and architecture, yet little is known about the intersecting pathways and how they are rewired during development. Here, we show that a single mutation in a gene encoding an AP1/FUL-like MADS-box transcription factor significantly delays flowering time and disrupts multiple levels of meristem determinacy in panicles of the C 4 model panicoid grass, Setaria viridis. Previous reports of AP1/FUL-like genes in cereals have revealed extensive functional redundancy, and in panicoid grasses, no associated inflorescence phenotypes have been described. In S. viridis, perturbation of SvFul2, both through chemical mutagenesis and gene editing, converted a normally determinate inflorescence habit to an indeterminate one, and also repressed determinacy in axillary branch and floral meristems. Our analysis of gene networks connected to disruption of SvFul2 identified regulatory hubs at the intersection of floral transition and inflorescence determinacy, providing insights into the optimization of cereal crop architecture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Decreases in polyunsaturated fatty acid content improve heat stress tolerance during flowering and silicle development in pennycress (Thlaspi arvense L.)

Introduction: Pennycress (Thlaspi arvense L.) is an emerging intermediate oilseed crop grown in the offseason between primary summer crops to produce three cash crops in two years. Previous efforts to improve seed oil quality produced Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome-edited lines with decreased polyunsaturated fatty acids (PUFAs) levels, through loss of function of the FATTY ACID DESATURASE 2 (FAD2), REDUCED OLEATE DESATURATION1 (ROD1), and FATTY ACID ELONGASE1 (FAE1) genes. While seed oil compositions were previously characterized, it remains unknown how vegetative and reproductive tissue compositions might differ and affect tolerance to high temperature (HT) conditions.Methods: In four growth chamber experiments, we explored HT tolerance during flowering and silicle development. Plants were subjected to a 34 °C day/28 °C night regime and compared to control plants maintained at 20 °C. Pollen grain viability at a range of temperatures, lipid peroxidation and proline content in leaves and silicles following HT, and seed yield were measured.Results: Both fad2 and rod1 mutant lines had relatively higher pollen viability (71% and 54% respectively) under moderately elevated temperature (28 °C) compared to wild-type controls (37%). They also showed smaller decreases in seed yield (0% and 40% for fad2 and rod1 respectively, compared to 61% for wild type), following HT exposure during late flowering and early silicle development. Silicles of fad2 plants experienced 65% less lipid peroxidation under HT and 55% less buildup of proline, signifying less stress.Discussion: The differential results of fad2 and rod1 are likely due to the role of FAD2 in membranes in all tissues, whereas ROD1 predominantly affects triacylglycerol (TAG) composition in oil-accumulating tissues including pollen. Our results indicate that decreasing PUFAs, through gene editing, can increase heat tolerance in reproductive tissues as an auxiliary benefit accompanying improved seed oil quality.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Scalable and automated CRISPR-based strain engineering using droplet microfluidics

Abstract We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system’s capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase ( galK ) in E. coli and (2) targeted engineering of the glutamine synthetase gene ( glnA ) and the blue-pigment synthetase gene ( bpsA ) to improve indigoidine production in E. coli .

42 ENGINEERING↗
DOE OSTI2020

Machine learning predicts new anti-CRISPR proteins

The increasing use of CRISPR–Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR–Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR–Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metabolic Pathway Engineering

Modern microbial and enzyme engineering and their advancement are increasingly dependent on the marriage of a wide range of sophisticated technologies. For students entering the field of biotechnology, the outlook is indeed daunting. Expertise at levels beyond that of simple familiarity will be needed to conduct competitive research. It goes without saying that to be competitive, all new researchers in this field will need basic preparation in molecular biology, biochemistry, and genetics. Further, experience working with concepts and experimental tools in enzyme biochemistry and kinetics, gene editing, computational metabolic pathway modeling, experimental pathway flux analysis, and computational clustering tools to process complex data sets will be vital for success. We speculate that most biotechnology researchers in early career at this time will build teams of collaborators to address these disparate science fields rather than attempt to become experts in one lab.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗